tox/2010/29 committee on toxicity of chemicals in …

This is a background paper for discussion. It does not reflect the views of the Committee and should not be cited.

TOX/2010/29 COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT CHRONIC TOXICITY OF METHANOL – FOLATE LEVELS AND THE IMPLICATIONS OF METHANOL EXPOSURE DURING PREGNANCY Introduction

1. Methanol is sequentially oxidised to carbon dioxide via formaldehyde and formate. It is the accumulation of formate which leads to the toxicity of methanol as a result of formate acidosis and the subsequent hypoxic damage. The last step of methanol metabolism is the breakdown of formate to carbon dioxide which requires a folate co-factor. In the light of this, Members asked whether pregnant women could be more vulnerable to methanol toxicity because of their increased requirement for folate, as illustrated by the recommendation to take folic acid supplements during the early stages of pregnancy. Any increased vulnerability could also apply to other people deficient in folate.

2. Annex A to this paper is a review by Kavet and Nauss (1990) which discusses methanol toxicity with a particular focus on folate metabolism. Annex B reviews the available data on methanol and reproductive toxicity.

Mechanism

3. Formate is oxidised to carbon dioxide through the action of formyl-THF synthetase, whereby formic acid combines with tetrahydrofolic acid (THF) to form 10-formyl-THF which is subsequently converted to carbon dioxide by formyl-THF-dehydrogenase. THF is derived from folic acid in the diet and is also regenerated in the folate pathway (see Fig 1 below, taken from Kavet and Nauss, 1990).

4. Rats metabolise formate more rapidly than humans and non-human primates. Following infusion of 100 mg/kg body weight (bw) formate, it is cleared from rats with a half time of 12 minutes and from humans with a half time of 31 minutes. The clearance time increases with dose in both suggesting that the pathway is saturable. At very high doses (approximately 4 g/kg) monkeys appreciably accumulate methanol but rats do not. This occurs because the maximum rate at which rats oxidise formate exceeds the supply of substrate, whereas in non-human primates metabolism of methanol to formate occurs at a rate (1.4-1.8 mmol/kg/h) that exceeds the capacity of the degradation pathway (0.75 mmol/kg/h). At low doses of methanol, however, the formate generating pathway is considerably less efficient than the clearing one and it does not accumulate.


Fig 1.Metabolism of forrmate by folate dependent reactions.

Key

DHF = Dihydrofolic acid

THF = Tetrahydrofolic acid

SAM= S-adenosylmethionine

Reaction [1] requires prior activation of formate by ATP

Reaction [2] involves [10N] Formyl-THF dehydrogenase and uses NADP as a hydrogen acceptor.

Reaction [3] is catalysed by 5-methylene-THF-reductase and is essentially irreversible.

Reaction [4] is catalysed by 5-methyl-THF homocysteine methyl transferase (methionine synthetase) and requires catalytic amounts of vitamin B12 and SAM.

5. The efficiency of formate metabolism is related to hepatic THF concentration, this is in turn controlled by dietary levels and the dynamic equilibrium within the loop that regenerates THF. Experimentally, folate deficient monkeys are more sensitive to methanol toxicity. Additionally, N2O inhibits the folate feedback loop and increases sensitivity to methanol in all species by reducing formate oxidation. The critical variable is the concentration of hepatic THF rather than total folate.


6. Large increases in circulating formate are linked to the acute toxic manifestations that result from exposure to large doses of methanol (>0.3 g/kg bw) (Kavet and Nauss, 1990). This increase would be expected with saturation of the folate pathway. Kavet and Nauss estimated the amount of methanol that would saturate the pathway, using the Michaelis- menten equation (see p 42 of paper attached at Annex A) and estimated that 210 mg/kg bw methanol (12.6 g methanol in a 60 kg adult) would saturate the pathway; this dose is comparable to the lower end of the range associated with toxicity.

Pregnancy and folate status

7. Folate requirements are increased during pregnancy as folate is required for one-carbon transfer reactions including those required for DNA synthesis for the growing fetus and placenta and for increased production of maternal red blood cells (Bailey, 2000). The increased demand for folate during pregnancy means that pregnant women are at greater risk of developing folate deficiency than non-pregnant women. Low red cell folate levels during pregnancy are associated with increased risk of neural tube defects (NTD). Red cell folate levels mirror hepatic levels of THF so these may also be lower (Caudill et al, 1997).

