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Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
EPA OTS 798.5450 (Rodent Dominant Lethal Assay)
Version / remarks:
GLP compliance:
yes (incl. certificate)
Type of assay:
rodent dominant lethal assay

Test material

Details on test material:
purity: 99.89%
Specific details on test material used for the study:
- Purity: 99.89%

Test animals

Fischer 344
Details on test animals and environmental conditions:
All animals were housed in BRRC animal room number 103 throughout the study (from quarantine until scheduled sacrifice). The males were received approximately two weeks before the initiation of the treatment period; shipment one of the females was received approximately three weeks prior to mating, ane shipments five and eight of the females were received two weeks prior to mating. All other shipments of females were received approximately one week prior to their respective mating periods. The animals were observed regularly for general health status and ability to adapt to the watering system, body weights, were measured at least once during the weeks prior to the mating period. Only male rats in good health whose weights were at least 200 g were used for the study.

Administration / exposure

Route of administration:
oral: drinking water
- Vehicle(s)/solvent(s) used: water
Details on exposure:
Drinking water solutions containing 5000, 25000 and 5000 ppm were prepared twice by adding the appropriate amount of processed tap water to the appropriate amount of the test substance placed in Malgene carboy, and mixing manually by swirling. Amber glass bottles with Malgene stoppers were filled from the carboys and placed on the animal's caging. All test substance solution concentrations were verified using a gas chromatographic procedure. Concurrent homogeneity and stability analyses were also conducted on the dosing solutions using the same methods.

One-hundred (100) virgin male rats were assigned to three treatment groups, one positive control group and one vehicle control group using a randomization procedure stratified by body weight, such that all five groups did not differ substantially in mean body weight or body weight range. There were twenty males per group. Two hundred (200) virgin female Fischer 344 rats from each shipment were similarly assigned to the five groups by stratified randomization. The shipments of females were staggered (approximately one per week for ten weeks) to ensure that each set of females were approximately equivalent in age and weight when bred to the treated males. All males assigned to the vehicle control and the test substance treatment groups were weighed immediately prior to the first exposure, immediately after the last exposure and then weekly as any remaining females were removed from the cage and two new females were added. Water consumption was measured during the 5 dosing days for control and test substance-treated groups and dosage was calculated. Urine from control and treated males was collected during the last 24 hours of the treatment period, and then analyzed for total volume, specific gravity, osmolality, pH, protein, glucose, ketones, bilirubin, occult blood and urobilinogen. Males assigned to the positive control group were weighed immediately prior to the first exposure of treated males, prior to dosing, and then weekly as the remaining females were removed from the cage and two new females were introduced. All males were examined daily for any clinical signs of toxicity.

On the day following the last exposure of the study males receiving the test substance (and 24 hours after injection of the positive control males), two naive (untreated) females were added to each male's cage. The females were checked twice daily for dropped or vaginal copulation plugs, and examined, if necessary, once daily (a .m.) by vaginal smear for detection of vaginal sperm. The date each female exhibited evidence of copulation was designated as gestational day (gd) 0 and recorded. The female was then removed from the male's cage and housed singly until scheduled sacrifice on gd 15. The females remained in the male's cage until there was evidence of copulation or until seven days elapsed, whichever came first. If there was no evidence of copulation then the sixth day of cohabitation was designated gd 0. After completion of the first week of breeding, two new naive females were added to each male's cage and observed and measured as described above. Two new females were added to each male's cage weekly for a total of ten weeks to encompass the entire spermatogenic cycle. All study females were examined daily for any clinical signs.
Duration of treatment / exposure:
5 days
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
5 000 ppm (nominal)
Dose / conc.:
25 000 ppm (nominal)
Dose / conc.:
50 000 ppm (nominal)
No. of animals per sex per dose:
Control animals:
Positive control(s):
A concurrent positive control group was included in the study in order to validate the experimental design. The positive control group consisted of 20 male Fischer 344 rats which received a single intraperitoneal injection of 0.5 mg/kg triethylenemelamine (TEM) 24 hours prior to commencement of the 10-week mating regimen.


