mammalian in vivo and in vitro cytogenetic assays: a report of the u.s. epa's gene-tox program

Mutation Research, 87 ( 1981 ) 143-188 143 Elsevier/North-Holland Biomedical Press

Mammalian in vivo and in vitro cytogenetic assays:

A report of the U.S. EPA's Gene-Tox Program I

R. Julian Preston 2, William Au 3, Michael A Bender 4, J. Grant Brewen 5, Anthony V. Carrano 6, John A. Heddle 7, Alfred F. McFee 8, Sheldon Wolff 9 and John S. Wassom 10

2. Biologv Division and Io Information Division, Oak Ridge National Laboratory *, Oak Ridge, TN 37830," "~ Division of Cell Biology, University of Texas System Cancer Center, Houston, TX 77030 **; 4 Brookhaven

National Laboratory ***, Upton, N Y 11973; "~ Allied Chemieal Corporation, Morristown, NJ 07960; Lawrence Livermore Laboratory 4, Livermore, CA 94550; 7 York University, Downsview, Ontario M3J 1P3

(Canada); s Comparative Animal Research Laboratory t,t, Oak Ridge, TN 37830, 9 University of California, San Franciseo, CA 94143 (U.S.A.)

(Received 25 May 1981) (Accepted 5 June 198 I)

Contents

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 II. General considerations used in the interpretation of data . . . . . . . . . . . . . . . . . . . . . . . . . 146

A. Differential sensitivity of cells in different stages of the cell cycle . . . . . . . . . . . . . . . . . . 146 B. Metabolic activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 C Suitability of protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 D. Tabular presentation of data base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

I Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, United States Environmental Protection Agency, Washington, DC). The authors are members of the Gene-Tox Work Group on Mammalian In Vivo and In Vitro Cytogenetics Assays.

* Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.

** Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830. *** Work supported by contract 79-D-X-0533 with the U.S. Environmental Protection Agency.

t A portion of this work was provided under the auspices of the U.S. Department of Energy contract No. W-7405-eng-48.

t t Work supported by the U.S. Environmental Protection Agency under Interagency Agreement No. 79-D-X-0533.

By acceptance of this article, the publisher or recipient acknowledges the U.S. Government's fight to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

0165-1110/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press

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I l i . B o n e - m a r r o w assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

A. Protocol for testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

1. N u m b e r o f a n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

2. N u m b e r o f cel ls s co red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

3. D o s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

4. R o u t e o f a d m i n i s t r a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

5. S a m p l i n g t i m e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

6. Data !bresentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

B. C h e m i c a l s r e v i e w e d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

IV. S p e r m a t o g o n i a assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149



V. S p e r m a t o c y t e a s say . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

A. Treated spermatogonial s t em cel ls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

1. P r o c o t o l for testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 I

2. Chemicals reviewed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

B. Treated spermatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

I. Protocol for testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

2. Chemicals reviewed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

VI. Oocytes and ea r ly e m b r y o assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158


1. O o c y t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

2. Male post-meiotic g e r m cel ls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

3. E a r l y e m b r y o s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164


VII . I n v i t r o cel l c u l t u r e assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164



V I I I . Peripheral leukocyte or lymphocyte assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165



IX. O v e r a l l a s s e s s m e n t o f c y t o g e n e t i c assay s y s t ems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 I

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Summary

This report presents an assessment made by the U.S. Environmental Protection Agency Gene-Tox Program's Work Group on mammalian cytogenetics of the clastogenic effects of chemicals in in vivo and in vitro mammalian cell assays. This assessment is based on information provided by the Environmental Mutagen Infor- mation Center, Oak Ridge National Laboratory, with the proviso that the experimental protocol used in these papers was adjudged to be acceptable by standards outlined by the Work Group. Some data were accepted as "qualitative only" because the protocol used was fairly close to that proposed as suitable. Using these criteria, 177 papers were selected for review.

6 assays were reviewed: bone marrow (32 papers, 31 chemicals), spermatogonial (10 papers, 10 chemicals), spermatocyte (25 papers, 25 chemicals), oocyte or early embryo (18 papers, 19 chemicals), in vitro cell culture (30 papers, 66 chemicals), and leukocyte (66 papers, 53 chemicals). Each assay was considered separately, and

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comparisons were then made between them for their similarities or differences in producing a positive or negative clastogenic effect of a particular chemical or chemical class.

A large proportion of the available cytogenetic data was not suitable for inclusion in the final data base because of poor experimental design or unsatisfactory reporting of the information. It was not possible to recommend any one assay for determining potential clastogenicity because each had its own particular advantages and limitations and provided unique information. For demonstrating in vivo effects, the bone-marrow assay is probably the simplest and most economical. If only in vitro exposures were considered, leukocytes or cultured mammalian cell lines would be suitable. However, there are advantages to using leukocytes because they are a synchronous population, at least through their first cell division, and because of the ready availability of human cells. In general, there was good agreement between clastogenicity and carcinogenicity.

I. Introduction

The initial papers selected for evaluation by the Mammalian In Vivo and In Vitro Cytogenetic Assay Work Group were obtained from Environmental Mutagen Infor- mation Center, Oak Ridge National Laboratory, searches through October 1978. References added to the file after this date were not considered. The total number of references (1896) was reduced to 254 by use of rejection criteria established either by the Gene-Tox Program or the Work Group.

Papers were not selected for evaluation if they fell into one or more of the following categories:

1. Papers dealing with cytological phenomena other than cytogenetics; 2. Abstracts; 3. Review articles with no new data; 4. Papers written in foreign languages where sufficient detail could not be ascer-

tained without a complete translation of the whole article; 5. Papers reporting sister chromatid exchange experimental results (this topic was

considered by another Work Group); 6. Papers where radiation was the damaging agent; 7. Papers having insufficient or no dosimetry; 8. Papers where the test system was plant or insect; 9. Papers where the data were for distribution of aberrations and only damaged

cells were reported; 10. Papers where 2 or more chemicals were given simultaneously and insufficient

information was reported on any one; 11. Papers where chromosome aberrations were studied in induced tumors.

The Work Group considered these 254 references as their initial data base. The Group established an experimental protocol that they felt was the best available one for detecting mutagens for each assay (bone marrow, spermatogonia, spermatocytes, oocytes, early embryos, leukocytes, and in vitro cell cultures). Papers that did not

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follow these protocols and would, therefore, provide problems with the interpretation of the results were rejected. Application of this final rejection criterion left 139 references to provide the best data base for use in the Work Group's review.

However, some of the papers that were rejected because they did not follow the recommended desirable protocol showed a positive effect. The Group considered that these data should not be disregarded, and they were included in a category of "acceptable positive result, qualitative only". A very few negative results were also included in the group, "qualitative only", where the design of the experiments fit closely tO the suggested protocol but the data were not reported in a way that the Group felt was very informative (e.g., percentage of abnormal cells). There were 38 papers in this category. The number of references in each group were 1642 initial rejection, 139 acceptable (quantitative), 38 acceptable qualitative only, and 77 rejected because protocol could lead or led to erroneous data.

The Work Group's decision to include the results from the papers in the "qualitative only" category raised the total number of papers which comprised the mammalian cytogenetics data base to 177, with 1719 papers rejected.

II. General considerations used in the interpretation of data

A. Differential sensitivity of cells in different stages of the cell cycle Radiation induces aberrations in cells in all stages of the cell cycle: chromosome-

type aberrations are induced in GI and chromatid-type in S and G 2. There is, however, a difference in sensitivity of cells in the different stages of the cell cycle, with G 2 generally being the most sensitive stage and G I the least sensitive. The consequences of the various types of aberrations produced will also be very different. For example, a chromosome-type deletion will usually be cell lethal because both chromatids are deleted and both daughter cells will be deficient for a major portion of genetic material subsequent to mitosis. A chromatid deletion, however, results in only 1 of the 2 daughter cells receiving a deleted chromosome and being killed; the other will contain a normal chromosome. The same sort of argument applies to symmetrical exchanges (reciprocal translocations), which are important in terms of genetic hazard, in that 1 of 4 possible segregation products of a chromatid exchange is a balanced, viable translocation.

Chemical mutagens have different spectra of action. A few are described as S-phase-independent since they produce aberrations in all stages of the cell cycle (e.g., 8-ethoxycaffeine, streptonigrin, and cytosine arabinoside). The majority are said to be S-phase-dependent since aberrations are produced either in cells which are in the S phase at the time of treatment or require an intervening S phase between treatment and observation at metaphase. Furthermore, there are differences in sensitivity of cells in the different stages of the cell cycle.

For these reasons, it is important that cells in different stages of the cell cycle be treated and analyzed. For in vivo studies on cycling cells and for asynchronous cells in culture, a single exposure will expose cells in all stages of the cell cycle, and analysis of these different stages can be made by fixing cells at different times after treatment. The times will vary for each assay and a general guideline is given under

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each protocol. For synchronous cell populations, such as peripheral lymphocytes, treatment at various times after stimulation will allow cells in the different stages of the cycle to be treated, in which case a common fixation time can be used. In all instances it is imperative that first post-treatment mitoses be analyzed in order to determine the maximum quantitative effects.

B. Metabolic activation In order for some compounds to be mutagenic they need to be metabolized to an

active form. Also some compounds can be metabolized to a non-active form. For in vivo assays this activation is performed by the animal itself, but for in vitro systems the cells themselves are usually not capable of activation. The subject of metabolic activation is complex, and in many ways only superficially understood. It is not feasible to discuss the problem in any great depth here, so only some general recommendations will be given.