8. Variants in the genes for methylene tetrahydrofolate reductase, methylene tetrahydrofolate dehydrogenase 1, methionine synthetase and human folate carrier are associated with an increased neural tube defect (NTD) risk. However, the precise mechanism(s) by which folate supplementation reduced NTD risk is uncertain and an interaction between the genes involved in folate metabolism may be involved (SACN, 2006).

Reproductive effects of methanol

9. A detailed review is attached at Annex B.

10. There are few data on the reproductive effects of methanol. In rodents very high doses of methanol via inhalation (generally > 2g/kg bw) are associated with a range of teratogenic effects. In the only available primate study, where lower inhalation doses were used, no clear adverse effects were apparent at up to 1800 ppm (approximately 98 mg/kg bw). There are few data on methanol toxicity in humans during pregnancy.

11. Studies of aspartame in rodents did not indicate any adverse effects at levels of 4% in the diet (3500- 600 mg/kg depending on the stage of the study). Aspartame has not been studied for reproductive effects in non-human primates.

Discussion


12. Women are at greater risk of developing poor folate status during pregnancy because of increased requirements during this time. Should red cell folate levels decrease, it is likely that liver stores may also be depleted. Women are advised to take a supplement of 400 µg folic acid while trying to conceive and for the first trimester of pregnancy as this is associated with a decreased risk of having a NTD-affected pregnancy, Higher doses (5 mg/day) are recommended for women at high risk of an NTD-affected pregnancy (women with a previous pregnancy affected by NTD or the offspring of men or women with spina bifida as well as other medical conditions such as diabetes).

13. The pathway for the conversion of formate to carbon dioxide shows that tetrahydrofolate is regenerated, suggesting that the reaction would be slower rather than being abolished in pregnant women or anyone else with reduced folate levels.

14. It has also been estimated that a dose of 210 mg/kg bw methanol is required to saturate the formate to carbon dioxide pathway. Dietary methanol has been estimated to be approximately 1 g/day (16.7 mg/kg in a 60 kg adult) a level comparable to endogenous production. The methanol provided by ADI level of aspartame consumption would be 4 mg/kg bw. Therefore it seems unlikely that dietary levels of methanol exposure would result in adverse effects, even in subjects with lower folate status.

15. High levels of methanol by all routes of exposure have adverse effects on reproduction and development in rodents, though the majority of the studies have used inhalation exposure. Non-human primates are more sensitive to systemic methanol toxicity through reduced folic acid status, however, it is uncertain whether they would be more sensitive reproductive effects. In the one available primate study of methanol by inhalation no clear adverse effects were apparent.

Questions for the Committee

14. Members are asked to comment on the following:

a).Whether pregnant women or other subjects with reduced folate status might be more sensitive to methanol toxicity and, if so, whether that would be relevant at dietary levels.

b). It has been suggested by Kavet and Nauss (1990) that a dose of 210 mg/kg bw methanol would be sufficient to saturate the pathway converting formate to carbon dioxide which if sustained, would allow formate to accumulate. Is this a reasonable estimate and, if so, could it be used as a pragmatic indicator of methanol toxicity?

Secretariat


October 2010


REFERENCES

Bailey, L.B. (2000). New Standard for Dietary Folate Intake in Pregnant Women. Am J Clin Nutr, 71 (suppl 1): 1340S-7S.

Caudill, M.A, Cruz, A.C., Gregory, J.F., et al (1997)

Kavet, R., Nauss, K.M., (1990). The Toxicity of Inhaled Methanol Vapors. CRC Crit Rev Toxicol, 21 21-50.

SACN, 2006. Scientific Advisory Committee on Nutrition. Folate and Disease Prevention, TSO, London.


TOX/2010/29 ANNEX A

COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT CHRONIC TOXICITY OF METHANOL – FOLATE LEVELS AND THE IMPLICATIONS OF METHANOL EXPOSURE DURING PREGNANCY

Kavet, R., Nauss, K.M., (1990). The Toxicity of Inhaled Methanol Vapors. CRC Crit Rev Toxicol, 21 21-50.

For copyright reasons the paper in this Annex is not included in the published version on the COT website. The bibliographic details of the annexed material are listed above. The documents are all in the public domain and individuals can obtain them by application to appropriate sources.

