Experimental Animal Data

Appendix II

Table 35. Dominant Lethal Results from Tyl et al. (87)

Parameter Acrylamide dose (mg/kg bw/day)

0.5 2.0 5.0

Plug/sperm-positive females ↔ ↔ ↔

Implantations/dam ↔ ↔ ↓14%

Live implants/litter ↔ ↔ ↓20%

Postimplantation loss ↓40% ↔ ↑2.3-fold

↑,↓ Statistically increased, decreased compared to control value.

↔ Not statistically different from control value.

Utility (Adequacy) for CERHR Evaluation Process:These data are of high quality, and are adequate and informative for the Evaluation Process. They are adequate to identify a male dominant lethal dose (~5 mg/kg bw/d), to show that the neurotoxicity seems to occur at doses similar to those that affected fertility, and to infer that female fertility might be affected as well. These data shed no light on mechanism, but the study was not designed to do so.

Appendix II

groups and was transiently depressed in the fi rst week of life in the 25 ppm group. Full litter loss and decreased pup weight were considered by the Expert Panel to represent developmental toxicity rather than female reproductive toxicity (see Section 3.4.2).

Other studies that included evaluations of female reproductive function (124, 125) were reported with insuffi cient detail to be used in the evaluation.

4.4.2.2 Male reproduction

The Expert Panel notes that there are a number of genotoxicity studies with endpoints that might be considered reproductive (e.g., pre-implantation loss after parental treatment). Studies that were designed to evaluate genotoxicity were grouped in Section 2. These studies include those with dominant lethal (Section 2.3.2.2), heritable translocation (Section 2.3.2.4), and specifi c locus mutation (Section 2.3.2.5) endpoints. Although these studies are placed for organizational purposes under the heading of genotoxicity rather than developmental or reproductive toxicity, the Expert Panel considers these studies important in evaluating the reproductive and developmental effects of acrylamide. Because genotoxicity studies were reviewed and summarized in Section 2, attention in this section is focused on studies for which the primary endpoints were not genotoxic, although some reproductive outcomes from genotoxicity studies are repeated here.

Three studies of adequate design were reported in suffi cient detail to permit an evaluation of acrylamide effects on male reproduction (75, 81, 129). Zenick et al. (81) gave acrylamide in drinking water to male rats at 0, 50, 100, or 200 ppm ( 5, 7–8, and 12 mg/kg/day, respectively) for up to 10 weeks. Mating was visually monitored and sperm count, motility, and morphology determined during week 9. Sperm in the uterus were also assessed. During week 10, males in the 0 and 100 ppm acrylamide groups were mated with intact estrous females, which were killed on GD 17 for evaluation of intra-uterine contents. Males were killed at week 11 for histology of one testis and epididymal fl uid, and homogenization resistant spermatid count determination from the other testis.

Morbidity and mortality led to termination of the high dose males at week 6. This group displayed neurological toxicity (hindlimb splay). There was a decrease in vaginal sperm in females mated to 100-ppm males compared to control males and only 1/15 females mated to a 100-ppm male had sperm in the uterus. Only 5/15 100-ppm males established a pregnancy, compared to 11/14 in the control group. Postimplantation loss was higher in females that were mated to 100-ppm sires (31.7 ± 3.8%) compared to 0-ppm sires (8.0 ± 1.1%). Testicular histology did not show abnormalities associated with 100-pm acrylamide treatment. The authors concluded that the male reproductive toxicity of acrylamide at 100 ppm (~,7–8 mg/kg bw/day) was due to abnormalities of copulatory behavior, delivery of sperm to the female genital tract, and postimplantation loss.

Sublet et al. (75) used a dominant lethal design plus an evaluation of mating and fertilization success to confi rm and extend the work of Zenick et al. (81). Male rats were treated with acrylamide 0, 15, or 45 mg/kg bw/day for 5 days by gavage and mated to untreated hormonally primed, ovariectomized females. Females were killed 10–15 minutes after ejaculation and sperm were recovered and examined by light microscopy. No abnormalities of male copulatory behavior were observed at any dose, and all females had sperm in the vagina, but there was a signifi cant decrease in the proportion of females with uterine sperm after mating with males in the 45 mg/kg bw/day group 1 week after treatment. There were no differences in the proportions of females with uterine sperm 2, 3, or 4 weeks

Appendix II

after treatment of males with either dose of acrylamide. Uterine sperm were evaluated by computer-assisted sperm analysis after females mated with 0 and 45 mg/kg bw/day acrylamide-treated males.

The only difference was a decrease in motile sperm and curvilinear velocity 3 weeks after treatment of males with acrylamide at the high dose. In an experiment where males were gavage dosed with 0, 15, or 45 mg/kg bw/day acrylamide for 5 days, the percent oocytes fertilized per female was reduced in a dose-dependent manner in weeks 1 (both acrylamide doses) and 3 (45 mg/kg/day acrylamide).