The raost commonly used activation system is the $9 fraction Of a liver micro- some preparation obtained from rats or mice whose liver metabolism has been stimulated by treatment of the animals with, for example, aroclor or phenobarbital. There are, however, cytotoxic effects associated with treatment of cells with $9 fraction, particularly in the case of lymphocytes. This problem can be overcome to some extent by using alternative treatment methods. For long-lived metabolites it is possible to preincubate the test compound with the $9 fraction, centrifuge, and add the supernatant to the cell culture. It has also been reported that the $9 mix can be contained in dialysis bags and then added to the cell cultures. This has been particularly advantageous with lymphocyte cultures where treatment for several hours with $9 mix in dialysis bags has not produced marked cytotoxic effects.

It is also possible to avoid the use of $9 mix altogether. This has been achieved by using cocultivation of the test cell with feeder cells that have maintained a high metabolic activity. Such feeder cells include lethally X-irradiated early passage embryonic cell cultures and primary cultures of liver cells from adult rats, with the latter being particularly successful. Primary hepatocytes or hepatoma cells can be used as the test cell, thus avoiding the use of a separate metabolic activation system in the form of $9 mix or feeder cells.

It is not suggested that all compounds be routinely tested with and without activation. If a compound gives a negative result in the absence of activation in an in vitro assay, then it should be tested in the same assay with activation or alternatively tested in an in vivo assay. It is not recommended that hepatocyte or hepatoma cell cultures be used as a routine test system because the cultures are more difficult to utilize than a permanent cell line such as the Chinese hamster ovary (CHO) cell discussed under the in vitro cell culture assay.

C. Suitability of protocols A protocol is described for each of the assay systems considered under the

category of in vivo and in vitro cytogenetics. It is not suggested that this is the only suitable protocol or that it is a panacea for all situations. We do feel, however, that for each assay, the protocol described is the best available for most compounds, or compound classes, to be tested. Furthermore, it is not possible to describe here

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changes in protocols that will take care of every alternative. The treatment and sampling times given in the protocols for in vivo assays refer to the mouse since this is the animal most commonly used in cytogenetic studies. Some variations might prove to be necessary for studies with other animals; this applies particularly to germ-cell studies.

D. Tabular presentation of data base 2 tables accompany the description of each assay system and desired protocol.

One table presents the results for the individual chemicals tested with a particular assay system and reference to the original data. The other table presents the data for each chemical class, showing the number of chemicals in a particular class that were tested and the results. In some cases where individual tests showed conflicting results a consensus result is presented. Where such a consensus result was not attainable, the outcome is given as inconclusive results (both positive and negative responses reported). In order to cross-reference between the 2 tables, the Chemical Abstracts Service (CAS) Registry Number can be used.

A summary table that compares the results from different assay systems is given at the end of the report.

III. Bone-marrow assay

A. Protocol for testing

1. Number of animals. While this is arbitrary, the use of too few animals increases the chance of false negatives. No fewer than 3 animals should be examined for each experimental point.

2. Number of cells scored. A minimum of 300 cells should be scored for each experimental point, with at least 50 cells from each animal.

3. Dose. There should be at least 3 doses and a positive and negative control. The doses selected should extend over at least a single,log range, with the maximum dose no less than a factor of 2 less than a dose producing a significant level of toxicity. Lower doses are acceptable if a positive response is obtained.

4. Route of admin&tration. The agent should be injected intraperitoneally in a single dose to simplify the estimation of the dose. Supplementary studies using another route of administration (e.g., inhalation or per os) may be desirable in some cases in order to approximate the anticipated route of exposure.

5. Sampling time. Different chemicals produce effects at different parts of the cell cycle and cause different amounts of mitotic delay. Multiple post-treatment sampling times are necessary to increase the probability of showing whether or not a compound is mutagenic. Early (1-3 and 6-8 h), middle (12-16 h), and late (24 h) sampling times will allow cells exposed in all stages of the cell cycle to be analyzed. If there is considerable mitotic delay, a 48-h sample time should be included.

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6. Data presentation. Aberrations should be expressed as the frequency per cell and not as percentage of aberrant cells because different aberrations have different genetic consequences and dose relations. Aberrations should be subdivided into whether they are chromosome- or chromatid-type. Within each of these classes the aberrations should be further subdivided into deletions and exchanges. Chromatid gaps should be scored but presented separately. The distinction between a chromatid deletion and a gap is arbitrary. A useful dividing line for routine scoring purposes is to score displaced fragments and all undisplaced fragments separated by a non- staining region equal to or greater than 1 chromatid width as deletions. If the non-staining region is less than 1 chromatid width or does not extend across the chromatid and is not displaced, it is scored as a gap.

B. Chemicals reviewed The Work Group reviewed 32 papers on the bone-marrow assay which resulted in

evaluations of 31 chemicals. Qualitative results of these studies are shown by compound in Table 1 and by chemical class in Table 2. Table 1 also correlates these results with the reported carcinogenicity of each compound when this information was available to the Group.

IV. Spermatogonia assay

A. Protocol for testing The protocol for the analysis of spermatogonial cells is the same as that for the

bone-marrow assay. It is desirable to sample at least 3 animals per experimental point and to score 50 cells per testis, giving a total of 300 cells per experimental point.

B. Chemicals reviewed 10 papers in which this assay was used were reviewed by the Work Group and 10

chemicals were evaluated. Table 3 summarizes the qualitative results obtained with these 10 compounds together with their correlation to carcinogenicity when the latter information was available to the Work Group. Table 4 shows the qualitative responses with these 10 chemicals according to their chemical classification.

V. Spermatocyte assay

The description and presentation of data using this assay has been divided into 2 sections that indicate cell stage at the time of treatment.

A. Treated spermatogonial stem cells Cells analyzed at diakinesis-metaphaseI 50-100 days after treatment were

spermatogonial stem cells at the time of treatment. Because there are several cell divisions between treatment and observation, the only aberrations observed will be

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TABLE 1

OVERALL QUALITATIVE RESULTS OF COMPOUNDS REVIEWED IN THE MAMMALIAN CYTOGENETIC BONE-MARROW ASSAY

Chemical Rcferencc Results a Carcino- (CAS registry number) genicity b

Mitomycin C Michelmann et al. (1978) + + (50-07-7)

Cyclophosphamide Mialler et al. (1972) + d + (50-18-0) Goetz et al. (1975)

6-Mercaptopurine Holden et al. (1973) + d * (50-44-2)

Triethylenemelamine Michel and Legator (1974) + + (51-18-3) Steelman and Schreiner (1977)

Thio-TEPA Malashenko and Surkova (1974) + d + (52-24-4)

Myleran Leonard and L6onard (1978) + d + (55-98- I )

METEPA Das and Manna (1974) + *

(57-39-6) Barbital Manna and Das (1973) + •

(57-44-3) 7,12-DMBA Kurita et al. (1969) + +

(57-97-6) Caffeine ROhrborn and Buckel (1976) cm •

(58-08-2) Nitrofurazone Goodman et al. (1977) - •

(59-87-0) Ethyl methanesulfonate Ray et al. (1974) + +

(62-50-0) Dichlorvos Dean and Thorpe (1972) -

(62-73-7) Monomeric acrylamide Shiraishi (1978) - *

(79-06-1) 2,4,5-T Majumdar and Hall (1973) + d •

(93-76-5) 2,4-D Pilinskayzi (1974) + *

(94-75-7) Cyclohexylamine Brewen et al. ( 1971 ) - •

( 108-91-8) Calcium cyclamate Majumdar and Solomon ( 1971 ) + *

(139-06-0) Triflupromazine Ray et al. (1973) - *

(146-54-3) ENT-50787 Manna and Das (1976) • + •

(302-48-7) Trimethylphosphate Adler et al. ( 1971 ) + +

(512-56-1) TEPA Adler et al. ( 1971 ) + •

(545-55-1) Ethylnitrosourea Soukup and Au (1975) + +

(759-73-9)

TABLE 1 (continued)

Chemical Reference Results a Carcino- (CAS registry number) genicity b

cis-fl-4-Methoxybenzoyl-fl- Goetz et al. (1976) + d • bromacrylic acid, sodium salt (21739-91-3)

Hycanthone Green et al. (1973) ? • (3105-97-3)

Lead acetate Jacquet et al. (1977) ÷ (301-04-2)

Aluminum chloride Manna and Das (1972) + • (7446-70-0)

Ozone Gooch et al. (1976) - • (10028-15-6)

Bleomycin Bornstein et al. ( 1971) + • (11056-06-7)

Cartap hydrochloride Kikuchi et al. (1976) - * (15263-52-2)

Rubidomycin Krogh Jensen and Philip (1971) + + (20830-81-3)

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a Results: (÷) positive; (+d) positive with dose response; ( - ) negative; ( ± ) positive and negative; (?) inconclusive; (cm) comutagen.

b The reported carcinogenicity results were obtained from Searle (1976), Tomatis et al. (1978), or Griesemer and Cueto (1980); (+) some evidence of carcinogenicity from animal studies; ( - ) no evidence of carcinogenicity from animal studies; ( , ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

rec iprocal t rans locat ions . These will appea r as mul t iva len t conf igura t ions (e.g., Cha in IV, Ring IV) at d i a k i n e s i s - m e t a p h a s e I . The p robab i l i t y of recover ing a t r ans loca t ion is very low after chemical t rea tment . The aber ra t ions induced by near ly all chemicals are of the chromat id- type . Cells con ta in ing dele t ions or asym- metr ica l exchanges will usual ly be ki l led at the first or second divis ion because of f r agment loss; if such a cell also conta ins a t rans locat ion , this too will be lost. Symmetr ica l ch roma t id exchanges a lone will have a high p robab i l i t y of not reaching meiosis because segregat ion will result in dup l ica t ions and deficiencies that are usual ly cell lethal. Even though a par t icu la r chemical p roduces ch romosomal damage in spe rmatogon ia l cells, the p robab i l i t y of recovering a rec iprocal t r ans loca t ion is low. There are no publ i shed repor ts o f chemicals which unambiguous ly induce a s ignif icant f requency of t rans loca t ions in spe rmatogon ia l s tem cells when observa- t ions are m a d e at d i a k i n e s i s - m e t a p h a s e I. Therefore a negat ive resul t in this assay does no t imp ly tha t the c o m p o u n d does not p roduce ch romosome aber ra t ions ; it is no t r e c o m m e n d e d as a screening assay for po ten t ia l mutagens .