Secretariat October 2010


TOX /2010/29 ANNEX B COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT CHRONIC TOXICITY OF METHANOL – METHANOL IN PREGNANCY

Review of human and animal studies on methanol and aspartame in pregnancy

Methanol

Introduction

1. The general population is exposed to low levels of methanol through environmental sources such as air, water and through contact with methanol-containing consumer products. Dietary sources, including fruit, fruit juices, aspartame, vegetables and alcoholic beverages, are thought to be the primary sources of current exposure in the general population. Methanol is also produced endogenously.

2. Acute exposure to high levels of methanol can result in damage to the nervous system and ocular damage as a result of tissue hypoxia. There have been several studies looking at the reproductive toxicity of methanol in animal models; however there has been little research in the area of methanol and its possible effect during human pregnancy.

Animal Studies

Rodents- Inhalation exposure

3. Nelson et al., (1985) studied the effects of prenatal methanol exposure in Sprague-Dawley rats in an attempt to assess teratogenic effects of industrial solvents. Pregnant rats (15 per exposure group) were exposed to 0, 5000 (1365 mg/kg/bw/day)1, 10,000 (2730 mg/kg/bw/day) or 20,000 (5460 mg/kg/bw/day) ppm methanol in air for 7 hours/day. The two lower dose groups were exposed on

1 The calculation was done as follows, assuming that 1ppm methanol = 1.3 mg/m3 and that the respiratory volume of a rat is 290 L/day or 0.29/24 = 0.012 m3 per hour: 0.012 x no hrs exposure X methanol mg/m3 divided by body wt (0.4 kg).


gestation day (GD) 1–19 whereas the 20,000 ppm group was exposed on GD 7–15. Control groups were exposed to air only. Blood methanol levels in concurrently-exposed, non-pregnant rats on days 1, 10, and 19 of exposure were measured by gas chromatography (GC) at 1000–2170, 1840–2240, and 5250–8650 mg/L in the low- to high-dose group, respectively. Background levels of blood methanol were not provided. The study authors assumed that blood methanol levels in pregnant rats were similar to those determined in non-pregnant rats. Maternal toxicity was evidenced by a slightly unsteady gait only in the high dose group during the first few days of exposure; there were no effects on bodyweight or food intake at any dose. The number of litters evaluated included 30 in the control group, 13 in the low dose group, and 15 in the two highest dose groups. Statistically significant and dose-related reductions in fetal weight were observed in the two highest dose groups. The increased number of litters with skeletal or visceral malformations was statistically significant at the 20,000 ppm dose. A range of visceral malformations were observed including exencephaly and encephalocele. Rudimentary and extra cervical ribs were observed at the greatest frequency at the 20,000 ppm dose. The authors concluded that methanol was a definite teratogen at 20,000 ppm (5460 mg/kg/bw/day), a developmental toxicant (decreased fetal weight) and possible teratogen (numerical elevation of some malformations) at 10,000 ppm (2730 mg/kg/bw/day), with a fetal no effect level of 5000 ppm (1365mg/kg/bw/day).

4. Slikker and Gaylor (1997) evaluated the developmental toxicity data from the above study by Nelson et al, (1985) using a quantitative dose-response risk assessment model. It was determined that excess risks of 1 in 1000 for reduced fetal weight and increased fetal brain malformations would occur from exposure to methanol vapour at concentrations of 980 and 1100 ppm, respectively. Slikker and Gaylor (1997) concluded that adjustment of the risk values by 10 for interspecies sensitivity (intraspecies sensitivity accounted for in the model) would result in values (98 and 110 ppm) comparable to those obtained by adjustment of the NOAEL (5000 ppm) with 100 (50 ppm) for intra-and interspecies variability.

5. Groups of (30-114) CD-1 mice were exposed to 1000, 2000, 5000, 7500, 10000, or 15000 ppm methanol for 7 hours/day on GD 6-15 (equivalent to 585, 1165, 2925, 4388, 5850 and 8775 mg/kg bw/day)2 (Rogers and Mole, 1997). Dams were observed twice daily and weighed on alternate days during the exposure period. Blood methanol concentrations were determined in some mice on gestation days 6, 10, and 15. On day 17, the remaining mice were weighed and killed and the gravid uteri removed. Implantation sites, live and dead fetuses and resorptions were counted, fetuses were examined externally and weighed as a litter. Half of each litter

2 Calculation as previously but assuming that a respiratory volume of 43L and a body weight of 28g for a mouse.


was examined for skeletal morphology and the other half of each litter was examined for internal soft tissue anomalies. Significant increases in the incidence of exencephaly and cleft palate were observed at 5000 ppm and above, increased embryo/fetal death at 7500 ppm and above (including an increasing incidence of full-litter resorptions), and reduced fetal weight at 10,000 ppm and above. A dose-related increase in cervical ribs or ossification sites lateral to the seventh cervical vertebra was significant at 2000 ppm and above. The authors concluded that the NOAEL for the developmental toxicity in this study was 1000 ppm, equivalent to approximately 585 mg/kg/bw/day.

