flavouring group evaluation 4 2-ethylhexyl derivatives ... · flavouring group evaluation 4:...

The EFSA Journal (2008) 929, 1-46


Flavouring Group Evaluation 41:

2-Ethylhexyl derivatives from chemical group 2

Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC)

(Question No EFSA-Q-2003-147)

Adopted on 3 April 2008

SUMMARY

The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (the Panel) is asked to advise the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the Member States. In particular, the Scientific Panel is asked to evaluate three flavouring substances in the Flavouring Group Evaluation 04 (FGE.04), using the Procedure as referred to in the Commission Regulation (EC) No 1565/2000. These three flavouring substances belong to chemical group 2, Annex I of the Commission Regulation (EC) No 1565/2000.

FGE.04 deals with one branched-chain aliphatic aldehyde, one branched-chain aliphatic acid and one ester of a branched-chain alcohol.

All three flavouring substances possess a chiral centre. The stereoisomeric composition has not been specified for any of the substances. Developmental and metabolism studies for 2- ethylhexanoic acid have demonstrated different biological activities for the two enantiomers of the substance.

All the three flavouring substances belong to structural class II according to the decision tree approach presented by Cramer et. al. (1978).

All the three substances have been reported to occur naturally in a wide range of food items.

In its evaluation, the Panel as a default used the Maximised Survey-derived Daily Intake (MSDI) approach to estimate the per capita intakes of the flavouring substances in Europe. However, when the Panel examined the information provided by the European Flavour Industry on the use levels in various foods, it appeared obvious that the MSDI approach in a number of cases would grossly

1 For citation purposes: Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the European Commission on Flavouring Group Evaluation 4: 2-Ethylhexyl derivatives from chemical group 2. The EFSA Journal (2009) 929, 1-49.

Flavouring Group Evaluation 4: 2-Ethylhexyl derivatives from chemical group 2


underestimate the intake by regular consumers of products flavoured at the use level reported by the Industry, especially in those cases where the annual production values were reported to be small. In consequence, the Panel had reservations about the data on use and use levels provided and the intake estimates obtained by the MSDI approach.

In the absence of more precise information that would enable the Panel to make a more realistic estimate of the intakes of the flavouring substances, the Panel has decided also to perform an estimate of the daily intakes per person using a modified Theoretical Added Maximum Daily Intake (mTAMDI) approach based on the normal use levels reported by Industry. In those cases where the mTAMDI approach indicated that the intake of a flavouring substance might exceed its corresponding threshold of concern, the Panel decided not to carry out a formal safety assessment using the Procedure. In these cases the Panel requires more precise data on use and use levels.

According to the default MSDI approach, the three flavouring substances in this group have estimated European daily per capita intakes from use as flavouring substances ranging from 0.0012 to 0.24 microgram, which are below the threshold of concern value for a structural class II substance of 540 microgram/person/day.

The available data on genotoxicity of the three flavouring substances in this group do not preclude their evaluation through the Procedure.

From the metabolism data of the three flavouring substances and the structurally related substance it is not possible to conclude that the metabolites are innocuous.

The toxicity of the flavouring substances in the present group as well as the structurally related substance has been well studied. A No Observed Adverse Effect Level (NOAEL) of 50 mg/kg body weight (bw) per day has been established based on a valid carcinogenicity study in rats. Also in rats, a valid developmental study provided a NOAEL of 100 mg/kg bw/day.

It is considered that on the basis of the default MSDI approach these three flavouring substances would not give rise to safety concerns at levels of intake arising from their use as flavouring substances.

When the estimated intakes were based on the mTAMDI approach they ranged from 1600 to 3900 microgram/person/day for the three flavouring substances from structural class II, which are above the threshold of concern for structural class II of 540 microgram/person/day.

Thus, for the three substances allocated to structural class II, the intakes, estimated on the basis of the mTAMDI approach, exceed the relevant threshold for the structural class. Therefore, more reliable exposure data are required. On the basis of such additional data, these flavouring substances should be reconsidered using the Procedure. Subsequently, additional data might become necessary.

In order to determine whether this evaluation could be applied to the materials of commerce, it is necessary to consider the available specifications:

Specifications including complete purity criteria and identity tests for the materials of commerce have been provided for the three flavouring substances. Information on stereoisomerism is missing for all three substances. Thus, the evaluation of the three substances [FL-no: 05.147, 08.078 and 09.381], cannot be finalised pending further information on specifications.

Keywords

2-ethylhexyl derivatives, 2-ethylhexanol, 2-ethylhexanal, 2-ethylhexanoic acid, 2-ethylhexyl acetate, FGE.04.


TABLE OF CONTENTS Summary ............................................................................................................................................................................... 1 Keywords............................................................................................................................................................................... 2 Background........................................................................................................................................................................... 3 Terms of reference................................................................................................................................................................ 3 Assessment............................................................................................................................................................................. 3

1. Presentation of the Substances in the Flavouring Group Evaluation FGE.04 ........................................................... 3 1.1. Description ........................................................................................................................................................... 3 1.2. Stereoisomers ....................................................................................................................................................... 4 1.3. Natural Occurrence in Food................................................................................................................................. 4

2. Specifications .............................................................................................................................................................. 4 3. Intake data.................................................................................................................................................................... 4

3.1. Estimated Daily per Capita Intake (MSDI approach) ........................................................................................ 5 3.2. Intake Estimated on the Basis of the Modified TAMDI (mTAMDI)................................................................. 6

4. Absorption, Distribution, Metabolism and Elimination ............................................................................................. 7 5. Application of the Procedure for the Safety Evaluation of Flavouring Substances .................................................. 8 6. Comparison of the Intake Estimations based on the MSDI Approach and the mTAMDI Approach....................... 9 7. Considerations of Combined Intakes From Use as Flavouring Substances .............................................................. 9 8. Toxicity...................................................................................................................................................................... 10

8.1. Acute Toxicity Studies....................................................................................................................................... 10 8.2. Subacute, Subchronic, Chronic Toxicity and Carcinogenicity Studies............................................................ 10 8.3. Developmental/Reproductive Toxicity Studies................................................................................................. 11 8.4. Genotoxicity Studies .......................................................................................................................................... 12

9. Conclusions ............................................................................................................................................................... 13 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 4 ..................................... 15 Table 2a: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach)............................................................................................................................................................................. 16 Table 2b: Evaluation Status of Hydrolysis Products of Candidate Esters.................................................................. 17 Table 3: Supporting Substances Summary ..................................................................................................................... 18 Annex I: Procedure for the Safety Evaluation................................................................................................................ 19 Annex II: Use levles / mTAMDI ....................................................................................................................................... 21 Annex III: Metabolism ...................................................................................................................................................... 24 Annex IV: Toxicity ............................................................................................................................................................. 31 References: .......................................................................................................................................................................... 40 Scientific Panel Members .................................................................................................................................................. 45 Acknowledgement .............................................................................................................................................................. 45



BACKGROUND

Regulation (EC) No 2232/96 of the European Parliament and the Council (EC, 1996) lays down a Procedure for the establishment of a list of flavouring substances, the use of which will be authorised to the exclusion of all other substances in the EU. In application of that Regulation, a Register of flavouring substances used in or on foodstuffs in the Member States was adopted by Commission Decision 1999/217/EC (EC, 1999a), as last amended by Commission Decision 2008/478/EC (EC, 2008). Each flavouring substance is attributed a FLAVIS-number (FL-number) and all substances are divided into 34 chemical groups. Substances within a group should have some metabolic and biological behaviour in common.

Substances which are listed in the Register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) No 1565/2000 (EC, 2000a), which is broadly based on the Opinion of the Scientific Committee on Food (SCF, 1999). For the submission of data by the manufacturer, deadlines have been established by Commission Regulation (EC) No 622/2002 (EC, 2002b).

After the completion of the evaluation programme the positive list of flavouring substances for use in or on foods in the EU shall be adopted (Article 5 (1) of Regulation (EC) No 2232/96) (EC, 1996).

TERMS OF REFERENCE

The European Food Safety Authority (EFSA) is requested to carry out a risk assessment on flavouring substances prior to their authorisation and inclusion in a positive list according to Commission Regulation (EC) No 1565/2000 (EC, 2000a).

ASSESSMENT

1. Presentation of the Substances in the Flavouring Group Evaluation FGE.04

1.1. Description

The present Flavouring Group Evaluation 04 (FGE.04), using the Procedure as referred to in the Commission Regulation (EC) No 1565/2000 (the Procedure – shown in schematic form in Annex I), deals with one branched-chain aliphatic aldehyde, one branched-chain aliphatic acid and one ester of a branched-chain alcohol. These flavouring substances belong to chemical group 2, Annex I of Commission Regulation (EC) No 1565/2000. The three flavouring substances (candidate substances) are 2-ethyl substituted hexyl derivatives. The group includes 2-ethylhexanal [FL-no: 05.147], 2-ethylhexanoic acid [FL-no: 08.078] and 2-ethylhexyl acetate [FL-no: 09.381].

The three candidate substances are closely structurally related to 2-ethylhexan-1-ol [FL-no: 02.082] (supporting substance) evaluated at the 49th meeting of the Joint FAO/WHO Expert Committee on Food Additives (the JECFA) in the group “Saturated aliphatic acyclic branched-chain primary alcohols, aldehydes and acids” (JECFA, 1998a; JECFA, 1999b). The JECFA has previously allocated an Acceptable Daily Intake (ADI) to 2-ethylhexan-1-ol of 0-0.5 mg/kg bw (JECFA, 1993a), which was maintained at the 49th meeting.



The three candidate substances under consideration, as well as their chemical Register names, FLAVIS-(FL-), Chemical Abstract Service- (CAS-), Council of Europe- (CoE-) and Flavor and Extract Manufacturers Association- (FEMA-) numbers, structure and specifications, are listed in Table 1 and Table 2a. The hydrolysis products (including their evaluation status) of the candidate ester are listed in Table 2b.

The name and structure for the supporting substance is listed in Table 3, together with its evaluation status (CoE, 1992; SCF, 1995; JECFA, 1999b).

1.2. Stereoisomers

It is recognised that geometrical and optical isomers of substances may have different properties. Their flavour may be different, they may have different chemical properties resulting in possible variation of their absorption, distribution, metabolism, elimination and toxicity. Thus, information must be provided on the configuration of the flavouring substance, i.e. whether it is one of the geometrical/optical isomers, or a defined mixture of stereoisomers. The available specifications of purity will be considered in order to determine whether the safety evaluation carried out for candidate substances for which stereoisomers may exist can be applied to the material of commerce. Flavouring substances with different configurations should have individual chemical names and codes (CAS number, FLAVIS number, etc.).

The three candidate substances possess a chiral centre [FL-no: 05.147, 08.078 and 09.381]. The stereoisomeric composition has not been specified for any of the substances.

1.3. Natural Occurrence in Food

The three candidate substances have all been reported to occur naturally in a wide range of food items. The food sources in which the substances occur are vegetables and fruits (potatoes, soybean, cherry, papaya, raspberry, tea, cranberry, mango, elderberry (juice), mushroom), meat (lamb, mutton, pork), and processed foods (many different cheeses, bread, milk powder, mutton (cooked), pork (cooked), rice (cooked). Quantitative data are only available for 2-ethylhexanoic acid. The levels range from trace amounts in blue cheese up to 1.29 mg/kg in raw mutton (TNO, 2000).

2. Specifications

Purity criteria for the three candidate substances have been provided by the Flavour Industry (EFFA, 2000b; EFFA, 2001a), (Table 1).

Judged against the requirements in Annex II of Commission Regulation (EC) No 1565/2000 (EC, 2000), this information is adequate for all three candidate substances, except that information on chirality is needed for all three substances (see Table 1).

3. Intake data

Annual production volumes of the flavouring substances as surveyed by the Industry can be used to calculate the “Maximised Survey-derived Daily Intake” (MSDI) by assuming that the production figure only represents 60 % of the use in food due to underreporting and that 10 % of the total EU population are consumers (SCF, 1999).



However, the Panel noted that due to year-to-year variability in production volumes, to uncertainties in the underreporting correction factor and to uncertainties in the percentage of consumers, the reliability of intake estimates on the basis of the MSDI approach is difficult to assess.

The Panel also noted that in contrast to the generally low per capita intake figures estimated on the basis of this MSDI approach, in some cases the regular consumption of products flavoured at use levels reported by the Flavour Industry in the submissions would result in much higher intakes. In such cases, the human exposure thresholds below which exposures are not considered to present a safety concern might be exceeded.

Considering that the MSDI model may underestimate the intake of flavouring substances by certain groups of consumers, the SCF recommended also taking into account the results of other intake assessments (SCF, 1999).

