Acrylamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented report of a guideline study conducted to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

rodent dominant lethal assay

Test material

Test material information

Constituent 1

Reference substance name:

Acrylamide

EC Number:

201-173-7

EC Name:

Acrylamide

Cas Number:

79-06-1

Molecular formula:

C3H5NO

IUPAC Name:

prop-2-enamide

Details on test material:

- Name of test material (as cited in study report): Acrylamide Monomer
- Physical state: white odourless crystalline solid
- Supplied by: Cytec Industries, Botlek, The Netherlands
- Analytical purity: 99.7%
- Lot/batch No.: MA90-5016
- Storage condition of test material: room temperature (below 80°F, equivalent to 45°C)

Test animals

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI.
- Age at study initiation: males: 11 weeks, females: 9 weeks at the start of the mating period
- Weight at study initiation: males: 306.8 to 383.6 g, females: 191.5 to 250.9 g
- Assigned to test groups randomly: yes, under following basis: One hundred fifty (150) males were assigned to six (6) groups, 25 males per group, by a stratified randomisation procedure to provide uniform body weights across groups at the initiation of the study. One hundred fifty (150) females, in the appropriate stage of estrus, were also assigned to the six (6) groups, 25 females per group, by a stratified randomization procedure to provide uniform body weights across groups at the initiation of mating.
- Fasting period before study: yes
- Housing: solid bottom polycarbonate cages with stainless steel wire lids, with Sani-Chips animal bedding
- Diet: ad libitum No. 5002 Purina Certified Rodent Chow
- Water: ad libitum tap water
- Acclimation period: 4 to 6-day quarantine period for the males and the 0 to 3 day quarantine period for the females
ENVIRONMENTAL CONDITIONS
- Temperature: 70.8 - 73.8°F.
- Humidity (%):46.4 - 64.5%.
- Photoperiod : 12:12 hour Iight/dark cycle
IN-LIFE DATES: From: May 7, 1997 To: June 7, 1997

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: test article is totally water-soluble
- Purity: sterile and distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Acrylamide monomer in vehicle or vehicle alone was administered by gavage, at a volume of 5.0 ml/kg, based on each animal's most recent body weight, to each male once daily for five consecutive days. The dosing solutions were administered in a syringe of appropriate volume attached to a 16 gauge, 2 inch curved dosing needle (Perfektum, Popper and Sons, New Hyde Park, NY). Gavage was chosen by the Sponsor as the route of administration to duplicate the route employed by Sublet et al. (1989).

Duration of treatment / exposure:

5 days

Frequency of treatment:

Daily

Post exposure period:

8 days prior to mating

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 15, 30, 45 and 60 mg/kg/day
Basis:
nominal in water
equivalent to 0.0, 1.0, 3.0, 6.0, 9.0 and 12.0 mg/ml at a dosing volume of 5.0 ml/kg

No. of animals per sex per dose:

25 males and 25 females (females were not dosed)

Control animals:

yes, concurrent vehicle

Positive control(s):

No

Examinations

Tissues and cell types examined:

All study males in all groups were weighed and subjected to a complete gross necropsy. The gross necropsy included examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. Sperm motility (motile and progressively motile) and sperm beat cross frequency were assessed immediately after necropsy; number of sperm (per sample and per g epididymis) was evaluated at a later date using appropriately retained sperm samples. For the males not perfused in vivo (20 per group), one cauda epididymis was immediately removed, weighed and seminal fluid from the cauda was assessed for sperm number and motility.
Mated females were weighed and sacrificed at mid-pregnancy, on gd 15. The gravid uterus with attached oviducts and one ovary was dissected free and removed. The total numbers of ovarian corpora lutea were counted. Total implantation sites RTI-65C-6B21-200 were also counted and recorded. The status of each implantation site was also documented: resorption (early, late and/or total) or live implantation. If any uterus appeared non-gravid upon initial inspection, it was placed in 10% ammonium sulphide for confirmation of pregnancy status. If implantation sites appeared after staining, the total number in the uterus was recorded. Each female and all her tissues were discarded. If a female was not pregnant at scheduled sacrifice, her pregnancy status was included in the calculation of the various reproductive indices, but her data were not entered or summarized since these data were reported only for pregnant females.

Details of tissue and slide preparation:

Gross lesions were retained in fixative. Five (5) males per group were preserved by in vivo perfusion fixation with glutaraldehyde prior to necropsy. The testes and one sciatic nerve of all study males (both perfused and non-perfused) were retained in fixative for possible subsequent histopathology. The other epididymis and both testes per non-perfused male were retained in fixative for possible subsequent histopathologic examination. The sciatic nerves from the perfused males, five/group, were embedded in paraffn, sectioned at approximately 3-4 microns, stained with hematoxylin and eosin and examined histopathologically. Subsequently. additional sections of sciatic nerves from these perfused males were cut, stained with Holm's Silver/Luxol Fast Blue Stain (which gives comparable results as Bodian's Fast Blue Stain) to better differentiate the axon and myelin sheath, and examined histopathologically (second report, Appendix III). The testes and sciatic nerves from non-perfused males were not processed or examined; the testes from the perfused males were also not processed or examined histopathologically.