6. In a study sponsored by the Japanese New Energy Development Organization (NEDO, 1987) 36 Sprague-Dawley rats per group were exposed to 0, 200, 1000, or 5000 ppm methanol vapour on GD 7-17 for an average of 22.7 hours/day (on a body weight basis the dose is equivalent to 179, 897 or 4485 mg/kg/day). In the assessment of prenatal development, a total of 19-24 dams/group were sacrificed on GD 20 and examined for implantation sites and number of corpora lutea. Fetuses were assessed for viability, sex, weight, and external malformations. Half the fetuses of each litter were fixed in Bouin’s solution and examined for visceral malformations. Skeletons from the remaining fetuses were stained with alizarin Red S and examined. Dams in the 5000 ppm group had a reduction in bodyweight gain and food and water intake during the first 7 days of methanol exposure. Significant fetal effects were only observed at 5000 ppm and included increased late resorptions, reduced numbers of live fetuses, decreased fetal weight, and increased numbers of litters containing fetuses with malformations, variations, and delayed ossification. Malformations noted were ventricular septal defect, as well as variations in the thymus, vertebrae, and ribs (including cervical ribs).

7. Twelve dams/group were allowed to deliver and nurse their litters. The dams were sacrificed when pups were weaned and examined for implantation sites. Statistically significant effects noted in the 5000 ppm group included prolonged gestation period (21.9 ±0.3 vs. 22.6 ±0.5 days in control and treated group), reduced post-implantation embryo survival (96.3 ±4.2% vs. 86.2 ±16.2%), and number of live pups/litter (15.2 ±1.6 vs. 12.6 ±2.5). Survival rate on postnatal day (PND) 4 was significantly reduced (98.9% vs. 81.8%). Pups were monitored for survival, growth, and achievement of developmental milestones (eyelid opening, auricle development, incisor eruption, testes descent, vaginal opening). Treatment related effects involving developmental milestones were not present when the delay in parturition was taken into consideration. Several organs (brain, thyroid, thymus, and testes) in animals prenatally exposed to 5000 ppm (approximately 4.5 g/kg bw/day) methanol were decreased in weight at 8 weeks of age; overall bodyweight was not adversely


affected by methanol exposure. The authors noted the similarity of fetal abnormality type seen in their study with those reported by Nelson et al., (1985).

Rodents -Oral exposure

8. In a study to examine the effects of early pregnancy exposure to methanol, Holtzman rats were gavaged with water or 1600, 2400, or 3200 mg/kg bw/day methanol in water on GD 1–8 (Cummings, 1993). Using conversion factors set out by Mole et al., (1990), the author estimated that peak blood methanol levels would be 1875, 2800, and 3700 mg/L in the low- to high dose dams, respectively. These blood levels were estimated to equal blood levels resulting from exposure to 10000, 15500, or 21000 ppm methanol vapour respectively, for 6 hours. Eight rats/group were sacrificed on GD 9, 11, and 20. On GD 9, gravid uterine weight was significantly reduced in dams at all doses and a significant decrease in implantation site weight was first noted in the mid dose group. Also noted was a significantly decreased maternal body weight and an increased number of small implantation sites with extravasated blood in the high dose group. Methanol treatment had no effect on the number of implantation sites or corpora lutea, ovarian weight, or serum levels of progesterone, estradiol, luteinizing hormone, and prolactin on the day following the last dose of methanol. An examination of embryonic development on GD 11 revealed no effects on the yolk sac diameter, fetal size, number of somites, viability, or overall development. When litters were examined on GD 20 there were no effects noted on litter size, fetal weight, or resorptions. Fetuses were only assessed for external abnormalities and none were observed. Maternal ovary weight and corpora lutea counts were determined in dams sacrificed on GD 9 and 20 and there were no effects noted. In contrast to results obtained on GD 9, methanol did not affect uterine weight on GD 20. Additionally, the decreased maternal body weight observed at GD 9 after the highest dose of methanol was not observed on GD 20. The authors also studied decidual cell response (DCR) in pseudopregnant3 rats. Results indicated that effects on uterine weight and implantation sites on GD 9 may have resulted from methanol-induced inhibition of the DCR. The author concluded that chemical exposure may cause some impairment of the DCR without necessarily affecting implantation success.