1. Protocol for testing. A t least 500 cells should be scored with no more than 50 f rom any one testis. Each testis should be cons idered separately. The t ime be tween t r ea tmen t and cell p r epa ra t i on should be 5 0 - 1 0 0 days , so the cells ana lyzed will

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TABLE 2

QUALITATIVE RESULTS OF COMPOUNDS EVALUATED IN THE MAMMALIAN CYTO- GENETIC BONE-MARROW ASSAY ACCORDING TO CHEMICAL CLASS a

EPA Gene-Tox chemical classification [CAS registry number(s)]

Number of compounds reviewed

Summary of overall results b

+ -- ,) ±

Alcohols and phenols 3 (3105-97-3) (11056-06-7) (20830-81-3)

Aliphatic amines 6 (50-07-7)

(146-54-3) (3105-97-3) (11056-06-7) (15263-52-2) (20830-81-3) Alkyl sulfates, sulfoxides, sulfones, sulfonates 2

(55-98-1) (62-50-0)

Amides and sulfonamides 4 (59-87-0) (79-06-1) (139-06-0) (11056-06-7)

Amino acids and derivatives 1 (11056-06-7)

Anthraquinones 1 (20830-81-3)

Antibiotics 3 (50-07-7) (11056-06-7) (20830-81-3)

Aromatic amines 2 (3105-97-3) (11056-06-7)

Aryl halides 2 (93-76-5) (94-75-7)

Aziridines 6 (50-07-7) (51-18-3) (52-24-4) (57-39-6) (302-48-7) (545-55-1)

Benzene ring 3 (93-76-5) (94-75-7) (2126-70-7)

Carbamates 3 (50-07-7) (11056-06-7) (15263-52-2)

2 0

3 2

2 0

1 2

1 0

I 0

3 0

I 0

2 0

6 0

3 0

2 1

0 0

0 1

0 0

0 0

0 0

TABLE 2 (continued)

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+ - ? ___

Carbohydrates and derivatives (11056-06-7) (20830-81-3)

Heterocyclic rings not otherwise classified (50-07-7) (50-18-0) (51-18-3) (11056-06-7)

Hydrazides (59-87-0)

Metals and derivatives (139-06-6) (2126-70-7) (7439-92-1) (7446-70-0)

Nitrofurans (59-87-0)

Nitrogen and sulfur mustards (50-18-0)

Nitrosoureas (759-73-9)

Organic buffer compounds not otherwise classified (50-44-2) (52-24-4) (15263-52.2)

Phenothiazines (146-54-3)

Phosphoric acid esters and phosphamides (50-18-0) (52-24-2) (57-39-6) (62-73-7) (302-48-7) (512-56-1) (545-55-1)

Polycyclic aromatic hydrocarbons (57-97-6)

Purine derivatives (5o-44-2) (58-o8-2)

Pyrimidine derivatives (57-44-3) ( l 1056-06-7)

Quinones (50-07-7)

Saturated alkyl halides (146-54-3)

2 0 0 0

4 0 0 0

0 1 0 0

2 1 0 1

0 1 0 0

1 0 0 0

1 0 0 0

2 1 0 0

0 1 0 0

6 1 0 0

1 0 0 0

1 0 0 0 1 cm

2 0 0 0

1 0 0 0

0 1 0 0

154

TABLE 2 (continued)




Unsaturated alkyl halides 2 1 1 0 0 (62-73-7) (2126-707)

Ureas, thioureas 1 I 0 0 0 (57-44-3)

Xanthenes I 0 0 I 0 (3105-97-3)

Unclassified 1 0 I 0 0 ( 10028-15-6)

Experimental results on 29 compounds were reviewed by the Work Group in this assay system. A chemical may be found in more than one chemical class and the information in this table can be cross-referenced to the information in Table 1 by using the CAS registry number(s) provided.

b Results: (+) positive; ( - ) negative; (?) inconclusive: (±) positive and negative results, no clear distinction could be drawn regarding the response as the Work Group judged that good experimental data existed for each type of response; (cm) comutagen; (0) no results reported.

have been spermatogonial stem cells at the time of treatment. Reciprocal translocations will be the only aberrat ion type observed. The dose levels to be used are difficult to determine. A high dose, or one

approaching a significant level of toxicity, would minimize the probabi l i ty of recovering a translocation; whereas a low dose, 1 log lower than the high dose, would maximize this probabil i ty but reduce the induced yield to a very low level. The solution is to compromise with a study of both dose levels.

The route of administrat ion should be intraperitoneal for all assays discussed. In order to approximate the anticipated route of exposure, supplementary studies using other routes may be desirable.

2. Chemicals reviewed. 20 agents were evaluated f rom studies, reported in 15 papers, in which spermatogonial stem cells were treated and spermatocytes were observed. Results f rom these studies are shown in Table 5A with each agent 's carcinogenic response when this informat ion was available to the Work Group. Table 6 shows the overall qualitative results according to chemical class of compounds evaluated in the spermatocyte assay.

B. Treated spermatocytes It is also possible to s tudy the effects of chemicals on meiotic cells by analyzing

d iak ines i s -metaphase I cells at different times after treatment. In most cases, D N A damage produced by a chemical will not cause aberrations unless the cell is in the S phase between t reatment and sampling. Thus the most likely cells to show aberra- t ions would be those in pre-leptotene at the time of treatment. It is not suggested that this be used as a routine assay.

155

TABLE 3

OVERALL QUALITATIVE RESULTS OF C O M P O U N D S REVIEWED IN THE M A M M A L I A N CYTOGENETIC S P E R M A T O G O N I A L ASSAY

Chemical Reference Results a Carcinogenicity b (CAS registry number)

Mitomycin C Adler (1974) + + (50-07-7)

Cyclophosphamide Rathenberg (1975) + + (50-18-0)

LSD-25 Jagiello and Polani (1969) - • (50-37-3)

TEM Luippold et al. (1978) + + (5M8-3)

Dichlorvos Dean and Thorpe (1972) - - (62-73-7)

Acrylamide Shiraishi (1978) + • (79-06-1)

Cyclohexylamine .Legator et al. (1969) + • (108-91-8)

Chrysene Basler et al. (1977b) - + (218-01-9)

Thiopbanate-methyl Maldta et al. 0973) - * (23564-05-8)

Aroclor 1242 Green et al. (1975) - • (53469-21-9)

a Results: ( + ) positive; ( - ) negative. b The reported carcinogenicity results were obtained from Searle (1976), Tomatis et al. (1978), or

Griesemer and Cueto (1980): ( + ) some evidence of carcinogenicity from animal studies; ( - ) no evidence of carcinogenicity from animal studies; ( . ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

1. Protocol for testing. The type of aberrations observed would be chromatid deletions and translocations (seen as multivalents).

The sampling times necessary to analyze cells treated in the different meiotic stages are for the mouse: diplotene, 1 day after treatment; pachytene, 5 days after treatment; zygotene, 9 days after treatment; leptotene, ! 1 days after treatment; and pre-leptotene, 12-14 days after treatment.

It is desirable to score 300 cells at any experimental point with no more than 50 cells from any one testis.

The doses and routes of administration are the same as for the bone-marrow or spermatogonial cell assays.

2. Chemicals reviewed. 11 papers in which differentiating spermatogonia or spermatocytes were treated and spermatocytes were observed were reviewed by the Work Group. Results from these studies on 10 chemicals are shown in Table 5B. Carcinogenicity results are also shown in Table 5 when this information was available. Table 6 shows the overall qualitative results according to chemical class of compounds evaluated in the spermatocyte assay.

156

TABLE 4

QUALITATIVE RESULTS OF COMPOUNDS EVALUATED IN THE MAMMALIAN CYTO- GENETIC SPERMATOGONIAL ASSAY ACCORDING TO CHEMICAL CLASS a

EPA Gene-Tox chemical Number of Summary of overall results b classification compounds [CAS registry number(s)] reviewed + - ? ±

Aliphatic amines I 1 0 0 0 (50-07-7)

Alkaloids l 0 1 0 0 (50-37-3)

Amides and sulfonamides 2 1 1 0 0 (50-37-3) (79-06- I )

Antibiotics I 1 0 0 0 (50-07-7)

Aryl halides I 0 1 0 0 (53469-21-9)

Aziridines 2 2 0 0 0 (50-07-7) (51-18-3)

Benzene ring 2 0 2 0 0 (23564-05-8) (53469-21-9)

Carbamates 2 1 I 0 0 (50-07-7) (23564-05-8)

Heterocyclic tings not 4 3 1 0 0 otherwise classified (50-07-7) (50-18-0) (50-37-3) (51-18-3)

Mycotoxins I 0 1 0 0 (50-37-3)

Nitrogen and sulfur mustards 1 1 0 0 0 (50-18-0)

Phosphoric acid esters and 2 I 1 0 0 phosphamides (50-18-0) (62-73-7)

Polycyclic aromatic hydrocarbons 1 0 1 0 0 (218-01-9)

Quinones 1 I 0 0 0 (50-07-7)

Unsaturated alkyl halides 1 0 1 0 0 (62-73-7)

Ureas, thioureas I 0 1 0 0 (23564-05-8)

a Experimental results on 10 compounds were reviewed by the Work Group in this assay system. A chemical may be found in more than 1 chemical class and the information in this table can be cross-referenced to the information in Table 3 by using the CAS registry number(s) provided.

b Results: ( + ) positive; ( - - ) negative; (?) inconclusive; (_±) positive and negative, no clear distinction could be drawn regarding the response as the Work Group judged that good experimental data existed for each type of response; (0) no results reported.