One of the alternatives is the “Theoretical Added Maximum Daily Intake” (TAMDI) approach, which is calculated on the basis of standard portions and upper use levels (SCF, 1995) for flavourable beverages and foods in general, with exceptional levels for particular foods. This method is regarded as a conservative estimate of the actual intake in most consumers because it is based on the assumption that the consumer regularly eats and drinks several food products containing the same flavouring substance at the upper use level.

One option to modify the TAMDI approach is to base the calculation on normal rather than upper use levels of the flavouring substances. This modified approach is less conservative (e.g. it may underestimate the intake of consumers being loyal to products flavoured at the maximum use levels reported) (EC, 2000a). However, it is considered a suitable tool to screen and prioritise the flavouring substances according to the need for refined intake data (EFSA, 2004a).

3.1. Estimated Daily per Capita Intake (MSDI approach)

The intake estimation is based on the Maximised Survey-derived Daily Intake (MSDI) approach, which involves the acquisition of data on the amounts used in food as flavourings (SCF, 1999). These data are derived from surveys on annual production volumes in Europe. These surveys were conducted in 1995 by the International Organization of the Flavour Industry, in which flavour manufacturers reported the total amount of each flavouring substance incorporated into food sold in the EU during the previous year (IOFI, 1995). The intake approach does not consider the possible natural occurrence in food.

Average per capita intake (MSDI) is estimated on the assumption that the amount added to food is consumed by 10 % of the population2 (Eurostat, 1998). This is derived for candidate substances from estimates of annual volume of production provided by Industry and incorporates a correction factor of 0.6 to allow for incomplete reporting (60 %) in the Industry surveys (SCF, 1999).

The total annual volume of production of the three candidate substances from use as flavouring substances in Europe is approximately 3 kg (EFFA, 2000b; EFFA, 2001a) and for the one supporting substance 604 kg (IOFI, 1995).

On the basis of the annual volumes of production reported for the three candidate substances, the daily per capita intakes for each of these flavourings have been estimated. The estimated daily per

2 EU figure 375 millions. This figure relates to EU population at the time for which production data are available, and is consistent (comparable) with evaluations conducted prior to the enlargement of the EU. No production data are available for the enlarged EU.



capita intakes of the substances from use as a flavouring substance are: 0.0012 microgram/day of 2-ethylhexanal [FL-no: 05.147], 0.24 microgram/day of 2-ethylhexanoic acid [FL-no: 08.078] and 0.12 microgram/day of 2-ethylhexyl acetate [FL-no: 09.381](see Table 2a).

3.2. Intake Estimated on the Basis of the Modified TAMDI (mTAMDI)

The method for calculation of modified Theoretical Added Maximum Daily Intake (mTAMDI) values is based on the approach used by SCF up to 1995 (SCF, 1995). The assumption is that a person may consume a certain amount of flavourable foods and beverages per day. For the three candidate substances information on food categories and normal and maximum use levels3,4,5 were submitted by the Flavour Industry (EFFA, 2000b; EFFA, 2001a; EFFA, 2007a). The three candidate substances are used in flavoured food products divided into the food categories, outlined in Annex III of the Commission Regulation (EC) No 1565/2000 (EC, 2000a), as shown in Table 3.1. For the present calculation of mTAMDI, the reported normal use levels were used. In the case where different use levels were reported for different food categories the highest reported normal use level was used.

Table 3.1 Use of Candidate Substances

Food category Description Flavourings used Category 1 Dairy products, excluding products of category 2 All three Category 2 Fats and oils, and fat emulsions (type water-in-oil) All three Category 3 Edible ices, including sherbet and sorbet All three Category 4.1 Processed fruits All three Category 4.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and

legumes), and nuts & seeds None

Category 5 Confectionery All three Category 6 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses &

legumes, excluding bakery All three

Category 7 Bakery wares All three Category 8 Meat and meat products, including poultry and game All three Category 9 Fish and fish products, including molluscs, crustaceans and echinoderms (MCE) All three Category 10 Eggs and egg products None Category 11 Sweeteners, including honey None Category 12 Salts, spices, soups, sauces, salads, protein products etc. All three Category 13 Foodstuffs intended for particular nutritional uses All three Category 14.1 Non-alcoholic ("soft") beverages, excl. dairy products All three Category 14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts All three Category 15 Ready-to-eat savouries All three Category 16 Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be

placed in categories 1 - 15 All three

3 ”Normal use” is defined as the average of reported usages and ”maximum use” is defined as the 95th percentile of reported usages (EFFA, 2002i). 4 The normal and maximum use levels in different food categories (EC, 2000) have been extrapolated from figures derived from 12 model flavouring substances (EFFA, 2004e). 5 The use levels from food category 5 “Confectionery” have been inserted as default values for food category 14.2 “Alcoholic beverages” for substances for which no data have been given for food category 14.2 (EFFA, 2007a).



According to the Flavour Industry the normal use levels for the candidate substances are in the range of 1 - 20 mg/kg food, and the maximum use levels are in the range of 5 -100 mg/kg (EFFA, 2000b; EFFA, 2001a; EFFA, 2007a), Table II.1.2, Annex II.

The mTAMDI values range from 1600 to 3900 microgram/person/day for the three candidate substances from structural class II (see Section 5).

For detailed information on use levels and intake estimations based on the mTAMDI approach, see Section 6 and Annex II.

4. Absorption, Distribution, Metabolism and Elimination

From the data presented in Annex III it can be concluded that 2-ethylhexanoic acid [FL-no: 08.078], 2-ethylhexanal [FL-no: 05.147] and the supporting substance 2-ethylhexanol [FL-no: 02.082] are rapidly absorbed from the gastrointestinal (GI) tract. It may also be assumed that the ester 2-ethylhexyl acetate [FL-no: 09.381] is rapidly hydrolysed and that its hydrolysis products acetic acid and 2-ethylhexanol are rapidly absorbed.

With respect to the 2-ethylhexyl moiety, it has been demonstrated in vitro that 2-ethylhexanol is converted into 2-ethylhexanal. The oxidation of 2-ethylhexanal to 2-ethylhexanoic acid has not specifically been studied, but based on the observation that in vivo 2-ethylhexanoic acid and metabolites thereof are major metabolites of 2-ethylhexanol, it can be assumed that in vivo 2-ethylhexanal is oxidised to give 2-ethylhexanoic acid.

2-ethylhexanoic acid in turn is resistant to the normal fatty acid beta-oxidation pathway. Although some beta-oxidation may occur, the ultimate degradation of the molecule is blocked by the 2-ethyl side chain. After the first step in this beta-oxidation, carbon dioxide may be released (i.e. the C1 carbon atom), ultimately resulting in the formation of 2- or 4-heptanone. However, in rodents this seems to be a minor pathway, which may cover approximately 7 % of the dose. More important is omega and omega-1 oxidation, leading to the formation of 2-ethyladipic acid, 6-hydroxy-2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid and several further oxidised products such as 2-ethyl-delta5-hexenoic acid.

The major pathway for elimination of these metabolites is the urine in which large amounts of 2-ethylhexanoic acid and 2-ethyladipic acid can be found, mainly in the form of glucuronide conjugates. Conjugation with sulphate does not seem to occur. The other minor metabolites are usually found in the unconjugated form. 5-hydroxy-2-ethylhexanoic acid may also be found in the form of a lactone, but it is not entirely clear whether this is a real metabolite or an artefact generated during sample clean-up. The available data further show that excretion of 2-ethylhexyl metabolites is virtually complete within 24-48 hours.

The metabolism of 2-ethylhexyl in the forms of diethylhexyl adipate (a plasticiser) and of 2-ethylhexanoic acid, has also been studied in humans. In some studies the major identified human metabolites were 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid, 2-ethyladipic acid and 5-keto-2-ethylhexanoic acid. In one study, large (in comparison with other metabolites) amounts of 3-oxo-2-ethylhexanoic acid and 4-heptanone were observed in human urine samples. Therefore, it has been speculated that in humans beta-oxidation of 2-ethylhexanol (released after hydrolysis of diethylhexyl adipate), leading to these two metabolites, is more important than omega and omega-1 oxidation. In addition, the supposed interspecies difference may also be an effect of the relatively low exposure of the participants in the particular study, in comparison with the (much higher) dose



levels that are usually studied in experimental animals and human volunteer studies. It should also be noted that data on mass balance in the human studies are poor.

Apart from the small amounts of carbon dioxide and the acetic acid residue from 2-ethylhexanoic acid, none of the metabolites expected from the candidate chemicals is endogenous. Overall, it is not possible to conclude that the metabolites are innocuous.

5. Application of the Procedure for the Safety Evaluation of Flavouring Substances

The application of the Procedure is based on intakes estimated on the basis of the MSDI approach. If the mTAMDI approach indicates that the intake of a flavouring substance might exceed its corresponding threshold of concern, a formal safety assessment is not carried out using the Procedure. In these cases the Panel requires more precise data on use and use levels. For comparison of the intake estimations based on the MSDI approach and the mTAMDI approach, see Section 6.

For the safety evaluation of the three candidate substances from chemical group 2 the Procedure was applied. The evaluations of the three substances are summarised in Table 2a.

Step 1.

The three candidate substances are classified into structural class II according to the decision tree approach presented by Cramer et al. (1978).

Step 2.

The three candidate substances cannot be anticipated to be metabolised to innocuous products, and accordingly the evaluation proceeds to step B3.

Step B3 The estimated levels of intake from use as flavouring substances are 0.0012 microgram/capita/day for 2-ethylhexanal [FL-no: 05.147], 0.24 microgram/capita/day for 2-ethylhexanoic acid [FL-no: 08.078] and 0.12 microgram/capita/day for 2-ethylhexyl acetate [FL-no: 09.381]. These figures are all below the threshold of concern for the structural class II of 540 microgram/person/day. Accordingly, these three candidate substances proceed to step B4 of the Procedure.

Step B4.

Based on a carcinogenicity study of the supporting substance 2-ethylhexanol [FL-no: 02.082] in rats, a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg body weight (bw)/day has been established (Astill et al., 1996b). This NOAEL provides a safety margin of more than 8 x 106 in relation to the estimated levels of the combined intake of the three candidate substances of 0.4 microgram/capita/day corresponding to 0.007 microgram/kg bw/day.

Accordingly, it is concluded that 2-ethylhexanal [FL-no: 05.147], 2-ethylhexanoic acid [FL-no: 08.078] and 2-ethylhexyl acetate [FL-no: 09.381] do not pose a safety concern at the estimated levels of intake, based on the MSDI approach.



6. Comparison of the Intake Estimations based on the MSDI Approach and the mTAMDI Approach

The estimated intakes for the three candidate substances in structural class II based on the mTAMDI range from 1600 to 3900 microgram/person/day. For all three substances the mTAMDI values are above the threshold of concern of 540 microgram/person/day for structural class II. This means that further information is required for all three substances. This would include more reliable intake data and then, if required, additional toxicological data.

For comparison of the intake estimates based on the MSDI approach and the mTAMDI approach, see Table 6.1.

Table 6.1 Estimated intakes based on the MSDI approach and the mTAMDI approach

FL-no EU Register name MSDI (μg/capita/day)

mTAMDI (μg/person/day)

Structural class Threshold of concern (µg/person/day)

05.147 2-Ethylhexanal 0.0012 1600 Class II 540 08.078 2-Ethylhexanoic acid 0.24 3100 Class II 540 09.381 2-Ethylhexyl acetate 0.12 3900 Class II 540

7. Considerations of Combined Intakes From Use as Flavouring Substances

Because of structural similarities of candidate and supporting substances, it can be anticipated that many of the flavourings are metabolised through the same metabolic pathways and that the metabolites may affect the same target organs. Further, in case of combined exposure to structurally related flavourings, the pathways could be overloaded. Therefore, combined intake should be considered. As flavourings not included in this FGE may also be metabolised through the same pathways, the combined intake estimates presented here are only preliminary. Currently, the combined intake estimates are only based on MSDI exposure estimates, although it is recognised that this may lead to underestimation of exposure. After completion of all FGEs, this issue should be readdressed.

The total estimated combined daily per capita intake is estimated by summing the MSDI for individual substances.

On the basis of the reported annual volumes of production in Europe (EFFA, 2000b; EFFA, 2001a) the combined estimated daily per capita intake of the three flavouring substances as flavourings is 0.4 microgram/day.

The three candidate substances are structurally related to 2-ethylhexanol evaluated by the JECFA at its 49th meeting (JECFA, 1999b) for which the estimated intake (in Europe) was 74 microgram/capita/day. The total combined level of exposure resulting from the use as flavourings, of the three candidate and the supporting substance does not exceed the threshold of concern for a substance belonging to structural class II of 540 microgram per person per day.



8. Toxicity

8.1. Acute Toxicity Studies

Acute toxicity data are available for all three candidate substances as well as for the supporting substance. The acute toxicity data are summarised in Annex IV, Table IV.1.