Rodents - I.P exposure

3 This is a female rat that has been treated with hormones (or mated with a sterile male) so that the uterus is receptive to in vitro fertilised ova and blastocysts which implant and develop normally.


9. The effects of methanol exposure during gastrulation4 were looked at by Rogers et al., (2004) in C57BL/6J mice. Pregnant mice were administered two intra-peritoneal injections totalling 3400 or 4900 mg/kg methanol or distilled water four hours apart on GD 7. On GD 17. Litters were examined for numbers of live, dead and resorbed conceptuses, and all fetuses were double stained for skeletal analysis. No maternal intoxication was evident; however, the high dosage level caused a transient deficit in maternal weight gain. The number of fetuses per litter was reduced at both dosages of methanol, and fetal weight was lower in the high dosage group. Craniofacial defects were observed in 55.8% of fetuses in the low dosage group and 91% of fetuses in the high dosage group. These defects included micro/anophthalmia, holoprosencephaly, facial clefts and gross facial angenesis. Skeletal malformations, particularly of the cervical vertebrae, were observed at both dosages of methanol, which are similar findings to the craniofacial defects and skeletal malformations reported in the CD-1 mouse following methanol exposure via inhalation at 10000 ppm (Rogers and Mole, 1997).

Non-human primates.

10. Burbacher et al., (1999) conducted a reproductive and developmental study in Macaca fascicularis monkeys exposed to methanol on behalf of the Health Effects Institute. The animals (11-12/dose/group) were exposed in chambers to air only or to 200, 600 or 1800 ppm methanol for 2.5 h/day during a pre-mating and mating period (about 180 days) and during the entire pregnancy (about 168 days). It was estimated by Clary (2003) that the methanol exposures were equivalent to 11, 33 and 98 mg/kg bw/day. Maternal body weight measurements and clinical observations were conducted routinely throughout the study. There were two different cohorts of monkeys used. All 13 monkeys in cohort 1 were feral born and were 5.5-11 years old. Cohort 2 was made up of 15 feral born monkeys and 9 colony-bred monkeys aged 5-13 years.

11. A small dose related increase in maternal blood methanol but not formate was noted throughout the exposure. Serum folate concentrations were not affected by pregnancy and methanol exposure. There were no clinical signs of toxicity and methanol exposure had no effect on menstrual cycles, conception rate or live birth index.

12. All methanol-exposed animals had a decrease of 6-8 days in the duration of pregnancy compared to controls. The reduction may have occurred because five methanol-treated females were C-sectioned due to pregnancy complications such as uterine bleeding and prolonged unproductive labour. These observations were not observed in the control group. It was unclear whether this effect was due to methanol 4 This is an early stage of embryonic development.


exposure since there was no dose response relationship and no difference in parameters such as birth weight or was due to an outlier in the control group (Clary, 2003). There were no methanol related effects on offspring birthweight or newborn health status or infant growth and physical development in the first year of life Burbacher et al., (1999).

13. The same study appears to have been published subsequently in more detail as Burbacher et al., (2004)5. The authors concluded that these results suggest a modest but significant effect of methanol on the biochemical events that control the timing of birth.

Human Studies

Case reports,

14. There are several clinical case reports in the literature on the effects of methanol consumption/inhalation on pregnant women. One such report described the first human newborn with a documented methanol concentration resulting from maternal exposure, which suggests that methanol can pass from the mother through the placenta to the fetus. (Belson and Morgan, 2004). Clinicians have reported a wide range of clinical manifestations after exposure to methanol. Hantson et al., (1997) reported on a 26 year old woman who ingested 250-500 ml methanol (equivalent to 2800 – 5650 mg/kg/bw in a 70 kg pregnant woman) during the 38th week of pregnancy. After treatment and subsequent birth of the child, no complications were noted in the mother or the newborn infant. These findings are in contrast to a case presentation by Bharti (2003) in which the author described a pre term male infant who developed cerebral infarcts in utero, leading to large areas of bilateral frontal cortical leukomalacia following maternal inhalation of carburettor-cleaning fluid during pregnancy The level of exposure to carburettor cleaning fluid was not reported in this study. The infant presented with significant metabolic acidosis at birth.