157

T A B L E 5

O V E R A L L Q U A L I T A T I V E R E S U L T S O F C O M P O U N D S R E V I E W E D IN T H E M A M M A L I A N

S P E R M A T O C Y T E ASSAY

Chemica l Reference Resul t s a Carc ino-

(CAS registry number ) genici ty b

A. Treated spermatogonial stem cells." observed spermatocytes (see text for explanation of negative results)

M i t o m y c i n C - +

(50-07-7)

Cyc lophosphamide - +

(50-18-0)

T E M - +

(51-18-3) _+

Isoniaz id - +

(54-85-3)

My le r an - +

(55-98-1) Caf fe ine - .

(58-08-2) Ethyl me thanesu l fona te - +

(62-50-0) D ime thy ln i t r o samine - +

(62-75-9)

Die thy l sulfate - - +

(64-67-5)

Me thy l me thanesu l fona te - +

(66-27-3)

T r e n i m o n - +

(68-76-8) / v l N N G - +

(70-25-7)

Cyc lohexy lamine - ,

(108-91-8)

Sod ium cyc lamate - ,

(139-05-9) T E P A - ,

(545-55-1)

I sopropy l me thanesu l fona te - ,

(926-06-7) Af la tox in BI - +

(1162-65-8) E t h i d i u m b romide - ,

( 1239-45- 8) Propyl me thanesu l fona te - ,

(1912-31-8) Ozone - ,

(10028-15-6)

B. Treated differentiating spermatogonia or spermatocytes: observed spermatoeytes

M i t o m y c i n C Adle r (1976) + + .(50-07-7)

Adle r (1974)

Gi l l i avod and L~onard (1971 )

L6onard and Linden (1972b)

Ca t t anach and Wi l l i ams (1971)

Lu ippo ld et al. (1978)

Pecevski et al. (1978)

Mi l t enburger et al. (1978)

L6onard and Linden (1972b)

Adle r and R 6 h r b o r n (1969)

L~onard et al. (1971)

Savkovic et al. (1978)

L~onard et al. (1971)

L~onard and Linden (1972b)

L6onard and L inden (1972b)

L+onard et al. (1971)

Lega tor et al. (1969)

L6onard and Linden (1972a)

Sram and Z u d o v a (1973)

L+onard and L inden (1972b)



L+onard and L inden (1972b)

Gooch et al. (1976)

158

TABLE 5 (continued)

Chemical Reference Results ~ Carcino- (CAS registry number) genicity b

Cyclophosphamide Rathenberg (1975) + + (50-18-0)

Phenylbutazone Rathenberg and Miiller (1972) - • (50-33-9)

LSD-25 Skakkcbhk et al. (1968) •

(50-37-3) Jagiello and Polani (1969) 6-Mercaptopurine Generoso et al. (1975) + •

(50-44-2) TEM Luippold et al. (1978) + +

(51-18-3) Isoniazid Adler et al. (1978) +

(54-85-3) Dichlorvos Dean and Thorpe (1972) -

(62-73-7) Methyl methanesulfonate Moutschen (1969) + +

(66-27-3) Acrylamide Shiraisfii (1978) + •

(79-06- I)

a Results: ( + ) positive; ( - ) negative; (-+-) positive and negative. The reported carcinogenicity results were obtained from Searle (1976), Tomatis ct al. (1978), or Griesemer and Cueto (1980): ( + ) some evidence of carcinogenicity from animals studies; ( - ) no evidence of carcinogenicity from animal studies; ( , ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

VI. Oocytes and early embryo assay

These systems afford the opportunity to study cytogenetic effects on female germ cells and male post-meiotic germ cells as well as on the early embryo. As in other cytogenetic studies, care must be taken to analyze the first cell division after treatment to insure th~it the maximum effect may be seen. Since most chemical mutagens studied require replicative DNA synthesis to exert an effect, it is imperative that the experimental protocol be designed to enable cells to pass through one S phase between treatment and fixation.

A. Protocol for testing 1. Oocytes a. Superovulation is permissible. b. Metaphase I cells should be examined. In the event of a negative result, the

experiment should be extended to include fertilized oocytes analyzed at the first cleavage division.

c. 3 doses, a negative control, and a positive control should be used. The doses

159

TABLE 6

QUALITATIVE RESULTS OF COMPOUNDS EVALUATED IN THE MAMMALIAN CYTO- GENETIC SPERMATOCYTE ASSAY ACCORDING TO CHEMICAL CLASS a




+ - - 9 +

Aliphatic amines (50-07-7) (108-91-8)

Alkaloids (50-37-3)

Alkyl sulfates, sulfoxides, sulfones, sulfonates (55-98- ~) (62-50-0) (64-67-5) (66-27-3) (926-06-7) (1912-31-8)

Amides and sulfonamides (50-37-3) (79-06- I) (139-05-9

Antibiotics (50-07-7)

Aromatic ammes (1239-45-8)

Aziridines (50-07-7) (51-18-3) (68-76-8)

Benzene ring (50-33-9) (1239-45-8)

Carbamates (50-07-7)

Heterocyclic rings not otherwise classified (50-07-7) (50-18-0) (50-33-9) (50-37-3) (51-18-3) (54-85-3) (1162-65-8) ( 1239-45- 8)

Hydrazides (50-33-9) (54-85-3)

Lactones (1162-65-8)

2 0 I 0 1

1 0 1 0 0

6 0 5 0 1

3 1 2 0 0

I 0 0 0 I

1 0 1 0 0

3 0 1 0 2

2 0 2 0 0

1 0 0 0 I

8 0 5 0 3

2 0 2 0 0

I 0 I 0 0

160

TABLE 6 (continued)




Metals and derivatives 1 0 1 0 0 (139-05-9)

Mycotoxins 2 0 2 0 0 (50-37-3) (1162-65-9)


Nitrosamines 1 0 t 0 0 (62-75-9)

Nitrosoguanidines 1 0 1 0 0 (70-25-7)

Organic sulfur compounds I 1 0 0 0 not otherwise classified (50-44-2)

Phosphoric acid esters and 2 0 I 0 1 phosphamides (5O- 18-0) (62-73-7)

Purine derivatives 2 I 1 0 0 (50-44-2) (58-08-2)

Quinones 2 0 1 0 I (50-07-7) (68-76-8)

Unsaturated alkyl halides 1 0 1 0 0 (62-73-7)

Ureas, thioureas I 0 1 0 0 (23564-05-8)

Unclassified 1 0 1 0 0 (10028-15-6)

a Experimental results on 25 compounds were reviewed by the Work Group in this assay system. A chemical may be found in more than I chemical class and tl'/e information in this table can be cross-referenced to the information in Table 5 (parts A and B) by using the CAS registry number(s) provided.

b Results: (+) positive; (--) negative; (?) inconclusive; ( ~ ) positive and negative, no clear distinction could be drawn regarding the response as the Work Group judged that good experimenal data existed for each type of response; (0) no results reported.

s h o u l d s p a n a 10-fold r a n g e wi th the h ighes t dose w i th in a f ac to r o f 2 o f tha t w h i c h gives a s ign i f i can t tox ic effect .

d. A m i n i m u m of 150 cells shou ld be ana lyzed . Al l a b e r r a t i o n d a t a s h o u l d be

p r e s e n t e d as c h r o m a t i d - a n d c h r o m o s o m e - t y p e abe r r a t i ons , a n d w i t h i n these ca te -

gor ies they c a n be fu r t he r c h a r a c t e r i z e d as de l e t i ons (or f r agmen t s ) , exchanges , and

gaps .

e. 3 p o s t - t r e a t m e n t s a m p l i n g t imes shou ld be used. F o r the mouse , these s h o u l d

161

TABLE 7

OVERALL QUALITATIVE RESULTS OF C O M P O U N D S REVIEWED IN T H E M A M M A L I A N CYTOGENETIC OOCYTE A N D EARLY EMBRYO ASSAY


Mitomycin C Yamamoto (1975) + + (50-07-7)

Cyclophosphamide Hansmann (1974) ÷ d + (50-18-0)

LSD-25 Jagiello and Polani (1969) - • (50-37-3)

TEM Caine and Lyon (1977) + + (51-18-3) Brewen and Payne (1978)

Isoniazid Franz (1978) - + (54-85-3)

Methotrexate Hansmann (1974) + d • (59-05-2)

Methyl methanesulfonate Brewen et al. (1975) + d + (66-27-3)

Triaziquone R6hrborn et al. ( 197 I) + + (68-76-8) R6hrborn and Hansmann ( 1971)

Hansmann and Rrhrborn (1973) Hansmann et al. (1974) Basler (1978)

Dimethyl mercury Jagiello and Lin (1973) - , (593-74-8)

Isopropyl methanesulfonate Caine and Lyon (1977) + , (926-06-7)

Mercuric acetate Jagiello and Lin (1973) - , (1600-27-7)

Streptonigrin Jagiello (1967) + , (3930-19-6)