8.2. Subacute, Subchronic, Chronic Toxicity and Carcinogenicity Studies

Data are available for the two candidate substances, 2-ethylhexanal [FL-no: 05.147] and 2-ethylhexanoic acid [FL-no: 08.078], and for the supporting substance 2-ethylhexan-1-ol.

For 2-ethylhexanal only subacute data and for 2-ethylhexanoic acid additional subchronic data are available. For 2-ethylhexanoic acid No Observed Effect Levels (NOELs) of 61 and 71 mg/kg body weight (bw)/day for male and female rats and of 180 and 205 mg/kg bw/day for male and female mice, respectively, resulted from the available subchronic toxicity study (Juberg et al., 1998).

For the supporting substance, 2-ethylhexan-1-ol, subchronic toxicity study as well as carcinogenicity studies are available in rats and mice. A No Observed Adverse Effect Level (NOAEL) of 125 mg/kg bw/day was established for rats and mice in the subchronic study and NOAELs of 50 and 200 mg/kg bw/day for rats and mice, respectively, were derived from the carcinogenicity studies (Astill et al., 1996a; Astill et al., 1996b).

Chronic toxicity and carcinogenicity of 2-ethylhexan-1-ol was tested in a valid GLP study (Astill et al., 1996b) in F344 rats and B6C3F1 mice (50 animals per sex and group) treated by oral gavage. Rats received doses of 0 (water), 0 (vehicle), 50, 150 or 500 mg/kg bw on five days per week for 24 months and mice 0 (water), 0 (vehicle), 50, 200 or 750 mg/kg bw on five days per week for 18 months. Treatment-related adverse effects were observed predominantly in the forestomach and liver. In mice, dose-related changes in organ weights were observed in both sexes with significant increases at 200 and 750 mg/kg bw/day in the stomach, liver, kidneys and brain. Corresponding histopathological changes were limited to the highest dose group and were observed in both sexes in the liver. The incidence of hepatocellular carcinomas was marginally, but statistically significantly (p<0.05) increased in females at 750 mg/kg compared with vehicle controls but not with water controls; it was not significantly increased in males but was outside the normal range, i.e. there was a weak adverse trend in hepatocellular carcinoma incidence at high toxic doses. There were no treatment-related differences from controls in tumour incidence for any other organ in males and females. Testis weights were slightly but significantly increased at 50, 200 and 750 mg/kg bw, but without histopathological findings. In rats there were no changes seen at 50 mg/kg bw/day. Significantly increased organ weights of stomach, liver (in females only), kidney, brain and testis (at 500 mg/kg only) were observed at 150 and 500 mg/kg in males and females. The stomach weight was also slightly (6 %) but statistically significantly (p<0.05) increased in females at 50 mg/kg bw but in the absence of any pathological lesions, this was not considered of biological relevance. At the higher doses there was a dose-related increase in the incidence of focal lesions and discolorations in the glandular stomach in female rats, while the incidence was moderate in males. Microscopically the observed gross pathology findings in the glandular stomach of both male and female rats appeared as erosions, ulcers, horny or glandular cysts, or focal lymphoid infiltrations and were not considered neoplastic. There was no adverse trend in liver tumour incidence.

The Panel concluded that 2-ethylhexanol showed a very weak hepatocarcinogenic effect in female mice only and that this type of tumor in this mouse strain is generally considered not relevant for humans. Accordingly, NOAELs of 50 and 200 mg/kg bw/day could be derived from the rat and mice carcinogenicity studies.



The data are summarised in Annex IV, Table IV.2.

8.3. Developmental/Reproductive Toxicity Studies

Developmental studies have been performed for the candidate substance 2-ethylhexanoic acid [FL-no: 08.078] and for the supporting substance 2-ethylhexanol [FL-no: 02.082].

In two studies the effect of different enantiomers on teratogenic action of 2-ethylhexanoic acid (sodium salt) was studied (Hauck et al., 1990; Collins et al., 1992). In both studies the authors found a strong dependence of the teratogenic action on the configuration of the test substance. In the study by Hauck et al. (1990) mice were injected intraperitoneally (i.p.) four times during gestational days 7 and 8 with 500 mg sodium 2-ethylhexanoate either as the (R)-enantiomer, (S)-enantiomer or as the racemate. The (R)-enantiomer gave rise to 59 % exencephaly, the racemate 32 % and the (S)-enantiomer 1 %. In the study by Collins et al. (1992) mice were injected i.p. three times during gestational days 8.0 – 9.0 with 576 mg/kg bw. The (R)-enantiomer gave rise to 51.4 % exencephaly, the racemate to 43.8 % and the (S)-enantiomer to 0 %.

2-Ethylhexanoic acid [FL-no: 08.078] has been studied extensively to evaluate its potential to cause developmental effects. Several of these studies were conducted only at high doses and not according to regulatory guidelines resulting in strong maternal toxicity (Ritter et al., 1985; Ritter et al., 1987; Narotsky et al., 1989; Hauck et al., 1990; Narotsky et al., 1991; Scott et al., 1994), which limit their utility for hazard assessment and risk characterisation. The most appropriate studies for this evaluation are the rat and rabbit oral gavage developmental toxicity studies described below:

2-Ethylhexanoic acid [FL-no: 08.078] (as sodium salt) was administered to pregnant Han:Wistar rats on gestation days 6 through 19 via the drinking water at doses of 100, 300 or 600 mg/kg/day (Pennanen et al., 1993). At 600 mg/kg decreased maternal body weight gain was observed. No other clinical signs were seen and no effects were seen on implantation or foetal survival. At 300 and 600 mg/kg decreased foetal growth was observed and at the highest dose level an increase in the incidence of foetal clubfoot was seen. At all dose levels an increase in the incidence of wavy ribs and retarded cranial ossification was observed, but as the increases in occurrence of these effects were not dose-related and such effects do not persist postnatally, the developmental NOAEL was 100 mg/kg bw/day based on the incidence of clubfoot and the NOAEL for maternal toxicity was 300 mg/kg bw/day.

In a study by Hendrickx et al. (1993) Fischer 344 rats and New Zealand white rabbits were given 2-ethylhexanoic acid [FL-no: 08.078] by gavage during gestational days 6 through 15 (rats) or 6 through 18 (rabbits). Rats received doses 0, 100, 250, 500 mg/kg bw/day and rabbits 0, 25, 125, 250 mg/kg bw/day. Data suggest a developmental NOAEL of 100 mg/kg bw/day in rats, as reduced skeletal ossification occurred at 250 mg/kg bw/day. A NOAEL for maternal toxicity was determined to be 250 mg/kg bw/day for rats in the study. For rabbits there were no effects on foetal viability, growth or morphology at any dose level. NOAEL for developmental toxicity was considered to be ≥250 mg/kg bw/day, whereas a NOAEL for maternal toxicity was determined at 25 mg/kg bw/day (Hendrickx et al., 1993).

There is one NTP study (NTP, 1991; only study abstract available) on the supporting substance 2-ethylhexanol [FL-no: 02.082]. Mice were given 2-ethylhexanol in the diet in doses up to 191 mg/kg bw/day on gestational days 0-17. There was no teratogenicity, developmental toxicity or maternal toxicity noted in the dose-range tested (0, 17, 59, 191 mg/kg bw/day).

The developmental and reproductive toxicity data are summarised in Annex IV, Table IV.3.



8.4. Genotoxicity Studies

Only in vitro genotoxicity studies are available for two of the candidate substances, 2-ethylhexanal [FL-no: 05.147] and 2-ethylhexanoic acid [FL-no: 08.078]. Both in vitro and two in vivo studies are available for the supporting substance 2-ethylhexan-1-ol [FL-no: 02.082].

Candidate substances

For 2-ethylhexanoic acid [FL-no: 08.078], five bacterial mutation assays gave negative results. In a valid chromosomal aberration test in Chinese hamster ovary (CHO) cells, 2-ethylhexanoic acid induced statistically significant increases of cells with structural chromosomal aberrations, only in the presence of exogenous metabolic activation and at doses close to complete toxicity. In the same test system 2-ethylhexanoic acid induced weak dose related increases of sister chromatid exchange (SCE) when applied in subtoxic dose ranges, both with and without metabolic activation. A weak, dose related increase in SCE was also observed in a study in human lymphocytes, only performed without exogenous metabolic activation.

The Panel considered the increase of chromosomal aberrations produced by 2-ethylhexanoic acid at the highest non-lethal concentration of unclear biological relevance. The observed increases in SCE were also considered of limited relevance for the overall evaluation of the genotoxic potential, as this end-point may reflect unspecific interference with DNA replication. One study with negative findings has been performed on the aldehyde of the group (Ames test).

Supporting substance

2-Ethylhexan-1-ol is assumed to be metabolised to the corresponding aldehyde and consequently to the carboxylic acid, i.e. the two candidate substances 2-ethylhexanal [FL-no: 05.147] and 2-ethylhexanoic acid [FL-no: 08.078]. The third candidate substance in the group, 2-ethylhexyl acetate ester (2-EHac) [FL-no: 09.381] is expected to be hydrolysed and it can be expected that its hydrolysis products acetic acid and 2-ethylhexanol are rapidly absorbed. Thus, the genotoxicity and carcinogenicity data on 2-ethylhexan-1-ol cover the candidate substance in this group.

In vitro

Twelve studies including tests for gene mutations in bacterial and mammalian cells, chromosomal aberration and SCE in mammalian cells have been performed on 2-ethylhexan-1-ol. Of these only one study (8-azaguanine resistance assay in Salmonella typhimurium strain TA100), which is not considered valid, reported positive findings, all 11 other studies on bacterial or mammalian cells gave negative findings.

In vivo

In a dominant lethal assay (Rushbrook et al., 1982) using oral doses of 250, 500 or 1000 mg 2-ethylhexanol/kg bw administered for five consecutive days to ICR/SIM mice, negative results were reported. However, the validity of the study could not be evaluated as only a study abstract was available.

In another in vivo study, which was of limited validity, 2-ethylhexan-1-ol did not induce chromosomal aberrations in bone marrow cells after oral administration of 0.02, 0.07 or 0.2 ml/kg bw per day (approximately 16.7, 58.4, 167 mg/kg bw per day assuming a density of 0.8344) to groups of five Fischer 344 male rats for five days (Putman, 1983).



Conclusion on genotoxicity

Bearing in mind the outcome of the evaluation of the carcinogenicity studies on 2-ethylhexan-1-ol in rats and mice, the Panel concluded that the available genotoxicity data do not preclude evaluation of the flavouring substances in the present group using the Procedure.

More detailed information on genotoxicity is given Annex IV, Table IV.4

9. Conclusions

The present Flavouring Group Evaluation deals with one branched-chain aliphatic aldehyde, one branched-chain aliphatic acid and one ester of a branched-chain alcohol. These flavouring substances belong to chemical group 2.

All the candidate substances possess a chiral centre [FL-no: 05.147, 08.078 and 09.381]. The stereoisomeric composition has not been specified for any of the substances. Developmental and metabolism studies for 2-ethylhexanoic acid [FL-no: 08.078] have demonstrated different biological activities for the two enantiomers of the substance.

All three candidate substances belong to structural class II.

The available data on genotoxicity of the three candidate substances in this group do not preclude the evaluation through the Procedure.

From the metabolism data of the three candidate substances and the supporting substance it is not possible to conclude that the metabolites are innocuous.

The toxicity of the candidate substances as well as the supporting substance has been well studied. A NOAEL of 50 mg/kg bw/day has been established based on a valid carcinogenicity study in rats. Also in rats, a valid developmental study provided a NOAEL of 100 mg/kg bw/day.

According to the default MSDI approach, the three candidate substances in this group have estimated European daily per capita intakes from use as flavouring substances ranging from 0.0012 to 0.24 microgram, which are below the threshold of concern value for a structural class II substance of 540 microgram/person/day. It is considered that on the basis of this default MSDI approach these three flavouring substances would not give rise to safety concerns at levels of intake arising from their use as flavouring substances.

When the estimated intakes were based on the mTAMDI approach they ranged from 1600 to 3900 microgram/person/day for the three candidate substances from structural class II, which are above the threshold of concern for structural class II of 540 microgram/person/day.

Thus, for the three substances allocated to structural class II, the intakes, estimated on the basis of the mTAMDI approach, exceed the relevant threshold for the structural class. Therefore, more reliable exposure data are required. On the basis of such additional data, these flavouring substances should be reconsidered using the Procedure. Subsequently, additional data might become necessary.

In order to determine whether this conclusion can be applied to the materials of commerce it is necessary to consider the toxicological assessment in the light of the available specifications of purity:



Specifications including complete purity criteria and identity tests for the materials of commerce have been provided for the three candidate substances. Information on stereoisomerism is missing for all three. Thus, the evaluation of the three substances [FL-no: 05.147, 08.078 and 09.381] cannot be finalised pending further information on specifications.