The authors suggested that impaired fertilizing ability of acrylamide-exposed sperm may play a role in the apparent pre-implantation loss seen after treatment. They proposed that although copulatory behavior appeared normal in their experiments, and acrylamide doses were below those associated with gross neurologic impairment, there might be subtle abnormalities of copulation.

Tyl et al. (129) repeated a portion of the study of Sublet et al. (75), using a design that matched the fi rst week of the Sublet et al. study. Male rats were exposed by daily gavage for 5 consecutive days to acrylamide at 0, 5, 15, 30, 45, or 60 mg/kg bw/day, 25 animals/dose group. Three days after the treatment period, males were cohabited with untreated females for a single overnight period, then males underwent grip testing and were killed for evaluation of cauda epididymal sperm parameters.

Mated females were killed on GD 15 for evaluation of uterine contents. There was reduced grip-strength at 60 mg/kg bw/day, though without detectable histopathology. Fewer males in the 60 mg/

kg/day group than control group mated or produced litters. Pair-wise comparison showed statistically signifi cant decreases in weight gain at 15 mg/kg bw/day and higher. Trend tests on the pregnancy index showed signifi cance for trend at 15 mg/kg bw/day and above, and pair-wise statistically fewer litters and reduced litter size (and increased post-implantation loss) at 45 and 60 mg/kg bw/day. The fertility and pregnancy data are compelling, and the Panel believes an incremental effect exists at and above 15 mg/kg bw/day.

Other studies provided supporting information. Hashimoto et al. (126) treated male ddY mice by (126) treated male ddY mice by (126 gavage with acrylamide 35.5 mg/kg bw twice/week for 8 weeks and demonstrated a decrease in relative testicular weight. Sakamoto et al. (127) administered acrylamide to ddY mice as a single (127) administered acrylamide to ddY mice as a single (127 oral dose of 100 or 150 mg/kg at age 30 days (prepubertal) or 60 days (adult) and found the round spermatid to be the cell type most sensitive to acrylamide toxicity. Pacchierotti et al. (88) treated male B6C3F₁ mice with single i.p. acrylamide doses of 0, 75 , or 125 mg/kg bw or with 5 daily i.p.

doses of 50 mg/kg. Males were mated to untreated females 7 days after the last acrylamide dose and killed weekly for fl ow cytometric analysis of testicular cell populations. The percent females showing evidence of mating after cohabitation with treated males was reduced in all acrylamide groups without regard to acrylamide dose. The reduction in mating success resolved by 28 days after treatment in the 125 mg/kg group. Changes in relative germ cell populations indicated probable impairment of chromosome segregation during spermatogonial mitosis. Marchetti et al. (89) gave acrylamide i.p. to male B6C3F₁ mice at 50 mg/kg bw/day for 5 consecutive days (discussed in Section 2.3).

Treated males were mated with untreated females at different time intervals. Acrylamide treatment was associated with a decrease in fertilization except with treatment of males at the stem cell stage.

Sakamoto and Hashimoto (124) gave male ddY mice acrylamide in drinking water at 0.3, 0.6, 0.9, and 1.2 mM [21.3, 42.6, 64.0, and 85.2 mg/L, respectively] for 4 weeks prior to mating or prior to evaluation of reproductive organ weight and epididymal sperm. There were dose-related decreases in the number of fetuses/dam in the top two dose groups; at the top dose, there were decreases in the proportion of pregnant dams and offspring per dam, and increases in resorptions per dam. Epididymal

Appendix II

sperm count was decreased and abnormal sperm forms increased at the top dose.

Two studies by Costa et al. (128), (79) provided evidence that the male reproductive toxicity of acrylamide is mediated by glycidamide; however, these studies did not provide conclusive evidence to establish glycidamide as the proximate reproductive toxicant.

4.4.2.3 Continuous breeding or multigeneration designs

Chapin et al. (130) performed a Reproductive Assessment by Continuous Breeding in Swiss mice on acrylamide or one of three analogs. Acrylamide was given in drinking water at 0, 3, 10, and 30 ppm. Doses in F₀ females were estimated to be 0, 0.81, 3.19, and 7.22 mg/kg bw/day. Doses in males could not be estimated due to highly variable water intake. There were no effects of treatment on the proportion of fertile pairs (pairs delivering at least one litter), percentage of pairs delivering each litter, number of litters per pair (4.8–5.0 litters/pair), or proportion of pups born alive. Considering all litters together, there was a signifi cant decrease at the high dose in mean pups/litter. Early resorptions and postimplantation loss were increased on dominant lethal testing at the top dose as well. Cross-over matings (treated males to untreated females) suggested that the acrylamide effect on mean pups/

litter was mediated through the treated male. Reproduction in F₁ mice that were exposed during gestation and again from weaning was not impaired except for a decrease in live pups/litter that was signifi cant on pair-wise comparison in the top dose group and that showed a dose-related trend across all dose groups. Estimated adult acrylamide intake in the F₁ generation was 0, 0.86, 2.9, and 7.7 mg/

kg bw/day. The authors interpreted the reproductive effects of acrylamide in males as attributable to dominant lethal activity at 30 ppm.