Sodium nitrite Tsuda and Kato (1977b) + , (7632-00-0)

Trypaflavin Basler et al. (1977a) - , (65431-33-6)

Chromic chloride Tsuda and Kato (1977a) - , (10025-73-7)

Chromic chloride hexahydrate Tsuda and Kato (1977a) - • (10060-12-5)

Chromium (3) sulfate Tsuda and Kato (1977a) - , (10101-53-8)

Phleomycin Jagiello (1968) + , (11006-33-0)

Mercaptomerin Jagiello and Lin (1973) - , (20223-84-1)

a Results: ( + ) positive; ( - ) negative; ( + d ) positive with dose response. b The reported carcinogenicity results were obtained from Searle (1976), Tomatis et al. (1978), or

Griesemer and Cueto (1980): ( + ) some evidence of carcinogenicity from animal studies; ( • ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

162

TABLE 8

QUALITATIVE RESULTS OF COMPOUNDS EVALUATED IN THE M A M M A L I A N CYTO- GENETIC OOCYTE A N D EARLY EMBRYO ASSAY ~

EPA Gene-Tox chemical classification Number of Summary of overall results b [CAS registry number(s)] compounds

reviewed + - ? -4-

Acridines and quinacridines I 0 1 0 0 (8054-52-0)

Alcohol and phenols 1 1 0 0 0 (3930-19-6)

Aliphatic amines 1 1 0 0 0 (50-07-7)

Alkaloids 1 0 1 0 0 (50-37-3)

Alkyl sulfates, sulfoxides, sulfones, sulfonates 2 2 0 0 0 (66-27-3) (926-06-7)

Amides and sulfonamides 3 1 2 0 0 (50-37-3) (59-05-2) (20223-84-1)

Amino acids and derivatives 1 I 0 0 0 (59-05-2)

Antibiotics 3 3 0 0 0 (50-07-7) (3930-19-6) (I 1006-33-0)

Aromatic amines 3 2 1 0 0 (59-05-2) (3930-19-6) (8048-52-0)

Aziridines 3 3 0 0 0 (50-07-7) (51-18-3) (68-76-8)

Benzene ring 2 2 0 0 0 (59-05-2) (3930-19-6)

Carbamates 1 1 0 0 0 (50-07-0)

Heterocyclic rings not otherwise classified 7 5 2 0 0 (50-07-7) (5o-18-0) (50-37-3) (51-18-3) (54-85-3) (59-05-2) (3930-19-6)

Hydrazides I 0 1 0 0 (54-85-3)

TABLE 8 (continued)

163




+ - ? ___

Metals and derivatives 4 1 3 0 0 (7632-00-0) (10025-73-7) (10060-12-5) (10101-53-8)

Mycotoxins 1 0 1 0 0 (50-37-3)


Organic sulfur compounds not otherwise classified 1 0 1 0 0 (20223-84-1)

Organomercury compounds 3 0 3 0 0 (593-74-8) (1600-27-2) (20223-84-1)

Phosphoric acid esters and phosphamides 1 1 0 0 0 (50-18-0)

Quinones 3 3 0 0 0 (50-07-7) (68-76-8) (3930-19-6)

Sulfur and nitrogen oxides 2 1 1 0 0 (7632-00-0) (10101-53-8)


b Results: (+) positive; ( - ) negative; (?) inconclusive; (±) positive and negative, no clear distinction could be drawn regarding the response as the Work Group judged that good experimental data existed for each type of response; (0) no results reported.

be 0.5, 6.5, and 28.5 days so that Graaf ian follicle, stage 5 - 6 , and stage-3 oocytes are sampled.

2. Male post-meiotic germ cells a. Superovulat ion of females is permissible. b. First-cleavage division embryos should be analyzed. In addition, later-stage

embryos may be studied to detect aberrations which can arise at times after the first post- t reatment S phase.

c. Use same dose protocol as for oocytes. d. 3 post- t reatment mating times should be used. These should be days 3 -6 ,

10-13, and 18-21 to insure samples treated as spermatozoa, m i d - l a t e spermatids,and early spermatids.

e. At least 150 cells should be scored and the data presented as chromosome- or

164

chromatid-type aberrations. Further subdivision into deletions and exchanges should be made.

3. Early embryos a. Females may be superovulated. b. Any embryonic stage may be analyzed. c. 3 post-treatment fixation times should be used. These should be 6, 12, and 24 h

with a preceding 3-h treatment with colchicine or other mitotic inhibitor. d. A minimum of 200 cells from 25-50 embryos should be analyzed. e. Dose regimes are the same as for oocytes. f. Aberration data should be recorded as chromosome- or chromatid-type, with

further classification as deletions or exchanges.

B. Chemicals reviewed 18 papers that dealt with this assay were reviewed by the Work Group, and

experimental results on 19 chemicals were evaluated. Table 7 summarizes the qualitative results obtained with these chemicals and their carcinogenic response when the latter information was available. Table 8 shows these qualitative results according to the various chemical classes in which the 19 chemicals were placed.

VII. In vitro cell culture assay

For short-term cytogenetic analysis, an in vitro cell culture assay is the system of choice. In vitro cytogenetic assays usually employ the peripheral leukocyte system or the monolayer cell culture system. The former is described in a separate section.

There are several important features of monolayer cultures. When good cell culture techniques are used, there are few variations between cultures of the same line regarding parameters such as cell cycle time and plating efficiency. The cell line can be frozen and stored so that it can be used for repeated assays and future reference.

Of the various cell lines available, the CHO cell line is probably the most suitable and is also readily available to investigators. The line has a short generation time (12-14 h) and the chromosome number is low (20-22) compared with human (46) or mouse (40).

A. Protocol for testing The following protocol applies to CHO cells or other cell lines. (1) Cultures should be set up uniformly with a similar number of cells per culture

flask (e.g., for CHO 5 X 105 to 1 X 106 cells per T25 flask). (2) Chemical treatment should be given to cells in an exponential stage of growth

(12-24 h after culture initiation). There should also be controls with and without the appropriate solvent. It is strongly recommended that a repeat of the experiment be performed. There should be 3 doses, covering a 10-fold range, the highest within a

165

factor of 2 of that which gives a significant toxic effect. (3) In order to maximize the assay system's ability to identify chemicals with

different cell cycle specificities to aberration induction, more than 1 treatment is recommended. For example, a continuous treatment can be given with fixations at 3, 8, and 12 h after the beginning of treatment. This method will insure that cells will be analyzed in the late (G2/S), middle (S), and early (G1/S) stages of the cell cycle. Shorter treatments of 1-2 h with several fixation times can be given if one requires a more specific assessment of the response of cells in different stages of the cell cycle. There should be a 1-2-h colchicine treatment while the chemical is still present.

(4) 300 metaphases should be scored per experimental point. Aberrations should be recorded as chromosome- or chromatid-type with further classification into deletions and exchanges.

B. Chemicals reviewed The Work Group reviewed 30 papers that reported the experimental evaluation of

66 chemicals. Table 9 shows the qualitative results obtained with these chemicals and their reported carcinogenic response, whenever this information was available. It should be noted that most of the results were obtained without the use of a metabolic activation system. Negative results with this assay may not, therefore, be comparable with in vivo assays for those compounds that require metabolic activation. Table 10 summarizes these results according to the various chemical classifica- tions in which these 66 compounds were placed.

VIII. Peripheral leukocyte or lymphocyte assay

Circulating mammalian leukocytes cultured in vitro can be used for the cytogenetic testing of substances. The technique can also be used for in vivo testing or for providing information on possible occupational or environmental exposure of individuals or populations to hazardous agents. However, several important points should be borne in mind when interpreting the results from such in vivo studies.

Chemicals can be divided into 2 classes: S-dependent and S-independent. S- Dependent chemicals essentially produce aberrations in the S phase or when the cells pass through an S phase between treatment and observation. S-Independent chemicals can produce aberrations in all stages of the cell cycle. The stimulated human leukocytes can repair DNA damage proficiently, and thus the great majority of damage induced in a G O leukocyte will be repaired in G 1 following stimulation, before the cell reaches the S phase. Aberrations in this case will not be observed following exposure to S-dependent chemicals. Aberrations can be induced in the cycling stem cell; but once this cell has to divide, the majority of the aberrations will be lost by cell killing, thus considerably reducing the sensitivity of the assay. It should also be noted that the induced chromosomal damage will be of the chromatid -type, but, because of the cell division and subsequent DNA replication, the damage observed in the stimulated lymphocyte will be of the chromosome-type. On the other hand, chromosome damage can be identified when exposure is to chemicals of the

166

TABLE 9

OVERALL QUALITATIVE RESULTS OF C O M P O U N D S REVIEWED IN M A M M A L I A N CYTO- GENETIC IN VITRO CELL C U L T U R E ASSAY


Mitomycin C Hartley-Asp and Kihlman ( 1971 ) + + (50-07-7)

Cyclophosphamide Bishun ( 1971) + @ + (50-18-0) Huang (1977)

DDT, p , p ' Palmer et al. (1972) + - (50-29-3) Kelly-Garvert and Legator (1973)

3,4-Benzo[ a ]pyrene Abe and Sasaki (1977) - + (50-32-8)

LSD-25 Sturelid and Kihlman (1969) • (50-37-3)

Actinomycin D Ostertag and Kersten (1965) -+- d • (50-76-0) Benedict et al. (1977)

Thio-TEPA Benedict et al. (1977) + + (52-24-4)

DDD, o,p' Palmer et al. (1972) + • (53-19-0)

Diethylnitrosamine Natarajan et al. (1976) + @ + (55-18-5)