TABLE 1: SPECIFICATION SUMMARY OF THE SUBSTANCES IN THE FLAVOURING GROUP EVALUATION 4

Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 4

FL-no EU Register name Structural formula FEMA no CoE no CAS no

Phys. form Mol. formula Mol. weight

Solubility 1) Solubility in ethanol 2)

Boiling point, °C 3) Melting point, °C ID test Assay minimum

Refrac. Index 4)Spec. gravity 5)

Specification comments

05.147

2-Ethylhexanal 6) O

10331 123-05-7

Liquid C8H16O 128.21

Slightly soluble 1 ml in 1 ml

160 MS 95 %

1.414-1.420 0.818-0.824

CASrn in Register does not specify isomer.

08.078

2-Ethylhexanoic acid 6)

OH

O

149-57-5

Liquid C8H16O2 144.21

Slightly soluble 1 ml in 1 ml

220 MS 99 %

1.420-1.426 0.893-0.913


09.381

2-Ethylhexyl acetate 6)

O

O

103-09-3

Liquid C10H20O2 172.27

Insoluble 1 ml in 1 ml

198 MS 95 %

1.417-1.423 0.870-0.876


1) Solubility in water, if not otherwise stated. 2) Solubility in 95 % ethanol, if not otherwise stated. 3) At 1013.25 hPa, if not otherwise stated. 4) At 20°C, if not otherwise stated. 5) At 25°C, if not otherwise stated. 6) Stereoisomeric composition not specified.



TABLE 2A: SUMMARY OF SAFETY EVALUATION APPLYING THE PROCEDURE (BASED ON INTAKES CALCULATED BY THE MSDI APPROACH)

Table 2a: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach)

FL-no EU Register name Structural formula MSDI 1) (μg/capita/day)

Class 2) Evaluation procedure path 3)

Outcome on the named compound [ 4) or 5)]

Outcome on the material of commerce [6), 7), or 8)]

Evaluation remarks

05.147

2-Ethylhexanal O

0.0012

Class II B3: Intake below threshold, B4: Adequate NOAEL exists

4) 7)

08.078

2-Ethylhexanoic acid

OH

O

0.24


4) 7)

09.381

2-Ethylhexyl acetate

O

O

0.12


4) 7)

1) EU MSDI: Amount added to food as flavour in (kg / year) x 10E9 / (0.1 x population in Europe (= 375 x 10E6) x 0.6 x 365) = µg/capita/day. 2) Thresholds of concern: Class I = 1800, Class II = 540, Class III = 90 µg/person/day. 3) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot. 4) No safety concern based on intake calculated by the MSDI approach of the named compound. 5) Data must be available on the substance or closely related substances to perform a safety evaluation. 6) No safety concern at estimated level of intake of the material of commerce meeting the specification of Table 1 (based on intake calculated by the MSDI approach). 7) Tentatively regarded as presenting no safety concern (based on intake calculated by the MSDI approach) pending further information on the purity of the material of commerce and/or information on stereoisomerism. 8) No conclusion can be drawn due to lack of information on the purity of the material of commerce.



TABLE 2B: EVALUATION STATUS OF HYDROLYSIS PRODUCTS OF CANDIDATE ESTERS

Table 2b: Evaluation Status of Hydrolysis Products of Candidate Esters

FL-no EU Register name JECFA no

Structural formula SCF status 1) JECFA status 2) CoE status 3) EFSA status

Structural class 4) Procedure path (JECFA) 5)

Comments

02.082 2-Ethylhexan-1-ol 267 OH

Category 1 a) No safety concern b)

Class II A3: Intake below threshold

08.002 Acetic acid 81 O

OH

Category 1 a) No safety concern b) Category A c)

Class I A3: Intake above threshold, A4: Endogenous

1) Category 1: Considered safe in use, Category 2: Temporarily considered safe in use, Category 3: Insufficient data to provide assurance of safety in use, Category 4: Not acceptable due to evidence of toxicity. 2) No safety concern at estimated levels of intake. 3) Category A: Flavouring substance, which may be used in foodstuffs, Category B: Flavouring substance which can be used provisionally in foodstuffs. 4) Threshold of concern: Class I = 1800, Class II = 540, Class III = 90 µg/person/day. 5) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot. a) (SCF, 1995). b) (JECFA, 1999b). c) (CoE, 1992).



TABLE 3: SUPPORTING SUBSTANCES SUMMARY

Table 3: Supporting Substances Summary

FL-no EU Register name Structural formula FEMA no CoE no CAS no

JECFA no Specification available

MSDI (EU) 1) (μg/capita/day)

SCF status 2) JECFA status 3) CoE status 4)

Comments

02.082 2-Ethylhexan-1-ol OH

3151 11763 104-76-7

267 JECFA specification (JECFA, 1998b)

74 Category 1 a) No safety concern b)

ADI: 0-0.5 (JECFA, 1993a).

1) EU MSDI: Amount added to food as flavouring substance in (kg / year) x 10E9 / (0.1 x population in Europe (= 375 x 10E6) x 0.6 x 365) = µg/capita/day. 2) Category 1: Considered safe in use, Category 2: Temporarily considered safe in use, Category 3: Insufficient data to provide assurance of safety in use, Category 4: Not acceptable due to evidence of toxicity. 3) No safety concern at estimated levels of intake. 4) Category A: Flavouring substance, which may be used in foodstuffs, Category B: Flavouring substance which can be used provisionally in foodstuffs. a) (SCF, 1995). b) (JECFA, 1999b).



ANNEX I: PROCEDURE FOR THE SAFETY EVALUATION

The approach for a safety evaluation of chemically defined flavouring substances as referred to in Commission Regulation (EC) No 1565/2000 (EC, 2000a), named the "Procedure", is shown in schematic form in Figure I.1. The Procedure is based on the Opinion of the Scientific Committee on Food expressed on 2 December 1999 (SCF, 1999), which is derived from the evaluation procedure developed by the Joint FAO/WHO Expert Committee on Food Additives at its 44th, 46th and 49th meetings (JECFA, 1995; JECFA, 1996a; JECFA, 1997a; JECFA, 1999b).

The Procedure is a stepwise approach that integrates information on intake from current uses, structure-activity relationships, metabolism and, when needed, toxicity. One of the key elements in the Procedure is the subdivision of flavourings into three structural classes (I, II, III) for which thresholds of concern (human exposure thresholds) have been specified. Exposures below these thresholds are not considered to present a safety concern.

Class I contains flavourings that have simple chemical structures and efficient modes of metabolism, which would suggest a low order of oral toxicity. Class II contains flavourings that have structural features that are less innocuous, but are not suggestive of toxicity. Class III comprises flavourings that have structural features that permit no strong initial presumption of safety, or may even suggest significant toxicity (Cramer et al., 1978). The thresholds of concern for these structural classes of 1800, 540 or 90 microgram/person/day, respectively, are derived from a large database containing data on subchronic and chronic animal studies (JECFA, 1996a).

In Step 1 of the Procedure, the flavourings are assigned to one of the structural classes. The further steps address the following questions:

• can the flavourings be predicted to be metabolised to innocuous products6 (Step 2)?

• do their exposures exceed the threshold of concern for the structural class (Step A3 and B3)?

• are the flavourings or their metabolites endogenous7 (Step A4)?

• does a NOEL exist on the flavourings or on structurally related substances (Step A5 and B4)?

In addition to the data provided for the flavouring substances to be evaluated (candidate substances), toxicological background information available for compounds structurally related to the candidate substances is considered (supporting substances), in order to assure that these data are consistent with the results obtained after application of the Procedure.

The Procedure is not to be applied to flavourings with existing unresolved problems of toxicity. Therefore, the right is reserved to use alternative approaches if data on specific flavourings warranted such actions.

6 “Innocuous metabolic products”: Products that are known or readily predicted to be harmless to humans at the estimated intakes of the flavouring agent” (JECFA, 1997a). 7 “Endogenous substances”: Intermediary metabolites normally present in human tissues and fluids, whether free or conjugated; hormones and other substances with biochemical or physiological regulatory functions are not included (JECFA, 1997a).



Decision tree structural class

Can the substance be predicted to be metabolised to innocuous products?

Procedure for Safety Evaluation of Chemically Defined Flavouring Substances

Do the conditions of use result in an intake greater than the threshold of concern for the structural class?

Do the conditions of use result in an intake greater than the threshold of concern for the structural class?

Data must be available on the substance or closely related

substances to perform a safety evaluation

Does a NOAEL exist for the substance which provides an adequate margin of safety under conditions of intended use, or does a NOAEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances?

Does a NOAEL exist for the substance which provides an adequate margin of safety under conditions of intended use, or does a NOAEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances?

Substance would not be expected to be of safety concern

Is the substance or are its metabolites endogenous?

Additional data required

Step 1.

Step 2.

Step A3.

Step A4.

Step A5.

Step B3.

Step B4.

Yes No

Yes

No No

No

Yes

No

Yes

Yes

Yes

No

Figure I.1 Procedure for Safety Evaluation of Chemically Defined Flavouring Substances



ANNEX II: USE LEVLES / MTAMDI

II.1 Normal and Maximum Use Levels

For each of the 18 Food categories (Table II.1.1) in which the candidate substances are used, Flavour Industry reports a “normal use level” and a “maximum use level” (EC, 2000a). According to the Industry the ”normal use” is defined as the average of reported usages and ”maximum use” is defined as the 95th percentile of reported usages (EFFA, 2002i). The normal and maximum use levels in different food categories have been extrapolated from figures derived from 12 model flavouring substances (EFFA, 2004e).

Table II.1.1 Food categories according to Commission Regulation (EC) No 1565/2000 (EC, 2000a)

Food category Description

01.0 Dairy products, excluding products of category 02.0 02.0 Fats and oils, and fat emulsions (type water-in-oil) 03.0 Edible ices, including sherbet and sorbet 04.1 Processed fruit 04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds 05.0 Confectionery 06.0 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery 07.0 Bakery wares 08.0 Meat and meat products, including poultry and game 09.0 Fish and fish products, including molluscs, crustaceans and echinoderms (MCE) 10.0 Eggs and egg products 11.0 Sweeteners, including honey 12.0 Salts, spices, soups, sauces, salads, protein products, etc. 13.0 Foodstuffs intended for particular nutritional uses 14.1 Non-alcoholic ("soft") beverages, excl. dairy products 14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts 15.0 Ready-to-eat savouries 16.0 Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be placed in categories 01.0 - 15.0

The “normal and maximum use levels” are provided by Industry for the three candidate substances in the present flavouring group (Table II.1.2).

Table II.1.2 Normal and Maximum use levels (mg/kg) for candidate substances in FGE.04 (EFFA, 2000b; EFFA, 2001a

EFFA, 2007a)

Food Categories Normal use levels (mg/kg) Maximum use levels (mg/kg) FL-no

01.0 02.0 03.0 04.1 04.2 05.0 06.0 07.0 08.0 09.0 10.0 11.0 12.0 13.0 14.1 14.2 15.0 16.0 05.147 3

15 2 10

3 15

2 10

- -

4 20

2 10

5 25

1 5

1 5

- -

- -

2 10

3 15

2 10

4 20

5 25

2 10

08.078 3 15

2 10

3 15

2 10

- -

10 50

5 25

10 50

2 10

2 10

- -

- -

2 10

5 25

3 15

10 50

15 75

5 25

09.381 7 35

5 25

10 50

7 35

- -

10 50

5 25

10 50

2 10

2 10

- -

- -

5 25

10 50

5 25

10 50

20 100

5 25

II.2 TAMDI Calculations

The method for calculation of modified Theoretical Added Maximum Daily Intake (mTAMDI) values is based on the approach used by SCF up to 1995 (SCF, 1995). The assumption is that a person may



consume the amount of flavourable foods and beverages listed in Table II.2.1. These consumption estimates are then multiplied by the reported use levels in the different food categories and summed up.

Table II.2.1 Estimated amount of flavourable foods, beverages, and exceptions assumed to be consumed per

person per day (SCF, 1995)

Class of product category Intake estimate (g/day)

Beverages (non-alcoholic) 324.0 Foods 133.4 Exception a: Candy, confectionery 27.0 Exception b: Condiments, seasonings 20.0 Exception c: Alcoholic beverages 20.0 Exception d: Soups, savouries 20.0 Exception e: Others, e.g. chewing gum e.g. 2.0 (chewing gum)

The mTAMDI calculations are based on the normal use levels reported by Industry. The seven food categories used in the SCF TAMDI approach (SCF, 1995) correspond to the 18 food categories as outlined in Commission Regulation (EC) No 1565/2000 (EC, 2000a) and reported by the Flavour Industry in the following way (see Table II.2.2): • Beverages (SCF, 1995) correspond to food category 14.1 (EC, 2000a) • Foods (SCF, 1995) correspond to the food categories 1, 2, 3, 4.1, 4.2, 6, 7, 8, 9, 10, 13, and/or 16

(EC, 2000a) • Exception a (SCF, 1995) corresponds to food category 5 and 11 (EC, 2000a) • Exception b (SCF, 1995) corresponds to food category 15 (EC, 2000a) • Exception c (SCF, 1995) corresponds to food category 14.2 (EC, 2000a) • Exception d (SCF, 1995) corresponds to food category 12 (EC, 2000a) • Exception e (SCF, 1995) corresponds to others, e.g. chewing gum.