Epidemiology studies

15. Lorente et al., (2000) investigated the role of maternal occupational exposure in occurrence of cleft lip and palate. Data from the study were obtained from a multicenter European case-referent study utilizing 6 congenital malformation registers between 1989 and 1992. Occupational exposures during the first trimester were studied in 851 women; 100 cases had infants with oral clefts and 751 referents had infants without oral clefts. The subjects were interviewed to determine

5 Original not yet obtained by secretariat.


occupational history and the types of products used on the job. An industrial hygienist reviewed interview responses to determine the probability of chemical exposures. Confounding factors considered included maternal age, socioeconomic status, residence, urbanization, country of origin, and medical history. Subjects were interviewed about smoking, and alcohol intake but it is not clear if the analyses considered those factors. Data were analyzed by estimating an adjusted odds ratio for each type of exposure and then conducting a stepwise logistic regression on all exposures with P≤20%. Analyses determined that at least 10% of the subjects were likely exposed to methanol during the first trimester of pregnancy. Odds ratios of 3.61 (95% C.I.: 0.91–14.4) and 3.77 (95% C.I.: 0.65–21.8) were calculated for methanol exposure and occurrence of cleft palate only and cleft lip with or without cleft palate, respectively. Although these odds ratios appear elevated, they are not statistically significant and therefore are also consistent with the null hypothesis of no increased risk for orofacial clefts after occupational exposure to methanol. The authors reported no association between methanol exposure and oral clefts.

Conclusion

16. There is limited information available on the effects of maternal exposure to methanol on the developing fetus. Three of the cases presented above are single case studies provided by clinicians and few other data are available.

Aspartame

Introduction

17. Aspartame is a synthetic non-nutritive sweetener and is approximately 200 times sweeter than sucrose. It is a methyl ester of a dipeptide composed of aspartic acid and phenylalanine.

18. The intestinal absorption and metabolism of aspartame has been investigated in rodents, pigs, primates and humans. In all species examined, aspartame is metabolised in the gastrointestinal tract by esterases and peptidases into three components: the two constituent amino acids, aspartic acid and phenylalanine, and methanol (Stegink, 1987). Aspartame can be completely hydrolysed to these three components in the gastrointestinal lumen, which are then absorbed into the general circulation. Alternately it can be hydrolysed to methanol and aspartylphenylalanine dipeptide, in which case the dipeptide is absorbed into the gastrointestinal mucosa cells and then cleaved into the amino acids (Stegink, 1987). Metabolism of the three cleaved components of aspartame has been shown to be identical to the metabolism of the components given individually (Stegink, 1987). On the basis of molecular


weight calculations, it is generally assumed that 10% of aspartame can be released as methanol.

19. The generation of methanol from the breakdown and metabolism of aspartame has been raised as an issue regarding the safety of aspartame. This has been reviewed in the context of the total amount of methanol consumed in the diet (Magnuson et al., 2007). The total methanol consumption from natural sources is estimated to average 10.7 mg/day. For individuals at the 95th percentile of methanol-containing foods the estimated methanol consumption figures are 33.3 mg/day or 0.55 mg/kg bw/day for a 60 kg individual. However, these figures underestimate the total exposure due to lack of data for several exposures. The average content of methanol in some commonly consumed foods, such as potatoes, onions, and celery was not reported and therefore methanol from these foods was not included in the total estimated methanol consumption. Taucher et al., (1995) estimated that humans produce approximately 1000 mg of methanol daily from fruits and vegetables. Methanol generated from aspartame is estimated to average 33 mg/day or 0.55 mg/kg bw/day for a 60 kg individual. For individuals at the 95th percentile the estimated methanol consumption figures are 93.9 mg/day or 1.57 mg/kg bw/day for a 60-kg individual. Since the figures for methanol from aspartame are derived from worse case assumptions, these are likely to overestimate methanol consumption from aspartame (Magnuson et al., 2007).

20. Since aspartame is a permitted food additive, the manufacturers were required to submit a full package of safety data including a range of studies on reproductive and developmental toxicity before approval was granted. Previous reviews of the data by the COT as well as the EU Scientific Committee for Food (SC)F and Joint FAO/WHO Expert Committee on Food Additives (JECFA) have not raised concerns about the potential for adverse reproductive effects. These data have not been considered further.