Evaluation criteria:

Males:
Mating index (%) = (No. impregnating females / No. males paired) x 100
Fertility index (%) = (No. males siring litters / No. impregnating females) x 100
Pregnancy index = (No. pregnant females / No. males impregnating females) x 100
Females:
Preimplantation loss (%), = ((No. corpora lutea – No.implantations) / No.corpora lutea) x 100
Postimplantation loss = ((No. total implantations - No. live implantations) / No. total implantations) x 100
Frequency of dominant lethal factors, FL %, = (1- (No. live implantations per female of test group / No. live implantations per female of control group)) x 100

Statistics:

Quantitative continuous data were compared among treatment groups against vehicle control group using Bartlett's test for homogeneity of variances. If this indicated lack of homogeneity of variances (p<0.001), nonparametric statistical tests were employed for continuous variables. If Bartlett's test indicated homogeneous variances (p>0.001), parametric statistical tests were employed for continuous variables. Parametric statistical procedures were applied to selected measures from this study. Appropriate GLM for proposed Analyses of Variance was used to determine significance of dosage-response relationship (Test for Linear Trend) and determine if significant dosage effects had occurred for selected measures. When a significant (p<0.05) main effect for dosage occurred, Dunnett's Multiple Comparison was used to compare each dosed group to the control group for that measure. A 1- or 2-tailed test was used for all pairwise comparisons to vehicle control group. Nonparametric tests included Kruskal-Wallis Test to determine if significant differences were present among groups, followed by Mann-Whitney U test for pairwise comparisons to designated control group, if Kruskal. Wallis test was significant. Jonckheere's test for k independent samples was used to identify significant dose-response trends for nonparametric continuous data. Frequency data were analyzed by Chi-Square Test for Independence for differences among treatment groups, and by Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, a 2-tailed Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between each dosed and control group. For all statistical tests, the significance limit of 0.05 (1- or 2-tailed) was used as criterion for statistical significance. A test for statistical outliers was performed on body weights.

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Remarks:

at 15, 30, 45 and 60 mg/kg/day

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

This study was performed to duplicate, to the extent possible, a portion of the study design of Sublet et al. (1989). Their design involved male Long-Evans rats dosed with acrylamide monomer at 0, 5, 15, 30. 45 or 60 mg/kg/day for five days (sd 1-5), then serially mated to naive females for 10 weeks beginning on sd 8. Effects in the Sublet study included reduced fertility and increased pre- and post-implantation loss at 15 through 60 mg/kg/day, primarily over the first three weeks post-treatment. The effects on week 1 appeared to result from an interference with sperm transport since there were reductions in percent females with uterine sperm (but not vaginal sperm) at 15 mg/kg/day (not significant) and 45 mg/kg/day (statistically significant), following a single ejaculation by treated males. Effects on sperm motility and curvilinear velocity were present at 45 mg/kg/day on week 3; there were no effects of treatment on sperm count, sperm linearity or straight line velocity (Sublet et al., 1989). There were no reported parameters of general toxicity or neurotoxicity for the males. Male systemic toxicity was present at 15, 30, 45 and 60 mg/kg/day, based on body weight changes during the dosing period (sd 1-5) and for the entire evaluation period until mating (sd 1-8). Grip strength in the hind limbs (but not the forelimbs) was significantly reduced at 60 mg/kg/day and treatment-related clinical observations were present at 45 and 60 mg/kg/day. There was no evidence of the characteristic acrylamide-induced peripheral neuropathy in the sciatic nerves from perfused males, e.g., no evidence for axonal swelling or degeneration after hematoxylin and eosin staining or after Holm's Silver/Luxol Fast Blue Stain which is a better stain to identify these effects (e.g., Friedman et al., 1997). Male reproductive toxicity was clearly present at 45 and 60 mg/kg/day. The effects included reduced mating, fertility and pregnancy indices (no values statistically significantly different from control males), increased post-implantation loss/litter (statistically significantly different at 45 and 60 mg/kg/day), decreased live implants per litter at 45 and 60 mg/kg/day (none statistically significantly different), reduced percent progressively motile sperm (possibly at 60 mg/kg/day, not statistically significantly different), and increased beat cross frequency (possibly but not significantly increased at 45 mg/kg/day, significantly increased at 60 mg/kg/day). Each of these parameters was affected by acrylamide monomer at doses as low as 15 mg/kg/day, with no statistically significant pairwise differences to the concurrent control group values. Reproductive parameters that were not affected included pre-implantation loss/litter and percent motile sperm. Epididymal sperm concentration was increased, but not statistically significantly, in all acrylamide-dosed groups with the highest value at 60 mg/kg/day (Text Table B). The increased sperm concentration in the cauda epididymis (site of sampling) may be due to reduced ejaculatory behavior or to interference with sperm transport to the vas deferens in acrylamide-treated males. The effects of acrylamide monomer on mating and fertility do not change; effects are obvious at 60 mg/kg/day and apparent at 15-45 mg/kg/day. The overall mating performance of the males is improved, confirming that the females used on the first pairing day were probably in early proestrus when paired and did not cycle to estrus during the overnight cohabitation period. The values for fertility index did not change for all groups except the 5 mg/kg/day group in which one female became sperm positive (but was not pregnant) on the first mating day.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
Systemic and reproductive toxicity was observed at 15, 30, 45 and 60 mg/kg/day. Statistically significant indicators of systemic toxicity occurred at 15-60 mg/kg/day, and statistically significant indicators of reproductive toxicity occurred at 45-60 mg/kg/day (with effects present at 15-30 mg/kg/day with no statistically significant differences). Therefore, the No Observabed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity was 5 mg/kg/day in Long-Evans male rats under the conditions of this study.