Tissues and cell types examined:
testes, uterus, ovaries (including corpora lutea), cervix, vagina and abdominal organs and thoracic organs and cavities of each pregnant animal.
Details of tissue and slide preparation:
All study females were sacrificed on day 15 of gestation by carbon dioxide asphyxiation. The maternal body cavities were opened by mid-sagittal thoracolaparotomy. The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic organs and cavities of each pregnant animal were examined grossly. The uteri and attached ovaries and oviducts were removed from the peritoneal cavity and ovarian corpora lutea of pregnancy were counted. The uteri were palpated and, when necessary, dissected longitudinally to determine the status of the implantation sites. The number of live implants and early and late resorptions were recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for confirmation of pregnancy status. The females and their uterine contents were then discarded.
After the last (tenth) breeding period was completed, all study males were weighed, sacrificed by exsanguination following methoxyflurane anesthesia and subjected to a full necropsy. The testes were removed, weighed and preserved in 10% neutral buffered formalin. After the above procedures were completed, the males were discarded.
The unit of comparison was the male and the pregnant female. Results of the quantitative continuous variables were intercompared for the three TetraEG-treated groups and vehicle control group by use of Levene test for equal variances, analysis of variance and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test. The positive control group was compared to the vehicle control group using the previously described set of statistics. Nonparametric data were examined statistically using the Kruskal-Wallis test followed by the Mann-Whitney U test, when appropriate. Frequency data were compared using the Fisher's Exact Test. For all statistical tests, the probability value of 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Test results
not specified
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:
Concentration analyses: Mean analytical concentrations of the test substance in water for the five (5) days of exposure of the study males were 100.1 to 106.6% of nominal for the 5000, 25000 and 50000 ppm target concentrations. No test chemical was detected in the vehicle control (0 ppm) water with the minimum detection limit of approximately 500 ppm. Homogeneity and stability studies performed on the 5000, 25000 and 50000 ppm dosing solutions indicated that the test substance was uniformly distributed throughout the prepared solutions, and that solutions were stable for at least 7 days when stored at room temperature.

Males - Treatment and Necropsy Phases: There were no treatment-related clinical signs of toxicity during the 5-day period. The body weights of all males were equivalent. Water consumption was increased at 50000 and 25000 ppm. The increased urine volume observed at 50000 and 25000 ppm is consistent with the increased water consumption in these dose groups. Urine osmolality and pH were reduced at 50000 ppm. No treatment-related lesions were observed. There were no effects of treatment on terminal body weight or on testes weights.

Mating and Reproductive Phase: Weeks 1 and 2 - Mature Sperm Stages: There was no effect of the test substance treatment on the number of pregnant and plug- or sperm-positive females. Mating and fertility indices indicated no effects of treatment. No apparent gestational effects of the test substance were noted. There were no effects of the test substance on reproductive parameters for week 2 of mating, no effects of the test substance on gestational parameters and no apparent dominant lethal effects. Weeks 3, 4 and 5 - Spermatid Stages: There were no effects on reproductive parameters for week 3. There was no effect of the test substance on gestational parameters and no dominant lethal effects were indicated for weeks 3, 4 and 5. There were no effects on reproductive parameters. No effects were observed in the gestational parameters for weeks 3, 4 and 5. Weeks 6, 7 and 8 - Spermatocyte Stages: No effects on reproductive parameters for weeks 6 and 7. For week 8, there were no effects on the number of males impregnating females or on the number of pregnancies. While the number of females with 2 or more non-viable implants appears increased in the 50000 ppm group, the percentages are within the ranges observed in the vehicle control group. There were no dominant lethal effects for week 8. Weeks 9 and 10 - Differentiating Spermatogonia Stages: For week 9, there were no effects on reproductive parameters. No statistical effects of treatment were observed for week 9. For week 10, there were no effects on reproductive parameters or on gestational parameters.

Applicant's summary and conclusion