Animal Studies

Rats

21. The effect of aspartame on reproduction was evaluated in a study by Brunner et al., (1979). Sprague-Dawley rats were fed diets containing aspartame at levels of 0, 2, 4, or 6% for at least 14 days before breeding, during breeding, pregnancy and lactation, and for 90 days postnatally. Diet containing 3% phenylalanine was also investigated. Food consumption measurements determined that these dietary levels of aspartame resulted in doses of approximately 0, 1600, 3500, and 5000 mg/kg bw/day during prebreeding and gestation; approximately 0, 4000, 7000, and 9600 mg/kg bw/day during lactation; and 0, 3000, 6000, and 9000 mg/kg bw/day post


weaning. Breeding pairs were enrolled in the study until 18 litters were available for each group. The number of animals used for various measures and parameters was highly variable; therefore, the total number of animals used in the study was not known. There was no effect on body weights during prebreeding or on maternal weight gain during gestation, but rats fed 6% aspartame during lactation lost more weight than other dietary groups. Increased offspring mortality was observed in rats fed the 6% aspartame. Gestation length and litter size was not affected by diet, however pups fed aspartame weighed significantly less than controls by PND 30 and remained lighter throughout the study. Eye opening was delayed by 1 day in pups in the 6% aspartame and 3% phenylalanine diet groups, but timing of pinnae detachment and incisor eruption were not affected. The NOAEL for this study is 4 % aspartame. Similar findings of no effect of maternal exposure to aspartame up to doses of 1614 mg/kg bw/day, and pup exposure of 3566 mg/kg bw/day on pup development were reported by Holder (1989).

22. To assess potential post-coital anti-fertility effects of aspartame, groups of five female Charles River rats were administered 300 mg/kg bw/day aspartame in corn oil or corn oil alone intragastrically for 7 days following mating (Lennon et al., 1980). A similar experiment was conducted in female hamsters with a group of 15 treated with corn oil only, and a group of 5 given daily doses of 300 mg/kg bw aspartame in corn oil. There were no differences noted in the number of rats that became pregnant, or the implantation and regression of corpora lutea in hamsters. However, the number of animals used in these studies was very small.

23. Lederer et al., (1985) conducted a study in which female Wistar rats were mated and then fed a commercial rat diet containing 10% aspartame (n = 14), 3% diketopiperazine (DKP) a breakdown product of aspartame (n = 10), 1.0% DKP (n = 15), 0.3% DKP (n = 18), or the base diet only (n = 27) for the next 20 days of gestation. Rats were then killed and embryonic development evaluated. There was no effect of 10% aspartame in the diet on maternal weight gain, number of implantations or foetal resorptions. The evaluation of teratogenic effects was focused on ocular lesions, and included cataracts, retinal coloboma, retinal dysplasia, major microphthalmos, anophthalmos, aberrant nerve fibres, and anarchic globes. The incidence and severity of ocular lesions observed in embryos were tabulated to establish a teratology index for each litter. The teratology indices of litters of rats fed 10% aspartame were similar to controls. Details on the specific lesions that contributed to the teratology indices were not provided.

Rabbits

24. The effect of aspartame added to feed during gestation in rabbits was assessed in a study by Ranney et al., (1975). Thirty mature New Zealand female rabbits were inseminated, and starting on day 6 of pregnancy were fed either the


control rabbit diet or diet containing 6% aspartame (15/group) until autopsy on day 20. Fetal and maternal tissues were collected. No effect on maternal body weight, litter size or foetal weights was observed. Based on food consumption measurements, the dose in this study was 1600 mg/kg bw/day and was a no observed effect level (NOEL).

Hormonal studies in rats, mice and rabbits

25. Aspartame was evaluated for possible estrogenic, androgenic, progestational, and glucocorticoid activities (Saunders et al., 1980). Doses of 300 mg/kg bw/day (number of days not defined) given orally to female SCH: ARS(ICR) mice did not affect mouse uterine weight in either an estrogenic or estrogen antagonism assay. There was no evidence of progesterone-like activity or progesterone antagonism in female New Zealand rabbits dosed with 300 mg/kg bw/day for 5 days. Castrated male Sprague-Dawley rats administered 300 mg/kg bw/day for 7 days did not display evidence of androgen activity by aspartame, or when treated with testosterone, evidence of androgen antagonism of aspartame. Adrenalectomized male Sprague-Dawley rats dosed with 300 mg/kg bw did not have increased liver glycogen, indicating aspartame does not have cortisone-like activity. One ovary was removed from female Sprague-Dawley rats, and then rats were dosed with vehicle or aspartame (300 mg/kg bw/day) for 14 days. The finding that the weight of the remaining ovary was similar in both groups was interpreted as evidence of lack of an effect of aspartame on pituitary regulation (Saunders et al., 1980). The estrogenic activity of more than 90 chemicals, including aspartame, was examined using the estrogen receptor-dependent breast cancer cell line, MCF-7 (Okubo and Kano, 2003). No evidence of estrogenic activity was observed with aspartame at concentrations up to 1000 μg/ml.