3-Methylcholanthrene Abe and Sasaki (1977) - + (56-49-5)

4-Nitroquinoline 1-oxide Stich et al. (1973) - d + (56-57-5) Abe and Sasaki (1977)

N a m b a et al. (1977) 7,12-DMBA Kato (1968) + +

(57-97-6) Caffeine Kihlman et al. (1977) + •

(58-08-2) Methotrexate Rodriguez Murcia and + d •

(59-05-2) Arroyo Nombela (1972) Benedict et al. (1977)

Dimethylni trosamine Natarajan et al. (1976) + d@ + (62-75-9)

Ethanol Abe and Sasaki (1977) - • (64-17-5)

Acetone Abe and Sasaki (1977) - • (67-64- l)

M N N G Howard et al. (1972) + + (70-25-7) Stich et al. (1973)

DDD,p,p' Palmer et al. (1972) + ? (72-54-8)

DDE, p,p' Palmer et al. (19725 + + (72-55-9) Kelly-Garvert and Legator (1973)

DDA, p,p' Palmer et al. (1972) - • (83-05-6)

Phenanthrene Kato (1968) - • (85-01-8)

6-Methylcoumarin Kihlman et al. ( 1971) + • (92-48-8)

TABLE 9 (continued)

Chemical Reference Results ~ Carcino- (CAS registry number) genicity b

4-Nitro-o-phenylenediamine Benedict (1976) + d - (99-56-9)

Cyclohexylamine Green et al. (1970) + d •

(108-91-8) N-Dibutylamine Abe and Sasaki (1977) + d •

(111-92-2) Anthracene Abe and Sasaki (1977) - •

(120-12-7) Pyrene Abe and Sasaki (1977) - •

(129-00-0) Triflupromazine Green et al. (1970) + ,

(146-54-3) Melphalan Benedict et al. (1977) + +

(148-82-3) Patulin Umeda et al. (1977) + ÷

(149-29-1) ENT-50787 Sturelid ( 1971 ) + •

(302-48-7) Dimethylamine hydrochloride Abe and Sasaki (1977) - •

(506-59-2) TEPA Sturelid ( 1971 ) + ,

(545-55-1) 8-Ethoxycaffeine Kihlman et al. (1971) + •

(577-66-2) N, N-Butylurea Abe and Sasaki (1977) ~ •

(592-31-4) N-Methyl-N-nitrosourea Kaina et al. (I 977) + +

(684-93-5) DDT, o,p' Palmer et al. (1972) + ,

(789-02-6) N, N-Butyl-N-nitrosourea Abe and Sasaki (1977) + d •

(869-01-2)

N-Dibutylnitrosamine Abe and Sasaki (1977) - + (924-16-3)

Aflatoxin B t Umeda et al. (1977) + + (I 162-65-8)

ENT-50991 Sturelid ( 1971) • + , (!195-67-1)

ENT-50990 Sturelid ( 1971 ) + • (1195-69-3)

Chromic trioxide Tsuda and Kato (1977a) + , (1333-82-0)

ENT-51254 Sturelid (I 971) + , (2275-61-8)

ENT-51253 Sturelid (1971) + , (2275-81-2)

167

168

TABLE 9 (continued)


4-Aminoquinoline 1-oxide Abe and Sasaki (1977) + d • (2508-86-3)

Hycanthone Benedict et al. (1977) ? •

(3105-97-3) DDE, o,p' Palmer et al. (1972) - •

(3424-82-6) Butylbutanolnitrosamine Abe and Sasaki (1977) - •

(3817-11-6) Bruneomycin Sinkus (1969) + •

(3930-19-6) ENT-51028 Sturelid ( 1971 ) + •

(4238-92-0) Butylbutanolamine Abe and Sasaki (1977) - •

(4543-95-7) 2-Nitro-p-phenylenediamine Benedict (1976) + d •

(5307-14-2) ENT-51256 Sturelid ( 1971 ) + •

(5774-35-6) Sodium nitrite Abe and Sasaki (1977) + d •

(7632-00-0) Tsuda and Kato (1977b) Saline Abe and Sasaki (1977) - •

(7647-14-5) Sodium hypochlorite Mickey and Holden Jr. ( 1971 ) + d •

(7681-52-9) Potassium permanganate Tsuda and Kato (1977a) - •

(7722-64-7) Potassium dichromate Tsuda and Kato (1977a) + •

(7778-50-9) Sterigmatocystin Umeda et al. (1977) + +

(10048-13-2) Bleomycin Paika and Krishan (1973) + •

(11056-06-7) Benedict et al. (1977) Rubidomycin De Grouchy and De Nava (1968) + d +

(20830-81-3) Mycophenolic acid Umeda et al. (1977) + •

(24280-93- I) Methylnitrosocyanamide Inui and Taketomi (1977) + •

(33868-17-6) Nitrosation products of

methylguanidine Lo and Stich (1975) + •

" Results: ( + ) positive; ( - - ) negative; ( ± ) positive and negative; (?) inconclusive; ( + d) positive with dose response; (@) metabolic activation.

b The reported carcinogenicity results were obtained from Searle (1976), Tomatis et al. (1978), or Griesemer and Cueto (1980): ( + ) some evidence of carcinogenicity from animal studies; ( , ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

169

TABLE 10

QUALITATIVE RESULTS OF CHEMICALS REVIEWED IN THE MAMMALIAN CYTOGENETIC IN VITRO CELL CULTURE ASSAY a




+ -- ?

Alcohols and phenols 10 (64-17-5) (149-29-1) (3105-07-3) (3817-1 I-6) (3930-19-6) (4543-95-7) (10048-13-2) (I 1056-06-7) (20830-81-3) (24280-93-1)

Aliphatic amines 9 (50-07-7) (108-91-8) (111-92-2) (146-54-3) (506-59-2) (3105-97-3) (4543-95-7) (11056-06-7) (20830-81-3)

Alkaloids 1 (50-37-3)

Amides and sulfonamides 3 (50-37-3) (59-05-2) (11056-06-7)

Amine-N-oxides 2 (56-57-5) (2508-86-3)

Amino acids and derivatives 4 (50-76-0) (59-05-2) (148-82-3) (11056-06-7)

Anthraquinones 1 (20830-81-3)

Antibiotics 5 (50-07-7) (50-76-0) (3930-19-6) (11056-06-7) (20830-81-3)

6 3 1 0

0 1 0 0

2 1 0 0

2 0 0 0

4 0 0 0

1 0 0 0

5 0 0 0

5 2 2 0

170

TABLE 10 (continued)



Summary of overall results ~'

Aromatic amines (50-76-0) (59-05-2) (99-56-9) (2508-86-3) (3105-97-3) (3930-19-6) (530% 14-2) (11056-06-7)

Aryl halides (50-29-3) (53-19-0) (72-54-8) (72-55-9) (83-05-6) (789-02-6) (3424-82-6)

Aziridines (50-07-7) (52-24-4) (302-48-7) (545-55-1) (1195-67-1) (1195-69-3) (2275-61-8) (2275-81-2) (4238-92-0) (5774-35-6)

Benzene ring (50-29-3) (53-19-0) (59-05-2) (72-54-8) (72-55-9) (83-05-6) (99-56-9) (148-82-3) (789-02-6) (3424-82-6) (3930-19-6) (5307-14-2)

Carbamates (50-07-7) ( 11056-06- 7)

Carbohydrates and derivatives (11056-06-7) (20830-81-3)

8

7

10

12

2

2

7 0 1 0

5 2 0 0

10 0 0 0

10 2 0 0

2 0 0 0

2 0 0 0



Halogens and inorganic derivatives (7681-52-9)

Heterocyclic rings not otherwise classified (50-07-7) (50-18-0) (50-37-3) (50-76-0) (59-05-2) (92-48-8) (149-29-1) (1162-65-8) (2508-86-3) (3930-19-6) (10048-13-2) (11056-06-7) (24280-93-1)

Lactones (50-76-0) (149-29-1) (1162-65-8)

Metals and derivatives (1333-82-0) (7632-00-0) (7647-14-5) (7681-52-9) (7722-64-7) (7778-50-9)

Mycotoxins (50-37-3) (149-29-1) (1162-65-8) (10048-13-2)

Nitriles, azides (33868-17-6)

Nitroaromatics (99-56-9) (5307-14-2)

Nitrogen and sulfur mustards (50-18-0) (148-82-3)

Nitroquinolines (56-57-5)

Nitrosamides (33868-17-6)

Nitrosamines (55-18-5) (62-75-9) (924-16-3) (3817-11-6)



+ - ? ~+

1

13

3

6

4

1

2

2

1

1

4

1 0 0 0

12 1 0 0

3 0 0 0

4 2 0 0

3 I 0 0

I 0 0 0

2 0 0 0

2 0 0 0

1 0 0 0

I 0 0 0

2 2 0 0

171

172



Number of Summary of overall results b

Nitrosoguanidines (No CAS No.) (70-25-7)

Nitrosoureas (684-93-5) (869-01-2)

Organic sulfur compounds not otherwise classified (52-24-4)

Phenothiazines (146-54-3)

Phosphoric acid esters and phosphamides (50-18-0) (52-24-4) (302-48-7) (545-55-1) (1195-67-1) (1195-69-3) (2275-61-8) (2275-81-2) (4238-92-0) (5774-35-6)

Polycyclic aromatic hydrocarbons (50-32-8) (56-49-5) (57-97-6) (85-01-8) (120-12-7) (129-00-0)

Purine derivatives (58-08-2) (577-66-2)

Pyrimidine derivatives (11056-06-7)

Quinones (50-07-7) (3930-19-6)

Saturated alkyl halides (50-29-3) (53-19-0) (72-54-8) (146-54-3) (789-02-6)

Sulfur and nitrogen oxides (7632-00-0)

Unsaturated alkyl halides (72-55-9) (3424-82-6)

Ureas, thioureas (592-31-4)

2

2

I

1

10

6

2

1

2

5

1

2

I

2 0 0 0

2 0 0 0

1 0 0 0

I 0 0 0

10 0 0 0

1 5 0 0

2 0 0 0

1 0 0 0

2 0 0 0

5 0 0 0

1 0 0 0

1 1 0 0

0 1 0 0

compounds reviewed + -- 9 +


173




+ - ? __+

Xanthenes 2 1 0 1 0 (3105-97-3) (10048-13-2)

Unclassified 1 0 1 0 0 (67-44-1)


b Results: ( + ) positive; ( - - ) negative; ( ± ) positive and negative, no clear distinction could be drawn regarding the response as the Work Group judged that good experimental data existed for each type of response; (?) inconclusive; (0) no results reported.