Table II.2.2 Distribution of the 18 food categories listed in Commission Regulation (EC) No 1565/2000 (EC, 2000a)

into the seven SCF food categories used for TAMDI calculation (SCF, 1995)

Food categories according to Commission Regulation 1565/2000 Distribution of the seven SCF food categories

Key Food category Food Beverages Exceptions 01 Dairy products, excluding products of category 02.0 Food 02 Fats and oils, and fat emulsions (type water-in-oil) Food 03 Edible ices, including sherbet and sorbet Food 04.1 Processed fruit Food

04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds Food

05 Confectionery Exception a

06 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery Food

07 Bakery wares Food 08 Meat and meat products, including poultry and game Food 09 Fish and fish products, including molluscs, crustaceans and echinoderms (MCE) Food 10 Eggs and egg products Food 11 Sweeteners, including honey Exception a 12 Salts, spices, soups, sauces, salads, protein products, etc. Exception d 13 Foodstuffs intended for particular nutritional uses Food 14.1 Non-alcoholic ("soft") beverages, excl. dairy products Beverages 14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts Exception c 15 Ready-to-eat savouries Exception b

16 Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be placed in categories 01.0 - 15.0 Food



The mTAMDI values (see Table II.2.3) are presented for each of the four flavouring substances in the present flavouring group, for which Industry has provided use and use levels (EFFA, 2000b; EFFA, 2001a; EFFA, 2007a). The mTAMDI values are only given for the highest reported normal use levels.

Table II.2.3 Estimated intakes based on the mTAMDI approach

FL-no EU Register name mTAMDI (μg/person/day) Structural class Threshold of concern (µg/person/day)

05.147 2-Ethylhexanal 1600 Class II 540 08.078 2-Ethylhexanoic acid 3100 Class II 540 09.381 2-Ethylhexyl acetate 3900 Class II 540



ANNEX III: METABOLISM

Introduction

This group of flavouring substances consists of three members: 2-ethylhexanal (2-EHal) [FL-no: 05.147], 2-ethylhexanoic acid (2-EHA) [FL-no: 08.078] and 2-ethylhexyl acetate (2-EHac) [FL-no: 09.381]. All group members can be considered as derivatives of 2-ethylhexanol (2-EHol) [FL-no: 02.082], and for this substance a lot of data have been obtained in studies related to the toxicokinetics of plasticisers, in particular di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA). As far as relevant, these studies will be discussed in this Annex. In addition, 2-ethylhexanol has previously been evaluated as a flavouring substance by JECFA (1998; 1999).

Most of the available studies did not pay attention to the chirality of 2-EHol and derivatives. In one study (Albro, 1975), it was indicated that mixture of stereoisomers was used. Another study addressed conjugation behaviour of separated 2-EHA enantiomers in liver microsomes of various mammalian species (Hamdoune et al., 1995).

III.1 Hydrolysis

Within this group of flavouring substances, hydrolysis is only relevant for the ester 2-EHac. Like with many other esters, it can be expected that 2-EHac is hydrolysed in the GI tract prior to absorption but there is no experimental evidence for this. However, the hydrolysis of the plasticisers DEHP and DEHA has been extensively studied. In particular hydrolysis data for the aliphatic DEHA can be used to evaluate the possibility of hydrolysis of 2-EHac, because in both esters the acid moiety is aliphatic.

(Carbonyl-14C-labelled)-DEHA was administered orally to rats at a dose level of 500 gm/kg bw as a solute in dimethyl sulphoxide (DMSO). Radioactive CO2 appeared in the exhaled air 6 hour (h) post dosing and ~ 80 % of the administered dose was excreted in exhaled air and urine after 24 h, while at 48 h some 97 % of the dose could be found in these excreta. The remainder could be found in the faeces. Urinary radioactivity consisted mainly of adipic acid. No parent DEHA or mono-2-ethylhexyl adipate (MEHA) was seen. Blood samples contained only adipic acid, while in the stomach DEHA, MEHA and adipic acid were found after 1 h. At later stages the amount of intragastric DEHA declined somewhat in favour of adipic acid and MEHA. In the intestine and liver only adipic acid was seen. Tissue homogenates of liver, pancreas and in particular intestine were able to hydrolyse MEHA to give adipic acid. With DEHA, the liver homogenate produced adipic acid and some MEHA, while the pancreas homogenate produced adipic acid and a considerable amount of MEHA. With the intestinal homogenate adipic acid, but hardly any MEHA was found. From these data it can be concluded that DEHA is rapidly hydrolysed after oral administration to rats. This hydrolysis starts in the stomach already, and any DEHA or MEHA that is absorbed intact will be hydrolysed by intestinal or hepatic enzymes (Takahashi et al., 1981).

Fox et al. (1984) studied the hydrolysis of DEHA in rat intestinal homogenates. The substance was hydrolysed to 2-EHol and adipic acid with a half-life of six minutes.

From in vivo studies with mice, rats and monkeys given 500 mg DEHA/kg bw, El Hawari et al. (1990) reported that in monkey urine, but not in mouse and rat urine, MEHA could be found together with 2-



EHol and DEHA and possibly also some other metabolites. However, lack of experimental data precludes firm conclusions to be drawn from this observation.

Conclusion

In rodents, it has been reported that DEHA can be assumed to be rapidly and completely hydrolysed shortly before or immediately after absorption. Limited data obtained in monkeys may indicate that in these species hydrolysis of DEHA could be slightly slower than in rodents. In analogy with the findings for DEHA in rats and mice, it may be assumed the monoester 2-EHac will be hydrolysed (or at least part of the dose) before absorption or shortly thereafter.

III.2 Absorption, Distribution, Metabolism and Excretion

As demonstrated above, the kinetics, among which absorption, of 2-EHol and of 2-EHA has been studied in several laboratory animal species and in humans. No studies were found with 2-EHal. Based on structural similarity to 2-EHol and 2-EHA, it may be assumed that the aldehyde will also be absorbed from the GI tract.

III.2.1 Studies with 2-ethylhexanol [FL-no: 02.082]

In vitro study

The in vitro oxidation of 2-EHol by mammalian alcohol dehydrogenase (not further specified) to yield 2-EHal proceeded with a Vmax of 0.30 μmol/min/mg protein and an apparent Km of 0.67 mM. Conversion of the aldehyde to 2-EHA was not studied but can be assumed to occur because of extensive formation of 2-EHA after oral administration of 2-EHol to rats (see below; Albro, 1975)

In vivo studies

1-14C-labelled 2-Ethylhexanol (2-EHol) was administered to rats by gavage at doses level of 29 μg and 278 mg/kg bw (same amount of radioactivity, the 2-EH was stated to show no optical activity) (Albro, 1975). At both levels of dosing, the majority of the radioactivity was excreted via the urine (25 % after 10 h, 65 % after 12 h and 81 % after 28 h). Faecal excretion accounted for 8.6 % of the administered dose within 20 h post dosing, and about 5 % of the administered radioactivity was excreted as carbon dioxide within about 7 h after administration. Total recovery of radioactivity was 96 % in excreta, 1.4 % in the carcass and 2.7 % in cage washings at 28 h after administration.

Urinary radioactivity consisted of 75 % and 3.4 % of acidic and neutral metabolites, respectively. 21.6 % of the urinary radioactivity was not characterised. No attempt was made to identify or quantify conjugated metabolites.

Sixty-one percent of the acidic radioactive fraction in the urine could be identified as 2-EHA and 22-27 % of this fraction was identified as 2-ethyl 1,6-hexanedioic acid (= 2-ethyl-adipic acid). Twelve to fifteen percent of this fraction could be attributed to 5-hydroxy-2-EHA, while 1 % of the urinary acidic metabolite fraction was identified as 5-keto-2-EHA. Indications were obtained that radioactive acetate was not produced during the metabolism of 1-14C-2-EHol.



Urinary neutral metabolites consisted of (non-radioactive) 4-heptanone and 2-heptanone (in total 7.5 % of the dose) and of unchanged 2-EHol (2.8 % of the dose).

Faecal radioactivity was not further identified (Albro, 1975).

1-14C-2EHol was also given to rats by gavage at dose levels of 50 and 500 mg/kg bw in a single dose study, and at 50 mg/kg bw in a repeated dose study after 14 daily oral doses of the same amount of unlabelled substance. In addition, 1 mg/kg bw 1-14C-2-EHol was also given as intravenous (i.v.) injection (Deisinger et al., 1994). Excretion products, including conjugates and exhaled radioactivity, were studied to draw up the excretion balance and, for the oral studies, to describe metabolic pattern. From the i.v. study also plasma kinetic data were obtained.

After i.v. administration, radioactivity in the blood showed a biphasic elimination pattern with a terminal plasma half-life of 60 h.

In all studies, recovery of radioactivity amounted to 94-100 % at 96 h post dosing. The majority of the radioactivity was excreted within 24 h after administration and in all cases renal excretion was most important (53-60 %; not including cage wash (9-20 % of the dose)). In the oral dose studies faecal excretion accounted for 13-15 % of the dose as compared to 3.8 % in the i.v. study. Exhaled air contained 11-14 % of the radioactivity in the low-dose and repeated dose studies, 8 % in the high-dose study and 23 % in the i.v. study. Exhaled radioactivity was predominantly CO2. Other volatile radioactivity comprised < 1 % of the dose and was not further studied.

Hardly any parent 2-EHol (or its glucuronide conjugate) was found in the urine. No differences in the pattern of urinary metabolites were observed between the low single dose and the low repeated dose (50 mg/kg bw). The major metabolites were 2-EHA, 2-ethyl-adipic acid, 6-hydroxy-2-EHA, and 5-hydroxy-2-EHA representing in total 28-30, 8.3, 6.3-8.7 and 2.4-3.6 % of the administered dose, respectively. No indications were obtained for induction of metabolism after repeated dosing.

At the high single oral dose (500 mg/kg bw), total urinary 2-EHA represented 25 % of the dose, while 2-ethyl-adipic acid comprised only 12 %. Virtually the entire urinary radioactivity was in the form of glucuronide conjugates. Sulphate or other conjugates could not be detected. Minor urinary components were 2-heptanone and 4-heptanone. In addition, 2-ethyl-delta5-hexenoic acid and lactones of 5-hydroxy-2-EHA were also observed, but possibly these might be analytical artefacts, at least in part (Deisinger et al., 1994).

III.2.2 Studies with 2-ethylhexanoic acid [FL-no: 08.078]

In vitro studies

The metabolism of 2-EHA was studied in rat, mouse and human liver microsomes in vitro. 2-ethyladipic acid was the main metabolite produced in rat, mouse and human liver microsomes. 2-ethyl-5-hexenoic acid was produced only in human liver microsomes. The metabolites were analysed also in vivo in urine of 2-EHA-exposed rats (single oral dose of 600 mg/kg bw) and in urine of sawmill workers exposed occupationally to 2-EHA. Both rat and human urine contained 2-ethyl-adipic acid as the main metabolite and also 2-ethyl-5-hexenoic acid. The data indicate that the same metabolites were formed in rats and man and that 2-ethyl-5-hexenoic acid can also be formed in the human liver (Pennanen et al., 1996).



Glucuronide conjugation of 2-EHA stereoisomers was studied in liver microsomal preparations of various mammalian species. (R)- and (S)-2-EHA were glucuronidated by rat microsomes with about the same Km of 2.09 - 2.35 mM and Vmax of 7.35 - 7.72 nmol/min×mg protein, showing no enantioselectivity. Dog liver microsomes were twice as active as rat liver microsomes, while human microsomes were approximately three or five times less active than rat and dog liver microsomes, respectively. Guinea pig and rhesus monkey liver microsomes were about as active as human liver microsomes. With racemic 2-EHA, guinea pig and rabbit microsomes were equally or half as active as human microsomes, respectively. In contrast to the other species, with guinea pig and rabbits, enantioselective glucuronidation was observed with R/S ratios of 3.2 and 2 (same order) (Hamdoune et al., 1995).

In vivo studies

The distribution of 2-14C-2-EHA was studied in rats with whole body autoradiography at 0.5, 1 and 6 h after i.p. injection. In addition, levels of radioactivity in the blood, brain, liver and kidney were determined at 2, 6 and 24 h following the i.p. administration to rats. With whole-body autoradiography, radioactivity was rapidly absorbed from the peritoneal cavity and the majority was found in the liver, kidneys and contents of the GI tract after 0.5 h. At 1 h, some radioactivity was also observed in the lungs and to a lesser extent in the salivary gland and skin. Radioactivity was also observed in the olfactory bulb but not in the brain. At 6 h post dosing radioactivity was still present in the liver and kidneys, but the intensity was clearly diminished.