Human Studies

26. A population-based case control study for paediatric brain tumour occurrence and aspartame consumption was conducted between 1984 and 1991 (Gurney et al., 1997). The authors commented that no elevated brain tumour risk to the child from maternal consumption of aspartame during pregnancy was evident, nor did they find any elevated risks during any trimester of pregnancy.

27. Halldorsson et al., (2010) carried out a prospective cohort study on 59,334 women from the Danish National Birth Cohort (1996 - 2002) in which they looked at an association between intakes of sugar sweetened and artificially sweetened soft drinks and preterm delivery. Soft drink intake was assessed mid-pregnancy by using a food frequency questionnaire. An association was reported between intake of artificially sweetened carbonated and noncarbonated soft drinks and an increased


risk of preterm delivery. No association was observed for sugar-sweetened carbonated soft drinks or for sugar-sweetened noncarbonated soft drinks. The authors concluded that a daily intake of artificially sweetened soft drinks may increase the risk of preterm delivery; however they did state that further studies are required to reject or confirm these findings. The study did not control for caffeine consumption, which is known to be associated with adverse reproductive outcomes. Since carbonated drinks will include colas this may have confounded the results.

Summary

28. Little is known about the placental transfer and fetal effects of methanol, and whether specific effects in addition to those characteristic of methanol toxicity occur. Ethanol does diffuse rapidly across the placenta and is evenly distributed throughout fetal body fluid. Whether this applies to methanol is uncertain, though case report data suggest that it may.

29. Developmental studies in rats and mice have shown a wide range of developmental anomalies at high levels of methanol exposure by inhalation (7 hours/day) in rodents with effects occurring at levels of 10,000 ppm and above in rats (2730 mg/kg) and 2000 ppm and above in mice (585 mg/kg/bw) (Nelson et al., 1985; Rogers and Mole, 1997). The effect of methanol on development and neurobehavior in primates has been examined by Burbacher et al., (1999). No clinical signs of toxicity were evident when monkeys were subjected to methanol via inhalation at up to 1800 ppm. The authors concluding that no clear cut treatment related effects were demonstrated in this study. There are currently no developmental data in humans exposed to methanol. However, there are several clinical reports in the literature on the effects of toxic levels of methanol consumption/inhalation on pregnant women.

30. The effect of aspartame during reproduction, development and lactation has been evaluated in rats, mice, hamsters and rabbits. No-effect levels of exposure during reproduction and gestation have been reported to range from 1600 mg/kg bw/day in rabbits to 4000 mg aspartame/kg bw/day in rodents. Adverse effects on pup development were observed in rat studies when doses exceeded 5000 mg/kg bw/day during reproduction and gestation. Consumption of up to 7000 mg aspartame/kg bw/day during lactation had no effect on pup development or maternal health, but higher doses affected body weights. Studies evaluating the estrogenic potential of aspartame have consistently been negative. Therefore, aspartame is considered to have no reproductive or teratogenic activity, and no effect on lactation. In these studies, effects have been observed at exceedingly high doses, and were secondary to reduced body weights. It should be noted that the ADI for aspartame was set on the basis of a range of animal studies which demonstrated a NOAEL of 4g/kg bw/day, the highest dose used,


31. There are few reliable human epidemiology data on this area.


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Abbreviations

ADI Acceptable Daily Intake C-Section Caesarian Section DCR Decidual Cell Response DKP Diketopiperazine DNA Deoxyribonucleic Acid EMEA European Medicines Agency GC Gas Chromatography GD Gestation Day JECFA Joint Expert Committee on Food Additives N2O Nitrous Oxide NOAEL No Observed Adverse Effect Level NTD Neural Tube Defects PND Postnatal Day PPM Parts Per Million SACN Scientific Advisory Committee on Nutrition SCF Scientific Committee on Food THF Tetrahydrofolic Acid

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