Tyl et al. (87) performed a two-generation reproduction and dominant lethal study in Fischer 344 (87) performed a two-generation reproduction and dominant lethal study in Fischer 344 (87 rats given acrylamide in drinking water. Drinking water concentrations were adjusted weekly based on animal body weight and water consumption to provide doses of 0, 0.5, 2.0, or 5.0 mg/kg bw/

day in 30 animals per dose group. Animals were mated after a 10-week exposure period. Drinking water concentrations were adjusted during lactation. Offspring were weaned to the same acrylamide exposure groups as their dams. Offspring were mated (30/sex/treatment group) to produce the F₂ generation. Some of the F₀ males were continued in a dominant lethal study. Increased incidences of head tilt, but not foot-splay, was signifi cant in the high-dose F₁ but not the F₀ animals. The number of live pups/litter was decreased and postimplantation loss was increased at the top dose for both F₀ and F₁ matings, although statistical confi rmation was not obtained for postimplantation loss in the F₁. This was evidence of reduced fertility and increased dominant lethality in the mice of both generations at the high dose.

Appendix II

Zenick et al. (81) Male ddY miceDrinking water: 21.3, 42.6, 64.0, 85.2 ppm [converted from mM]

Decreased litter sizeLOAEL=64.0 ppm (~14 mg/kg bw/day) Sakamoto (124)Decreased pregnancy rate and increased resorptionsLOAEL=85.2 ppm (~18 mg/kg bw/day) Male Long- Evans ratsDrinking water 0, 50, 100, 200 ppm

Impaired ejaculation, decreased vaginal and uterine sper

m and pregnancy rates and increased

postimplantation loss in cohabited females

LOAEL=100 ppm (about 7–8 mg/kg bw/day)Zenick et al. (81) Male Long- Evans rats

Gavage: 0, 0, 5, 15, 30, 45, 60 mg/kg bw/day × 5 days

Increased pre- and postimplantation loss

LOAEL=15 mg/kg bw/day Sublet (75) Gavage: 0, 15, 45 mg/kg bw/day × 5 days

Decreased uterine sperm in

cohabited females, decreased uterine sper

m motilityLOAEL=45 mg/kg bw/day Decreased fertilization of oocytesLOAEL=15 mg/kg bw/day (lowest tested level) Male Long- Evans rats

Gavage: 0, 5, 15, 30, 45, 60 mg/kg bw/day × 5 days

Decreased mating; fewer litters producedLOAEL=45 mg/kg bw/day Tyl (129) Swiss mice, cohabiting pairs Drinking water: 0, 3, 10, 30 ppmDecreased live litter size in two generationsLOAEL=30 ppm (7–8mg/kg bw/day)Chapin (130) Fischer 344 rats, 2-generationDrinking water: 0, 0.5, 2.0, 5.0 mg/kg bw/day

Decreased live litter size and

increased postimplantation loss in twLOAEL=5 mg/kg bw/dayTyl (87)(87)(87 o generations LOAEL lowest observed adverse effect level; NOAEL= no observed adverse effect level. a Considered by the Expert Panel to represent developmental toxicity rather than reproductive toxicity.

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The Expert Panel found no human data with which to directly evaluate possible reproductive toxicity of acrylamide. The data are suffi cient to conclude that acrylamide is a reproductive toxicant in male rats bred to untreated females manifested as impaired delivery of sperm to the female genital tract, and a reduction in litter size due to increased postimplantation loss at exposure levels of about 5–8 mg/kg bw/day in drinking water. The data suggest that acrylamide is not a female reproductive toxicant in rats at exposures up to 100 ppm in drinking water (about 10–14 mg/kg bw/day). Data are suffi cient to conclude that acrylamide is a reproductive toxicant in male mice bred to untreated females as manifested by a decrease in litter size with drinking water exposures of about 7–14 mg/kg bw/day. The data suggest that acrylamide has no effects on female reproductive function in mice at doses up to 7–8 mg/kg bw/day. The Expert Panel found the data suggestive that the male reproductive toxicity of acrylamide in rodents is due to multiple effects, including impairment of mating ability and genetic damage in sperm, and possibly sperm function. In addition, there are suffi cient data to conclude that acrylamide induces transmissible genetic damage in male germ cells of mice in the form of reciprocal translocations and gene mutations. Such effects can lead to genetic disorders and infertility in subsequent generations. An analysis of genetic risk associated with acrylamide was conducted by Dearfi eld et al. (58). Such risks were not considered by the Expert Panel in their evaluation of LOAELs because of the lack of testing at low dose levels where reproductive and developmental toxic effects are observed.

However, considering the incidence in treated and control animals of the response detected for heritable translocations at the lowest dose level tested (40 mg/kg bw/day × 5 days), it is likely that such effects would occur at lower dose levels. The rat and mouse data are assumed relevant to assessment of human reproductive risk.

Appendix II