At 2 h post dosing concentrations in the blood, liver and kidneys amounted to 0.3, 0.2 and 0.1 % dose/g tissue. About 10 times less activity was found in the brain. At 6 h tissue radioactivity content had decreased by a factor of 5 and at 24 h in the liver and kidneys radioactivity was 0.01 % dose/g tissue. In the blood and brain, the activity was below the detection limit (Pennanen et al., 1991a).

English et al. (1998) administered 2-14C-2-ethylhexanoic acid (2-EHA) to rats at dose levels of 100 or 1000 mg/kg bw in a single dose study. In addition, in a repeated dose study 100 mg/kg bw of radioactive 2-EHA was also administered to rats after 14 preceding daily oral doses of 100 mg of unlabelled 2-EHA/kg bw. The substance was dissolved in corn oil and given by gavage. The labelled substance was also given at a dose level of 1 mg/kg bw via i.v. injection. Urine, faeces and blood samples were collected for up to 96 h after dosing. Urinary excretion of conjugated and free metabolites was studied. Exhalation of radioactive carbon dioxide was not monitored.

After i.v. administration, in total 66 % of the radioactive dose was recovered in the urine (+ cage wash) and 3.6 % in the faeces. Of the urinary and faecal metabolites >95 % and >80 %, respectively, were excreted within the first 24 h. Elimination from the blood followed a biphasic pattern. Over 60 % of the radioactivity was eliminated from the blood within the first hour after administration. The terminal plasma half-life of 14C was 85 h. The estimated area under curve (AUC) amounted to 9 μg-equivalents/g×h.

As with the i.v. study, the majority of the dose was excreted in the urine and faeces within 24 h post treatment, also after single dose administration, with either low or high dose, with a total recovery of ca. 90 %. After the high dose, the faecal excretion of metabolites was diminished by about half, in favour of increased urinary elimination. In the low-dose group, peak blood levels were reached within 15-30 minutes. Radioactivity in the blood decreased by 70% within the first hour after dosing. After that a



terminal plasma half-life of 60 h was calculated. The estimated AUC amounted to 560 μg-equivalents/g×h.

In the repeated dose study, only 75 % of the radioactive dose was recovered at 96 h and 15 % of this dose was found in the faeces. Again, the majority of the radioactivity was excreted within the first 24 h after dosing.

Study of the urinary metabolites from the single high-dose animals revealed the presence of large quantities of glucuronide but not sulphate conjugates. The major metabolite was the glucuronide conjugate of 2-EHA, which accounted for 45 % of the administered dose. Glucuronide conjugates, probably of 6-hydroxy-2-EHA and 2-ethyl-adipic acid, accounted each for about 3.6 % of the dose. Unconjugated 2-EHA was found in a quantity of 2.4 % of the dose.

In the single low-dose animals, urinary 2-EHA and its glucuronide conjugate comprised 6.7 and 20 % of the dose, respectively, while 6-hydroxy-2-EHA and 2-ethyl-adipic acid comprised together 14.3 % of the dose. After repeated oral dosing, these percentages were further reduced to 4.5, 12 and 11.6 %, respectively.

Other minor metabolites were 5-hydroxy-2-EHA, ethylketohexanoic acid and delta5-2-heptenone. The structures of several other minor metabolites were not completely elucidated, but probably among these some isomeric lactones were present (English et al., 1998).

Remark: The study authors stated that 2-EHA was rapidly and extensively absorbed. Based on comparison of AUC estimates, it can be concluded that oral bioavailability is approximately 60 %. However, this does not match with the amount recovered in urine (+ cage wash) (~80 % after oral dosing vs. 67 % after i.v.). From the i.v. study it can be concluded that some elimination occurs via the GI tract (including bile). The fact that the high-dose administration results in a relative reduction of (rather than an increase in) faecal excretion is another indication that GI uptake is probably not saturated and that a much higher percentage of the oral dose is actually absorbed.

Especially after the low doses a relatively small part of the urinary metabolites has been elucidated and exhalation of CO2 has not been determined. The author speculated that 2-14C-2-EHA is metabolised to 14CO2, but this is doubtful as the 2-carbon atom is not readily accessible to metabolism (see studies by Albro, 1975; Deisinger et al., 1994). The recovery in the studies by English et al. (1998) is fairly low, which hampers interpretation of quantitative differences in relationship to differences in dose levels and frequency.

Pennanen et al. (1991b) administered the sodium salt of 2-EHA to six-week old rats via their drinking water for 9 weeks, resulting in dose levels of 600 mg/kg bw/day. At the end of the study urine was collected and examined for 2-EHA metabolites. The main metabolite was 2-ethyl-adipic acid. Other identified metabolites were 2-ethyl-delta5-hexenoic acid, lactones of hydroxylated 2-EHA, 6-hydroxy-2-EHA, five other hydroxylated metabolites and 2-EHA-glucuronide (Pennanen et al., 1991b).

III.2.3 Studies with diethylhexyl adipate

Di-(1-14C-2-ethylhexyl)-adipate was administered to male rats and mice and to pregnant mice at a dose level of 843 μg or 84.3 μg/animal in rats and mice, respectively. The substance was given by gavage or by i.v. injection. At regular time intervals animals were sacrificed and the position of the radiolabel was followed using whole-body autoradiography. During the first 24 h, high levels of radioactivity were



observed in the body fat, liver and kidneys (after p.o. and i.v. dosing) and in the intestinal contents (after p.o.). Very little radioactivity could be found in the foetal urine, amniotic fluid, liver and intestine. Urinary excretion and considerable enterohepatic recirculation were also observed.

In the bile cannulation study, radioactivity appeared in the bile much faster when DEHA was given as a solute in DMSO than when corn oil was used. Also blood levels of radioactivity were much higher and rose more rapidly when DMSO was the vehicle, than with corn oil. The authors concluded the absorption of DEHA (or hydrolysis products) is much slower when administered in corn oil than when administered in DMSO (Bergman & Albanus, 1987).

(Hexyl-2-14C)- di-2-(ethylhexyl) adipate (DEHA) was administered by gavage to mice, rats and cynomolgus monkeys at a dose level of 500 mg/kg bw. Excreta were collected for 24 h (mice, rats) or 48 h (monkeys) before sacrifice.

Mice eliminated 91 %, 6-8 % and 1-2 % of the dose via urine, faeces and expired air, respectively. The GI tract, blood and tissues contained in total less than 1 %. In rats, the excreta contained 76 %, 17 % and 1-2 % of the dose, respectively, while tissues, GI tract and blood contained about 6 % of the radioactive dose. Monkeys excreted ~62 % in the urine and 32 % in the faeces. In their blood, GI tract and tissues only traces of radioactivity were found.

Urine contained metabolites of 2-EHol, including 2-EHA, its glucuronide, 5-hydroxy-2-EHA and 2-ethyl-adipic acid. No sulphate conjugates were found. Rat urine had more oxidised metabolites and less 2-EHA glucuronide. Monkey urine contained the mono-ester MEHA, its glucuronide and small amounts of 2-EHol and DEHA (El-Hawari et al., 1990)

The metabolism and pharmacokinetics of [2H10]-DEHA uniformly labelled on the ethyl side chains were determined in six male human volunteers. The dose administered was 46 mg [2H10]-DEHA given orally. No parent molecule was found in plasma. However, the only metabolite detectable in plasma was unconjugated [2H5]-2-EHA (peak plasma level: 1.6 μg/ml after 1-2 h). At 31 h no 2-EHA was detectable in the plasma anymore. The dominant urinary metabolite identified was 2-EHA, present as a conjugate and accounted for an average of 8.6 % of the administered dose (range 6.1 to 12.3 %). Further oxidative metabolism products detected in urine were [2H5]-2-ethyl-5-hydroxyhexanoic acid, [2H5]-2-ethylhexanedioic acid, [2H5]-2-ethyl-5-keto-hexanoic acid and [2H5]-2-ethylhexanol, which together accounted for a further 3.5 % of the dose. In faecal samples, only 0.43 and 0.27 % of the dose were recovered as unchanged DEHA and MEHA, respectively. The fate of the remainder was not determined, due to severe matrix interference. Peak urinary excretion occurred within 8 h after administration. At 36 h no urinary excretion could be observed any longer (Loftus et al., 1993).

2-Ethylhexanoic acid (EHA) was used as a urinary marker of the controlled dietary uptake of di-(2-ethylhexyl) adipate (DEHA) in a limited human population study with 112 participants (Loftus et al., 1994). Following the intake of a mean DEHA dose of 5.4 mg presented with food, the peak urinary elimination of 2-EHA occurred approximately 6 h after ingestion with a mean 2-EHA elimination of 0.43 (±0.16) mg corresponding to a mean of 10 % of the administered dose (range 6.3 to 14.7 %).

Walker and Mills (2001) investigated the urine from individuals with normal and increased plasticiser exposure (the intake was from food and medical devices and not precisely quantified). Using GC/MS, 3-oxo-2-EHA, the (mitochondrial) beta-oxidation product of 2-EHA, was identified as an enol in all samples. In addition, 2-ethyl-adipic acid and 5-hydroxy-EHA, omega and omega-1 oxidation products



of EHA respectively, and 2-EHol-glucuronide were found, but only in trace amounts in some samples from people with increased plasticiser exposure. The median concentrations of 3-oxo-EHA and total 4-heptanone (its decarboxylation product) of seven high plasticiser intake samples were 30-175-fold higher than in normal samples. The authors concluded that beta-oxidation is a major catabolic pathway of EHA in man (Walker & Mills, 2001).

III.3 Conclusion

From the data presented in Annex III it can be concluded that 2-ethylhexanoic acid (2-EHA) [FL-no: 08.078], 2-ethylhexanal (2-EHal) [FL-no: 05.147] and the supporting substance 2-ethylhexanol (2-EHol) [FL-no: 02.082] are rapidly absorbed from the GI tract. It may also be assumed that the 2-ethylhexyl acetate ester (2-EHac) [FL-no: 09.381] is rapidly hydrolysed and that its hydrolysis products acetic acid and 2-EHol are rapidly absorbed.

With respect to the 2-ethylhexyl moiety, it has been demonstrated in vitro that 2-EHol is converted into 2-EHal. The oxidation of 2-EHal to give 2-EHA has not specifically been studied, but based on the observation that in vivo 2-EHA and metabolites thereof are major metabolites of 2-EHol, it can be assumed that in vivo 2-EHal is oxidised to give 2-EHA.

2-EHA in turn is resistant to the normal fatty acid beta-oxidation pathway. Although some beta-oxidation may occur, the ultimate degradation of the molecule is blocked by the 2-ethyl side chain. After the first step in this beta-oxidation, carbon dioxide may be released (i.e. the C1 carbon atom), ultimately resulting in the formation of 2- or 4-heptanone. However, in rodents this seems to be a minor pathway, which may cover some 7 % of the dose. Much more important is omega and omega-1 oxidation, leading to the formation of 2-ethyl-adipic acid, 6-hydroxy-2-EHA, 5-hydroxy-2-EHA and several further oxidised products such as 2-ethyl-delta5-hexenoic acid.

The major pathway for elimination of these metabolites is the urine in which large amounts of 2-EHA, and 2-ethyl-adipic acid can be found, mainly in the form of glucuronide conjugates. Conjugation with sulphate does not seem to occur. The other minor metabolites are usually found in the unconjugated form. 5-hydroxy-2-EHA may also be found in the form of a lactone, but it is not entirely clear whether this is a real metabolite or a study artefact generated during sample clean-up. The available data further show that excretion of 2-EH-metabolites is virtually complete within 24-48 hours.

The metabolism of 2-EH, in the forms of diethylhexyl adipate (DEHA; a plasticiser) and of 2-EHA, has also been studied in humans. In some studies the major identified human metabolites were 2-EHA, 5-hydroxy-2-EHA, 2-ethyl-adipic acid and 5-keto-2-EHA. In one study, in human urine samples large (in comparison with other metabolites) amounts of 3-oxo-2-EHA and 4-heptanone were observed. Therefore, it has been speculated that in humans beta-oxidation of 2-EHol (released after hydrolysis of DEHA), leading to these two metabolites, is much more important than omega and omega-1 oxidation. In addition, the supposed interspecies difference may also be an effect of the relatively low exposure of the participants in the particular study (only “background” exposure), in comparison with the (fairly high) dose levels that are usually studied in experimental animals and human volunteer studies. It should also be noted that data on mass balance in the human studies is poor.

Apart from the small amounts of carbon dioxide and the acetic acid residue from 2-EHac, no endogenous metabolites are formed from the candidate chemicals. From the chemical structures, it is not possible to conclude that the intermediate or final metabolites are innocuous.