S-independent type since damage induced in a G O lymphocyte can be converted into an aberration during G O or G~ following stimulation. The situation here is similar to that for ionizing radiation, which is S-independent, where there are many docu- mented reports of estimations of occupational and accidental exposures.

An additional complication arises since aberrations induced in stem cells will have a high probability of being lost as a result of cell division. This means that if individuals are studied some months after exposure there will have been a turnover of much of the lymphocyte population, and repopulation will have been largely from normal cells. Aberration frequencies will be considerably reduced. This is apparent from studies of individuals receiving chemotherapy. Chromosome aberrations are observed in samples taken during and immediately after treatment, but after 2 or more months the aberration frequency is often not significantly different from the control values. Therefore blood samples should be taken as soon as possible following an accidental exposure.

In order to determine whether a population has been exposed to a hazardous agent, it is essential to compare the aberration frequency of this population with a control population. Also, there are many factors which could influence aberration yields (e.g., age, sex, occupation, geographic location, medical history, life-style), and this control population should be matched as closely as possible for these potentially influential factors to the "exposed" population.

Finally, it should be emphasized that an observation of increased chromosome aberration yields in the peripheral leukocytes of individuals or populations is an indication of exposure to a clastogen and is not a predictor of an increased genetic risk or of an increased risk of cancer.

The leukocyte assay can be used as an in vivo test provided that the above considerations are taken into account. It should also be appreciated that because of these same considerations the in vivo assay is rather insensitive. However, they do not detract from the usefulness of the system for doing in vivo testing. In the event

174

that in vivo exposures are utilized, it is recommended that in vitro experiments be done in parallel to avoid the possibility of obtaining false-negative results due to S-stage specificity of the substance.

Since the leukocytes are essentially all in the G o stage of the cell cycle at the time of blood withdrawal and spend at least the first 12 h of their culture life in the Gl stage, they can be readily used to detect the rare, truly radiomimetic chemicals by treatments during the first few hours of culture time. Subsequent stages (S and G2) can be studied as outlined in the protocol.

A. Protocol for testing For a test with peripheral leukocytes, freshly drawn heparinized venous blood or

a buffy coat obtained from this is added to a culture medium (many different ones have been shown to be suitable), usually in a ratio of l part blood to l0 parts medium. If strongly electrophilic substances are tested, treatment should be done in physiological saline to minimize the possibility of the substance interacting with serum and red blood cells. Often, in these cases, purified lymphocytes should be treated rather than whole blood. Exposures should be made at all phases of the cell cycle, i.e., G 0, G~, S, and G 2. After treatment, the cells should be cultured in medium without the test chemical. Duplicate cultures should be used for each treatment. Preferably, separate experiments should be carried out on blood from each of 2 donors. When G o or G~ cells are tested, fixation should be carried out at a time early enough after initiation to insure that the bulk of the cells sampled are in their first in vitro mitosis *. The fixation time will usually be no later than 52 h after culture initiation, unless it has been determined that the treatment produces a large cell-cycle delay. When there is delay, a sampling time that corresponds to the first mitotic division should be used. Subsequent stages of the cell cycle, S and G 2, can be tested for sensitivity by adding the substance between 36-48 h in a culture that would be fixed 52 h after initiation. Again, if there is cell-cycle delay, a later fixation will be necessary. As with all cytogenetic chromosome aberration tests, the aberrations should be presented as chromatid- and chromosome-type aberrations and further subdivided into deletions and exchanges. 300 cells should be analyzed for each experimental point. At least 3 doses should be used, covering a 10-fold range, with the highest dose being within a factor of 2 of that which gives a significant toxic effect. There should also be appropriate controls, i.e., a negative a n d / o r solvent control and a positive control.

B. Chemicals reviewed 53 agents were evaluated by the Work Group from experimental data from 66

papers. Table 11 summarizes the qualitative results obtained from these studies with

* It is recommended that BrdU (1 × 10-5 M) be added to the cultures at the time of stimulation and remain in the culture until fixation. The slides should be stained by the fluorescence plus Giemsa technique (or suitable alternative) for differential staining of the chromatids and only cells that do not show differential staining (i.e., first-division cells) should be analyzed.

175

TABLE 11

OVERALL QUALITATIVE RESULTS OF COMPOUNDS REVIEWED IN THE MAMMALIAN CYTOGENETIC LEUKOCYTE OR LYMPOCYTE ASSAY


Mitomycin C Shaw and Cohen (1965) + + (50-07-7)

Cyclophosphamide Chebotarev et al. (1976) + d / + + (50-18-0) Madle et al. (1978)

Bauchinger and Schmid (1969) Schmid and Bauchinger (1968) Dobos et al. (1974) Morad and El Zawahri (1977)

LSD-25 Abbo et al. (1968) - , (50-37-3) Sturelid and Kihlman (1969)

TEM Luippold et al. (1978) + + (51-18-3)

Nitrogen mustard Conen and Lansky ( 1961 ) + + (51-75-2)

Thio-TEPA Funes-Cravioto and Yakovenko (1972) + + (52-24-4) Kirichenko (1974)

Busulphan Honeycombe (1978) + d - (55-98-1)

4-Nitroquinoline I-oxide Hiragun and Nishimoto (1973) + + (56-57-5)

Urea Oppenheim and Fishbein (1965) + • (57-13-6)

7,12-DMBA O'Brien et al. (1971) + + (57-97-6)

Caffeine Lee (1971) + d • (58-08-2)

N-Nitrosomorpholine Newton et al. (1977) + + (59-89-2)

Methyl methanesulfonate Brewen et al. (1970) + + (66-27-3)

Trenimon Meist (1975) 9 + (68-76-8)

Benzene Tough and Court Brown (1965) + + (71-43-2) Khan and Khan (1973)

Koizumi et al. (1974) Funes-Cravioto et al. (1975) Ducatman et al. (1975) Szentesi et al. (1976) Purchase et al. (1978)

2,4-D Pilinskaya (1974) + , (94-75-7)

Styrene Meretoja et al. (1977) + + (100-42-5)

Epichlorohydrin Kucerova and Polivkova (1976) + + (106-89-8) Kucerova et al. (1977)

Vinyl chloride (75-01-4)

+ +

176



Cyclohexamine Brewen et al. ( 1971 ) - * ( 108-91-8)

Hydroxyurea Oppenheim and Fishbein (1965) + * (127-07-1)

Cytosine arabinoside Brewen and Christie (1967) + • (147-94-4)

Ethylenimine Chang and Elequin (1967) + + ( 151-56-4)

Lead acetate Schmid et al. (1972) - + (301-04-2) Deknudt and Deminatt i (1978)

Trimethylphosphate S6derman (1972) + d + (512-56-1)

TEPA Kucerova and Polivkova (1976) + • (545-55- I) Selezneva and Chebotarev (1976)

Acetohydroxamic acid Oppenheim and Fishbein (1965) - • (546-88-3)

8-Ethoxycaffeine Jackson (1964) + •

(577-66-2) N-Hydroxyurethane Oppenheim and Fishbein (1965) + •

(589-41-3) Ethylnitrosourea Soukup and Au (1975) + d ÷

(759-73-9) Cadmium sulfide Shiraishi et al. (1972) + +

(1306-23-6) Potassium arsenite Oppenheim and Fishbein (1965) + •

(1332-10-1) Aflatoxin Promchainant et al. (1972) ? +

(1402-68-2) Mari juana and D-9-

tetrahydrocannabinol Nichols et al. (1974) • (1972-08-3)

N-Hydroxy cyclohexylamine Brewen et al. (1971) • (2211-64-5)

3[ bis(2-Chloroethyl)amino]- 4-methylbenzoic acid Popescu et al. (1973) + •

(5977-35-5) l-Phenyl-3,3-dimethyltriazene Newton et al. (1977) ? •

(7227-91-0) Lead Schmid et al. (1972) ± •

(7439-92-1) Deknudt et al. (1973) Bauchinger et al. (1976) Deknudt and L+onard (1975) Deknudt et al. (1977)

Cadmium Deknudt et al. (1973) (7440-43-9) Bauchinger et al. (1976)

Deknudt and L6onard (1975)

+ +



Zinc Deknudt et al. (1973) ± • (7440-66-6) Bauchinger et al. (1976)

Deknudt and L~onard (1975) Zinc chloride Deknudt and Deminatti (1978) - •

(7646-85-7) Sodium hypochlorite + d •

(7681-52-9) Hydroxylamine - •

(7803-49-8) Ozone --+ •

(10028-15-6)

177

Mickey and Holden ( 1971 )

Oppenheim and Fishbein (1965)

Zelac et al. (1971) Merz et al. (1975) Gooch et al. (1976) McKenzie et al. (1977)

Cadmium chloride Deknudt and Deminatti (1978) - + (10108-64-2)

Bleomycin Hayez-Delatte and Feremans (1975) + d / + • (11056-06-7) Dresp et al. (1978)

Promchainant (1975) Fluoride L~onard et al. (1977) - •

(16984-48-8) Daunorubicin Vig et al. (1969) + +

(20830-81-3) Vig (1971b) Krogh Jensen and Philip ( 1971 )

Luteoskyrin Keutel and M6ckel (1969) + + (21884-44-6)

N, N, N'-tris-( 2-Chloroethyl)-N', o- propylene phosphoric acid (22089-22-1)

Adriamycin (23214-92-8)

Sodium azide (26628-22-8)

Fotrin (37132-72-2)

Hampel et al. (1968) + d •

Vig (1971a) + •

Sander et al. (1978) - ,

Tarusina and Yakovenko (1976) + •

a Results: ( + ) positive; ( - ) negative; ( ± ) positive and negative; (?) inconclusive; ( + d ) positive with dose response.

b The reported carcinogenicity results were obtained from Searle (1976L Tomatis et al. (1978), or Griesemer and Cueto (1980): ( + ) some evidence of carcinogenicity from animal studies; ( • ) there was either insufficient or no information available to the Work Group from the sources used regarding the carcinogenic response of this compound.