ANNEX IV: TOXICITY

Oral acute toxicity data are available for the three candidate substances and for the one supporting substance evaluated by the JECFA at the 49th meeting. The supporting substance is listed in brackets.

TABLE IV.1: ACUTE TOXICITY

Table IV.1: Acute Toxicity

Chemical Name [FL-no] Species Sex LD50 (mg/kg bw) Reference

(2-Ethylhexan-1-ol [02.082]) Rat M/F 3255 - 6400 (Smyth et al., 1969a; Scala & Burtis, 1973; Albro, 1975).

Rat NR 3078 (NTIS, 1991).

Rat NR 2732 (IUCLID Dataset, 2000f).

2-Ethylhexanal [05.147]

Rat NR 3730 (Smyth et al., 1969a; Lington & Bevan, 1994).

Rat NR 1600 to 3000 (Eastman Kodak Co., 1955a; Smyth & Carpenter, 1944; Eastman Kodak Co., 1987f).

Rat NR 3000 (RTECS, 1991).

Rat NR 3276 (IUCLID Dataset, 2000f).

Rat NR 2043 (Eastman Kodak Co., 1987a).

2-Ethylhexanoic acid [08.078]

Rat NR 3000 (Smyth & Carpenter, 1944).

2-Ethylhexyl acetate [09.381] Rat NR 3000 (Smyth & Carpenter, 1944).

M: Male. F: Female. NR: Not Reported.



Subacute / subchronic / chronic toxicity data are available for two candidate substances and for the one supporting substance evaluated at the 49th JECFA meeting. The supporting substance is listed in brackets.

TABLE IV.2: SUBACUTE / SUBCHRONIC / CHRONIC / CARCINOGENICITY STUDIES

Table IV.2: Subacute / Subchronic / Chronic / Carcinogenicity Studies

Chemical Name [FL-no] Species/Sex No./group Route Dose levels

mg/kg/bw Duration (days)

NOAEL (mg/kg/day) Reference Comments

Mouse/M, F 10

Gavage 0, 100, 300, 1000, 1500 11 100 (Astill et al., 1996a) 2, dose range finding study.

Mouse/M, F 10

Gavage 0, 25, 125, 250, 500 90 (5 days per week) 125 (Astill et al., 1996a) Published, well reported study. Significantly (p<0.05) increased relative liver and stomach weight in males at 250 and 500 mg/kg. Treatment related histopathological changes limited to forestomach (males and females) and highest dose group.

Rat/M, F 10

Gavage 0, 100, 300, 1000, 1500 11 100 (Astill et al., 1996a) 2, dose range finding study.

Rat/M, F 10

Gavage 0, 25, 125, 250, 500 90 (5 days per week) 125 (Astill et al., 1996a) Published, well reported study. Reduced body weight gain in the highest dose group. Significantly (p<0.01) increased relative liver, kidney, stomach, testis weight at 250 and 500 mg/kg in males and females. Treatment related histopahtological changes in liver and forestomach in males and females at 500 mg/kg.

(2-Ethylhexan-1-ol [02.082])

Mouse/M, F 50

Gavage 0, 50, 200, 750 540 (5 days per week for 18 months)

200 (Astill et al., 1996b) Valid, well reported published GLP-study carried out in accordance with Health Effect Guidelines of EPA 1987. The mortality was moderate (up to 30 %) until the end of the study. No treatment related clinical effects and haematological findings at 50 and 200 mg/kg. Body weight and food consumption reduced (p<0.01) in M and F at the highest dose. Effects on organ weights mainly limited to highest dose: increased (p<0.01) organ weight of liver (F), stomach (M,F), brain (M,F) and kidney (F); in males kidney weight decreased (p<0.01). Testis weight slightly but significantly increased at 50, 200 and 750 mg/kg. Treatment related and significant histopathological changes limited to liver and lung and highest dose group. No adverse findings reported for testis. Incidence of hepatocellular carcinomas significantly (p<0.05) increased in females at 750 mg/kg compared with vehicle controls but not with water controls; not significantly increased in males but was outside the normal range.







Rat/M, F 50

Gavage 0, 50, 150, 500 730 (5 days per week for 24 months)

50 (Astill et al., 1996b) Valid, well reported published GLP-study carried out in accordance with Health Effect Guidelines of EPA 1987. Mortality at 500 mg/kg markedly increased (up to 52 %) in females after 78 weeks. Body weight but not food consumption significantly (p<0.01) reduced in M and F at 150 and 500 mg/kg. At 150 and 500 mg/kg increased incidence of lethargy and unkemptness. Significantly increased organ weights at 150 and 500 mg/kg in liver (F), stomach (M,F), brain (M,F), kidney (M,F) and testis (M, at 500 mg/kg only). In females stomach weight slightly but significantly (p<0.05) increased at 50 mg/kg. Dose-related increase in incidence of focal lesions and discolorations in the glandular stomach in female rats, but not considered neoplastic. Treatment related and significant histopathological changes limited to the highest dose group with effects in the lung (congestion: M,F; foam cells: M, bronchopneumonia: M,F), liver (congestion: M,F), lymph nodes (hyperplasia: F), spleen (congestion: F) and prostate (atrophy). There was no adverse trend in liver tumour incidence.

Rat Gavage 14 130 (Rhodes et al., 1984) Rat Gavage 14 NR (Lake et al., 1975) Rat/M Diet 21 ND <1200 (Moody & Reddy, 1982) 2-Ethylhexanal [05.147] Laying hen/F Diet 28 ND <1% (Wood & Bitman, 1984) Rat/M, F Diet 0, 730, 1380, 2500

(0.75, 1.5, 3 % in the diet) 14 ND <1380 (Eastman Kodak Co.,

1987d; IUCLID Dataset, 2000f)

Rat/M Diet 2000 (2 % in the diet)

21 ND <2000 (Moody & Reddy, 1982; IUCLID Dataset, 2000f)

Rat/M, F Gavage 0, 200, 800, 1600 15 (11 doses) 2 weeks (5 days per week)

M: 200; F <200 (Eastman Kodak Co., 1987b; IUCLID Dataset, 2000f)

Rat/M, F Diet 0, 65, 330, 10003 (0, 0.1, 0.5, 1.5 % in diet)

90 M: 303; F: 360 (Eastman Kodak Co., 1988a; Eastman Kodak Co., 1987e; IUCLID Dataset, 2000f)

This unpublished study seems to be the same as the one published under Juberg et al., 1998. In high- and mid-dose group increased relative liver weight in males, hepatocyte hypertrophy and eosinophilia in the liver in males and females. All effects reversible within 28 days recovery period. Dose of 65 mg/kg referred to as NOEL and 330 mg/kg as NOAEL .


Rat/M Drinking water

20 NR (Manninen et al., 1989)







Rat/M, F 10

Diet 0, 61, 303, 917 (males) 0, 71, 360, 1068 (females) (0, 0.1, 0.5, 1.5 % in diet)

90 M: 61 F: 71

(Juberg et al., 1998) The publication refers to the studies by Eastman Kodak Co, 1987. The 0.5 % and 1.5 % diets associated with increased relative liver weight (p≤0.05) and histopathological changes in hepatocytes (hypertrophy). Effects reversible within 28 days recovery period. Effects on selected clinical chemistry parameters after 13 weeks: increased (p≤0.05) ALT and cholesterol levels and decreased bilirubin and triglycerides in male and female rats at 1.5 %. Cholesterol also increased in male rats at 0.5 %.

Mouse/M, F 10

Diet 0, 180, 885, 2728 (males) 0, 205, 1038, 3139 (females) (0, 0.1, 0.5, 1.5 % in diet)

90 M: 180 F: 205

(Juberg et al., 1998) The publication refers to the studies by Eastman Kodak Co, 1987. The 0.5 % and 1.5 % diets were associated with increased relative liver weight (p≤0.05) and histopathological changes in hepatocytes (hypertrophy). The effects were reversible following recovery for 28 days. Effects on selected clinical chemistry parameters after 13 weeks were increased (p≤0.05) ALT and cholesterol levels and decreased bilirubin and triglycerides in male and female mice at 1.5 % and in female mice at 0.5 %.

Mouse/M Diet 4 <1000 (Lundgren et al., 1988a; Lundgren et al., 1988b)

Mouse/M, F Diet 0, 1800, 3500, 75003

(0, 0.75, 1.5, 3 % in diet) 14 M<1608, F<1965 (Eastman Kodak Co.,

1987c; IUCLID Dataset, 2000f)

Mouse/M Diet 14 ND <2000 (Lundgren et al., 1987) Mouse/M, F Gavage 0, 200, 800, 1600 15 (11 doses) M: 800, F: 1600 (Eastman Kodak Co.,

1987a; IUCLID Dataset, 2000f)

Mouse/M, F Diet Approx. 190, 950, 29003 (0, 0.1, 0.5, 1.5 % in diet)

90 M: 180, F: 205 (Eastman Kodak Co. 1988b; IUCLID Dataset, 2000f)

This unpublished study is the same as the one published under Juberg et al., 1998.

Laying hen/F Diet 28 ND <1% (Wood & Bitman, 1984)

NR = not reported. ND = not determined. M = Male; F = Female. 1.This study was performed at a single dose level that produced no adverse effects. 2. Summarised by JECFA,49th meeting (JECFA, 1998a). 3 median dose as indicated in TSCATS/OTS 0525548, 1988.



Developmental and reproductive toxicity data are available for one candidate and the one supporting substance.

TABLE IV.3: DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES

Table IV.3: Developmental / Reproductive Toxicity Studies

Chemical name [FL-no] Study type Duration Species /sex Route/doses NOAEL

mg/kg/day Reference Effects/ Comments

(2-Ethylhexan-1-ol [02.082]) Dev. tox/ Gestation day 0-17 Mice Diet 0, 0.009, 0.03, 0.09 % (0, 17, 59, 191 mg/kg bw/day)

Offspring = 191 Maternal toxicity 191

(NTP, 1991) Negative for teratogenicity, developmental toxicity and maternal toxicity in the dose-range tested.

Dev. tox./Gestation day 12 Rat/F Gavage Offspring = 900 Maternal toxicity not reported

(Ritter et al., 1987) 1.

Dev. tox./gestation day 12 Rat/F Oral Offspring < 900 Maternal toxicity not reported

(Ritter et al., 1985) 2.

Dev. tox/gestation days 6-15 Rat/F Gavage Offspring and maternal < 900 (Narotsky et al., 1989; Narotsky et al., 1991)

3.

Dev. tox./gestation days 6-15 Rat/F 25/dosage group

Gavage 0, 100, 250, 500 mg/kg bw/day

Offspring = 100 Maternal = 250

(Hendrickx et al., 1993) 4. Well conducted, valid gavage study. At 100 mg/kg variations such as dilation of lateral ventricles but not statistically significant, at 500 mg/kg bw reduced skeletal ossification.

Dev. Tox./gestation days 6-19 Rat/F Drinking water 0, 100; 300; 600 mg/kg bw/day


(Pennanen et al., 1992a) 5. Decreased maternal body weights and slight decrease in foetal body weight at 600 mg/kg. Doses above 100 mg/kg caused skeletal malformations (clubfoot) while in all exposed groups statistical increases in skeletal variations were seen (not dose-related). The development of visceral tissues was less affected.

Repro./Premating (males 12 wk, females 2 wk), during mating and gestation

Rat/M, F Drinking water 0; 100; 300; 600 mg/kg bw/day

Fertility, develop. and male repro.= 100

(Pennanen et al., 1993) 6. Slight but dose-dependent decrease in fertility and increase in time to mating at 300 and 600 mg/kg. Slight decrease in sperm quality and motolity at 100 and 600 mg/kg. Abnormal sperm occurred at 300 and 600 mg/kg. Several pups were abnormal and physical development assessed by several landmarks was delayed at 300 and 600 mg/kg.

Dev. tox./single dose on gestation days 4, 5, 6 or 7

Rat/F Gavage 600 mg/kg bw

Offspring < 600 Maternal toxicity not reported

(Pennanen et al., 1993) 7. Number of implants were counted on day 10 of gestation. Administration of 600 mg/kg on day 6 decreased the number of implantations and caused resorptions. Effects when administered on other days not reported in study report. No information given on maternal effects.


Dev. tox./gestation days 6-18 Rabbit/F; 15/dosage group

Gavage 0, 25, 125, 250 mg/kg bw/day


(Hendrickx et al., 1993) 8. Well conducted, valid gavage study. Exposure during organogenesis gave caused maternal toxicity but did not give rise to developmental toxicity.