178

TABLE 12

QUALITATIVE RESULTS OF COMPOUNDS EVALUATED IN THE MAMMALIAN CYTO- GENETIC LEUKOCYTE OR LYMPHOCYTE ASSAY ACCORDING TO CHEMICAL CLASS a




+ - .9 +

Alcohols and phenols 5 (1972-08-3) (11056-06-7) (20830-81-3) (21884-44-6) (23214-92-8)

Aliphatic amines 5 (50-07-7) (108-91-8) (11056-06-7) (20830-81-3) (23214-92-8)

Alkaloids 1 (50-37-3)

Alkyl epoxides 1 (106-89-8)

Alkyl sulfates, sulfoxides, sulfones, sulfonates 2 (55-98-1) (66-27-3)

Amides and sulfonamides 3 (50-37-3) (546-88-3) (11056-06-7)

Amine-N-oxides 1 (56-57-5)

Amino acids and derivatives I (11056-06-7)

Anthraquinones 2 (20830-81-3) (23214-92-8)

Antibiotics 4 (50-07-7) (I 1056-06-7) (20830-31-3) (23214-92-8)

Aromatic amines 2 (147-94-4) (11056-06-7)

Aryl halides I (94-75-7)

Aziridines 7 (50-07-7) (51-18-3) (52-25-5) (68-76-8) ( 151-56-4) (545-55-1) (37132-75-2)

4 1 0 0

4 I 0 0

0 I 0 0

1 0 0 0

2 0 0 0

I 2 0 0

1 0 0 0

1 0 0 0

2 0 0 0

4 0 0 0

2 0 0 0

1 0 0 0

6 0 I 0



Benzene ring (71-43-2) (94-75-7) (100-42-5) (5977-35-5) (7227-91-0)

Carbamates (50-07-7) (589-41-3) (11056-06-7)

Carbohydrates and derivatives ( 147- 94-4) (11056-06-7) (20830-81-3) (23214-92-8)

Halogenated ethers and halohydrins (106-89-8)

Halogens and inorganic derivatives (7681-52-9)

Heterocyclic tings not otherwise classified (50-07-7) (50-18-0) (50-37-3) (51-18-3) (59-89-2) (1402-68-2) ( 1972-08- 3 ) (11056-06-7) (22089-22-1) (37132-72-2)

Hydroxylamines (127-07-1) (546-88-3) (589-41-3) (2211-64-5) (7803-49-8)

Lactones (1402-68-2)

Metals and derivatives (301-04-2) (1306-23-6) ( 7439-92-1 ) (7440-43-9) (7440-66-6) (7646-85-7) (7681-52-9) ( I 0108-64-2)

(26628-22-8)



4- -- 9 +

5

3

4

1

1

10

5

I

10

4 0 1 0

3 0 0 0

4 0 0 0

I 0 0 0

1 0 0 0

7 2 I 0

2 3 0 0

0 0 I 0

3 4 0 3

179

180




Summary of overall results h

@ '? __

Mycotoxins (50-37-3) (1402-68-2) (21884-44-6)

Nitriles, azides (26628-22-8)

Nitrogen and stllfur mustards (5O- 18-O) (51-74-2) (5977-35-5) (22089-22-1)

Nitroquinolines (56-57-5)

Nitrosamines (59-89-2)

Nitrosoureas (759-73-9)

Organic sulfur compounds not otherwise classified (52-24-4)

Organophosphorus compounds (37132-72-2)

Phosphoric acid esters and phosphamides ( 50-18-0) (52-24-4) (512-56-1) (545-55-1) (22089-22-1)

Polycyclic aromatic hydrocarbons (57-97-6)

Purine derivatives (58-08-2) (577-66-2)

Pyrimidine derivatives ( 147- 94-4) (I 1056-06-7)

Quinones (50-07-7) (68-76-8)

Saturated alkyl halides (106-89-8)

Triazenes (7227-91-0)

Unsaturated alkyl halides (75-01-4)

Ureas, thioureas (57-13-6) (127-07-1)

I 1 I 0

0 1 0 0

4 0 0 0

1 0 0 0

1 0 0 0

1 0 0 0

I 0 0 0

1 0 0 0

5 0 0 0

I 0 0 0

2 0 0 0

2 0 0 0

I 0 1 0

I 0 0 0

0 0 I 0

I 0 0 0

2 0 0 0


181

EPA Gene-Tox chemical classification Number of Summary of overall results b [CAS registry number(s)] compounds

reviewed + - ? ~+

Unclassified 2 0 1 0 1 ( 10028-15-6) (16984-48-8)

" Experimental results on 51 compounds were reviewed by the Work Group in this assay system. A chemical may be found in more than 1 chemical class and the information in this table can be cross-referenced to the information in Table 11 by using CAS registry number(s) provided.

b Results: ( + ) positive; (--) negative; (?) inconclusive; ( ± ) positive and negative, no clear distinction could be drawn regarding the response as the Work Group judged that good experimental data existed for each type of response; (0) no results reported.

each compound's reported carcinogenicity whenever this information was available. Table 12 shows these qualitative results among the various chemical classes in which these 53 agents were categorized.

IX. Overall assessment of cytogenetic assay systems

It is clear that each assay has particular advantages but also attendant disadvantages, and it is difficult to recommend a single cytogenetic assay as the most likely to show the clastogenic potential of compounds. Table 13 compares results of 37 compounds evaluated in more than 1 of the assays reviewed in this report. All results correlated well with the reported carcinogenicity response (in 17 of 18 cases); carcinogenicity data were not available to the Work Group on 19 of the chemicals in this group.

Of the in vivo assays, if a determination is required of whether or not a compound can produce chromosome aberrations, the bone-marrow assay is probably the simplest and cheapest to perform. It is more difficult to obtain the suggested number of analyzable spermatogonial cells. If some estimate is required of the potential genetic hazard of a compound, clearly Other assays are more appropriate. The frequency of mutations and translocations transmitted to the F~ is generally very much higher from chemically .treated post-spermatogonial cell stages than from spermatogonial cells. Even when the much shorter lifetime of post-spermatogonial cells is taken into account, it is still, in most cases, really these cells that are at greater risk in terms of genetic hazard. Therefore, studies of first-cleavage divisions or early embryos where the treated post-spermatogonial cells can be analyzed are more appropriate. Also considerations of the chromosome damage in oocytes would be required. These latter considerations fall outside the scope of testing at this point.

Of the in vitro assays, either peripheral leukocytes or cell lines are ~uitable. The leukocyte system has the advantage that the cells are synchronous, at least at the time of stimulation by phytohemagglutinin, and remain sufficiently synchronous

82

,..1

6 ~

~ 0 0 ,.m

>

o ~

0

0 0

©

z E~

<

<

e ~ ~ ~~

o ÷ o + I ÷ -r- o o ÷ o -t-

+ -+- + ÷ ; ÷ o o + -t- + +

o o I I I o o I o o o I

o o o o ÷ + o I o o o o o

o + o ~ I ÷ + I o + + o +

i I <

1 8 3

+ . + ~* + . . . + + ÷ . + + ,, + . .

+ ~ o o + 0 o + + o o ÷ + ÷1 + + o

o + o + + o o + o + q- +

÷ o

I ~ o o o I I ] o L o I o o

o o o o o I o o o q'- o q- ~ o o o o o

+ ¢-. ~ o I o + o ~ -I- o I + o I + o o

• ~ ~ ,.1~ ¢"1 ;,-'1 ' 0

184

U-1 ,-4

6~

J ~

.=

0

÷ .~ + + , +

+ + + ~. o +

,~ + + o + o

4- o ~ + o o

+ l I o I o c ~

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+ o o o o o

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. .='9, ~ _ < ~ '

185

throughout the first cell cycle to allow mainly S or G 2 cells to be treated. Also results

ob ta ined in vitro can be compared with those ob ta ined in the same cell type in vivo. The disadvantages of any cytogenetic assay are: (1) it is t ime-consuming; and (2)

in order to be convinced that a part icular compound shows a negative response,

studies mus t be conducted with $9 activation for in vitro assays and with mult iple fixations to take account of possible differential sensitivities of different stages of the cell cycle for in vivo and in vitro assays.

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mammalian in vivo and in vitro cytogenetic assays: a report of the u.s. epa's gene-tox program

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