Table IV.3: Developmental / Reproductive Toxicity Studies

Chemical name [FL-no] Study type Duration Species /sex Route/doses NOAEL

mg/kg/day Reference Effects/ Comments

Dev. tox, effects of enantiomers./gestation days 7-8

Mouse/ F I.p. (as Na-ethylhexanoate) either single injection or 500 mg/kg bw x 4

S-isomer = 1000 R-isomer < 1000 Not possible to determine NOAEL

(Hauck et al., 1990) 9. The authors found a strong enantioselectivity of the teratogenic action; 500 mg/kg bw x 4 gave rise to 59 % exencephaly for the R-isomer, 1 % for the S-isomer and 32 % for the racemate.

Dev. tox./ day 12 of gestation Rat/F Gavage Offspring < 1800 Maternal toxicity not reported

(Scott et al., 1994) 10.

Teratology,, dev. tox., effects of enantiomers/gestation days 7.5-9.0 or single dose day 8 or days 7-8; 7.5-8.5; 8-9; 8.5-9.5; 9-10

SWV mouse/F or C57BL/6NCrlBR mouse/F

I.p. or s.c. as (Na-ethylhexanoate) 403 – 1037 mg/kg/bw

Not possible to determine NOAEL

(Collins et al., 1992) Valid study. Object was to determine most sensitive time to induce exencephaly; to assess species differences in susceptibility; and to determine difference in effects of enantiomers. Days 8, 8.5, and 9 of gestation most sensitive time to induce exemcephaly. SWV mice were found to be more susceptible than C57BL6 mice. The R-enantiomer gave rise to 51.4 % exencephaly after 3 i.p. injections of 576 mg/kg bw (gestational days 8.0, 8.5, 9.0), the racemate gave rise to 43.8 % exencephaly, and the S-enantiomer to 0 % exencephaly, with the same dosing regime.

M = Male; F = Female. 1. 1800 mg/kg produced decreased foetal weight, increased dead and resorbed foetuses and increased incidence of malformed foetuses. No toxic or teratogenic effects were observed at the 900 mg/kg level. No information given on maternal effects. 2. At 1800 mg/kg, foetal resorptions, death and malformations including cardiovascular defects, hydronephrosis, deformities of tail and limbs. At 900 mg/kg, fewer resorptions and stillborn foetuses and lower incidence of hydronephrosis. No information given on maternal effects. 3. At 900 mg/kg, effects on development included delayed parturition, decreased progeny viability, reduced pup weights and induced malformations of the vertebrae and ribs. These effects occurred at highly maternally toxic doses. Maternal effects included mortality, decreased body weight or body weight gain, respiratory toxicity and transient signs of depressed motor activity. 4. Increased resorptions, dead foetuses and growth retardation, but no malformations, were observed at 500 mg/kg. Slight developmental toxicity (reduction in skeletal ossification) occurred in foetuses exposed to 250 mg/kg. No embryotoxic effects were noted. The compound was not teratogenic.Dams at 500 mg/kg experienced hypoactivity, ataxia and audible respiration and had increased liver weights (absolute and relative to body weight). 5. Decreased maternal body weights and slight decrease in foetal body weight at 600 mg/kg. Doses above 100 mg/kg caused skeletal malformations (clubfoot) while in all exposed groups statistical increases in skeletal variations were seen (not dose-related). The development of visceral tissues was less affected 600 mg/kg. Several pups were abnormal and physical development assessed by several landmarks was delayed at 300 and 600 mg/kg.. 6. Slight but dose-dependent decrease in fertility and increase in time to mating at 300 and 600 mg/kg. Slight decrease in sperm quality and motility at 100 and 600 mg/kg. Abnormal sperm occurred at 300 and 600 mg/kg. Decreased average litter size at 600 mg/kg. Several pups were abnormal and physical development assessed by several landmarks was delayed at 300 and 600 mg/kg. 7. Number of implants were counted on day 10 of gestation. Administration of 600 mg/kg on day 6 decreased the number of implantations and caused resorptions.Effects when administered on other days not reported in study report. No information given on maternal effects. 8. No foetal- or embryotoxicity was noted and no treatment-related malformation or developmental variations occurred. The compound was not teratogenic. Low incidence of maternal death and abortion at 125 and 250 mg/kg/day. 250 mg/kg/day females also experienced hypoactivity, ataxia and gasping, and showed slight reductions in body weight and feed consumption. Thickened epithelium and ulceration of the glandular portion of the stomach was observed at 250 mg/kg/day. No differences in liver weight were noted. 9. The R-isomer of 2-ethylhexanoic acid was highly teratogenic (exencephaly) and embryotoxic (lethality, foetal weight retardation) at 1000 mg/kg, while the S-isomer had no effects. No information given on maternal effects. 10. Increased resorptions, decreased foetal body weight, increased malformations, mainly of cardiovascular system and appendicular skeleton. No information given on maternal effects.



In vitro mutagenicity/genotoxicity data are available for two candidate substances one supporting substance evaluated at the 49th JECFA meeting. The supporting substance is listed in brackets.

TABLE IV.4: GENOTOXICITY (IN VITRO)

Table IV.4: Genotoxicity (in vitro)

Chemical Name [FL-no] Test System Test Object Concentration Result Reference Comments 8-azaguanine resistance assay S. typhimurium TA100 63, 126, 189 μg/ml

(0.5 – 1.5 mM) (-S9)

Weakly positive (Seed, 1982) 5, Published non-GLP study carried out not according to current guidelines. Insufficiently reported. Study not considered valid. Number of revertants max. 2.5-fold increased at the highest concentration that was highly cytotoxic. Carried out only in the absence of S9. S9 decreased the cytotoxicity and the mutagenic activity of other, related test substances in the same study considerably.

Ames S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

up to 2 ml urine/plate Negative (DiVincenzo et al., 1985) 5, Test performed on the metabolites of 2-ethylhexan-1-ol in urine from rats treated with 1 g/kg bw ethylhexanol for 15 days.

Ames S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

0.01 – 1.0 µl/plate Negative (Kirby et al., 1983) 1, 5.

Ames S. typhimurium TA98, TA100, TA1535, TA1537

33 – 220 μg/plate Negative (Zeiger et al., 1985) 1, 4, 5.

Ames S. typhimurium TA98, TA100, TA1535, TA1537, TA1538, TA2637

100 – 2000 μg/plate Negative (Agarwal et al., 1985) 5.

Ames (preincubation method)

S. typhimurium TA98, TA100, TA1535, TA1537

0, 3.3, 10, 33, 100, 220, 333 μv/plate (-/+S9)

Negative (±S9) (NTP, 1981) Valid study. Study detail available on NTP homepage. Substance was cytotoxic at the highest dose and slightly cytotoxic at 220 μg/plate.

Mouse lymphoma assay L5178Y/TK+/- mouse lymphoma cells 0.01-0.3 µl/ml Negative (Kirby et al., 1983) 1, 5. Rec-assay Bacillus subtilis 500 μg/disk Negative (Tomita et al., 1982) 5. CHO mutation assay Chinese hamster ovary cells 1.5-2.8 mM Negative (Phillips et al., 1982) 5. Chromosomal aberration test Chinese hamster ovary cells 0, 50, 108, 233, 500 μg/ml

(-/+S9) Negative (±S9) (NTP, 1989b) Valid study. Study detail available on NTP homepage.

Cytotoxic effects at the highest dose with and without S9. Sister chromatid exchange test Chinese hamster ovary cells 0, 1.7, 5, 17, 50, 167 μg/ml

(-S9) 0, 5, 17, 50, 167, 500 μg/ml (+S9)

Negative (±S9) (NTP, 1989b) Valid study. Study detail available on NTP homepage. Cytotoxic effects at the highest dose with and without S9.

(2-Ethylhexan-1-ol [02.082])

Unscheduled DNA synthesis assay

Primary rat hepatocytes Not specified Negative (Hodgson et al., 1982) 5.



Table IV.4: Genotoxicity (in vitro)

Chemical Name [FL-no] Test System Test Object Concentration Result Reference Comments 2-Ethylhexanal [05.147] Ames S. typhimurium TA97, TA98, TA100,

TA1535 0, 3, 10, 33, 100, 166, 333 μg/plate (-S9) 0, 3, 10, 33, 100, 166, 333, 666 μg/plate (+S9)

Negative (±S9) (NTP, 1984d) (Zeiger et al., 1988)

1, Valid study. Study details available on NTP homepage (study completed 1984). Cytotoxic effects at the highest dose with and without S9.

Ames S. typhimurium TA98, TA100 0.144-1440 µg/ml (0.001-10 mM)

Negative (Warren et al., 1982b) 1.

Ames S. typhimurium .various strains NR Negative (Haworth et al., 1983) 1. Ames (preincubation method)

S. typhimurium TA97, TA98, TA100, TA1535

0, 33, 100, 333, 1000, 3333, 6666 μg/plate

Negative (NTP, 1984b) Valid study. Cytotoxic effects observed at the highest dose.

Ames S. typhimurium TA97, TA98, TA100, TA1535

33 - 6666 μg /plate Negative (Zeiger et al., 1988) 1.

Gene mutation E. coli WP2 uvrA NR Negative (Hoechst AG, 1982) 1. Sister chromatid exchange test Human lymphocytes 91-361 μg/ml

(0.63 – 2.5 mM) (-S9)

Weakly positive (Sipi et al., 1992) 2, Published non-GLP study not in accordance with OECD guideline 479 (no metabolic activation). This study is not considered valid. Insufficient report of important details of method and results. Treatment for 48 hours, 30 metaphases scored per duplicate culture for SCE and 100 for replication index RI. Dose-dependent effect reported. Statistically significant increase in SCE at 0.63 mM (p<0.01) and 1.25-2.5 mM (p<0.001). Increase was approx. 1.5times the number of SCE in concurrent controls (no detailed data given). RI decreased dose-dependently from 2.7 to 1.0 in the concentration range tested. Concentrations of above 5 mM were clearly toxic.

Chromosomal aberration assay Chinese hamster ovary cells 0, 1030, 1500, 2060, 2530 μg/ml (-S9) 0, 2250, 2530, 3000, 3470 μg/ml (+S9, exp.1) 0, 2700, 3000, 3500, 4000 μg/ml (+S9, exp. 2)

Negative (-S9) Weakly positive (+S9)

(NTP, 1984c) Valid study. Study details available on NTP homepage. Weakly positive result with S9 reproducible in two independent trials. Cytotoxic effects were observed at the highest dose with and without S9. Total aberrations increased at second highest dose, no dose-related effect.


Sister chromatid exchange test Chinese hamster ovary cells 0, 199, 301, 505, 2500 μg/ml and 400-1000 μg/ml (-S9) (2 trials) 505, 753, 1000, 2500, 5200 μg/ml and 1000-3500 μg/ml (+S9) (2 trials)

Positive (±S9) (NTP, 1984c) Valid study. Study details available on NTP homepage. Positive result with S9 reproducible in two independent trials. Cytotoxic effects were observed at the highest dose with and without S9. Increase in SCEs was dose-related.

1. Assay performed with and without metabolic activation. 2. Treatment of cultures was reported to lower pH slightly (max. 0.2 pH units) immediately after introducing the test substance to cultures. 3. Studies are presented only in tables without providing data on dosing, cytotoxicity or pH values and, therefore, key information are not available. 4. Metabolic activation if the form of S9 derived from rat and hamster. 5.Studies summarised by JECFA 49th meeting (JECFA, 1998a).



In vivo mutagenicity/genotoxicity data are only available for the supporting substance evaluated by JECFA at the 49th meeting (JECFA, 1998a).

TABLE IV.5: GENOTOXICITY (IN VIVO)

Table IV.5: Genotoxicity (in vivo)

Chemical Name [FL-no] Test System Test Object Route Dose Result Reference Comments Dominant lethal assay ICR/SIM mice Gavage 250, 500, 1000 mg/kg bw/day

for five days Negative (Rushbrook et al., 1982) Validity cannot be evaluated (only

study abstract available). 2-Ethylhexan-1-ol [02.082]

Chromosomal aberration assay

Fischer 344 rat bone marrow cells

Gavage 16.7, 58.4 or 167 mg/kg bw/day for five days

Negative (Putman, 1983) Limited validity (Only male rats were used).



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SCIENTIFIC PANEL MEMBERS Fernando Aguilar, Herman Nybro Autrup, Susan Barlow, Laurence Castle, Riccardo Crebelli, Wolfgang Dekant, Karl-Heinz Engel, Nathalie Gontard, David Michael Gott, Sandro Grilli, Rainer Gürtler, John Christian Larsen, Jean-Charles Leblanc, Catherine Leclercq, F. Xavier Malcata, Wim Mennes, Maria Rosaria Milana, Iona Pratt, Ivonne Magdalena Catharina Maria Rietjens, Paul P. Tobback, Fidel Toldrá.

ACKNOWLEDGEMENT The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food wishes to thank Judith Amberg-Müller, Ulla Beckman Sundh, Wim Mennes, Jørn Gry, Harriet Wallin, Vibe Beltoft, Pia Lund and Karin Nørby for their contribution to the draft Opinion.

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