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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines, but was conducted according to GLPs and the report contains sufficient data for interpretation of study results.
Justification for type of information:
Please see read across justification document.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Chernoff test has been developed as a short-term screening technique for developmental toxicity (Chernoff & Kavlock, 1980). In general, a single dose level (1000 mg/kg/day, or lower) producing some measure of maternal toxicity (not to exceed 10% mortality or 30% lower weight gain than controls) is administered to pregnant animals during the period of major organogenesis. The dams are allowed to deliver and the pups are weighed and counted on days 1 and 3 postpartum (day 1 is the day of birth). The underlying hypothesis for this assay is that a wide spectrum of developmentally toxic effects will be expressed as intrauterine death or impaired neonatal growth or survival.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
DOWFAX C-6 Alkylate sodium sulfonate
Reference Number: Lot No. CR-130-1-36 (This same test material was utilized for the probe study.)
Sopurce: Specialty Chemicals Department, The Dow Chemical Company, Midland, MI
Purity: 41.9% active ingredients in aqueous solution (Freshour, 1985) and infrared analysis indicated spectral features supporting the proposed structure of a mixture of C-6 alkylated diphenyloxide sodium sulfonate (Putzig, 1985).
Stability & Homogeneity: Analyses of dosing solutions used for the probe study indicated that DCA was stable for up to 15 days, and the reproducibility of the results from repeated injections indicated the material was homogeneously dispersed (Freshour, 1985).

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
Test Animals
Stock supplies of adult male and female Fischer 344 rats were obtained from Charles River Laboratory, Kingston, NY. This strain was selected for use on the basis of its general acceptance and suitability for toxicity testing and the availability of a reliable commercial supplier. Upon arrival at the laboratory (Fully accrdited by the American Association for Accreditation of Laboratory Animal Care (AAALAC)), animals were examined for health status by the laboratory veterinarian and acclimated to the laboratory environment according to the Standard Operating Procedures of the Reproductive Toxicology Laboratory in rooms designed to control temperature (approx. 22°C), humidity (approx. 50%) and light cycle (12 hours light and dark). Virgin female rats weighing approximately 165-225 grams were bred with stock animals and day 0 of pregnancy was determined by the presence of sperm in vaginal smears. Randomization of test animals into groups was performed using computer generated tables of random numbers according to their day 0 of pregnancy. Rats placed on study were uniquely identified by inserting a numbered metal tag in the ear of each rat. Animals were allowed free access to Purina Certified Rat Chow #5002 (Ralston Purina Company, St. Louis, MO) and tap water. Analysis of the Purina Certified Rat Chow was performed by the Ralston Purina Company to confirm that the diet provide adequate nutrition, and to quantify the levels of selected contaminants associated with the formulation process. Analysis of municipal water is performed according to the Standard Operating Procedures of the Mammalian and Environmental Toxicology Research Laboratory.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Groups of 29 and 37 bred females were given DCA in water by gavage on days 6 through 15 of gestation at dose levels of 300 and 1000 mg/kg/day, respectively. Oral gavage was selected as the most appropriate route to administer an accurate dose over the limited period of dosing. DCA dose solutions were prepared in deionized water such that a dose volume of 4 ml/kg provided the appropriate dose and dose volumes were adjusted daily according to body weights. The top dose level of 1000 mg/kg/day was selected on the basis of the results of the probe study discussed previously, and as the maximum dose level required by the guidelines in the event that maternal toxicity is not apparent. A lower dose level was selected for this study as suggested by guidelines and to insure the presence of a no effect level. A control group of 43 bred females was administered the deionized water vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing solution prepared was analyzed once during the course of this study and the mean concentration for all solutions was calculated to be 100.7 + 1.0% of the targeted concentration (Freshour, 1985).
Details on mating procedure:
Virgin female rats weighing approximately 165-225 grams were bred with stock animals and day 0 of pregnancy was determined by the presence of sperm in vaginal smears.
Duration of treatment / exposure:
Days 6 through 15 of gestation.
Frequency of treatment:
Daily
Duration of test:
Not specified in report but at least up to post -partum day 3 when lactation body weights were taken.
No. of animals per sex per dose:
Groups of 29 and 37 bred females were given DCA in water by gavage on days 6 through 15 of gestation at dose levels of 300 and 1000 mg/kg/day, respectively.
Control animals:
yes, concurrent vehicle
Details on study design:
Test Procedure
All animals were examined daily for signs of toxicity. Body weights were recorded daily during the period of dosing, and on days 16 and 21 of gestation. Statistical analyses of maternal body weight and body weight gain were performed using data recorded on gestation days 6, 9, 12, 16 and 21. Food and water consumption were recorded at 3 day intervals during days 6 through 21 of gestation.

On day 21 of gestation, all pregnant females were transferred to plastic shoe box cages provided with corn cob nesting material and allowed to deliver their litters. All litters were examined as soon as possible after delivery for the presence of external anomalies, with the day of delivery considered day 1 post partum. The following parameters were recorded for each litter: ( 1 ) the date of parturition, (2) litter size on day 1 and day 3 postpartum, (3) the weight of the litter and lactating mother on day 1 and day 3 postpartum, ( 4 ) the sex of each pup; and (5) the number of live pups on day 1 and day 3 postpartum which had milk in their stomachs. Pups which were found dead during the course of the study were examined for anomalies t o the extent allowed by the condition of the pup.

Animals which did not deliver a litter within three days afte the expected date of parturition were sacrificed and the uterus examined for the presence of resorbed implantations. Uteri which did not show visible evidence of implantations were stained with a 10% solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of implantation sites. Adult animals which died spontaneously were submitted for gross pathologic examination and details of the condition of the uterus and contents were recorded. At the termination of the study, all maternal animals and neonates were sacrificed with no further examination.

Examinations

Maternal examinations:
All animals were examined daily for signs of toxicity. Body weights were recorded daily during the period of dosing, and on days 16 and 21 of gestation. Statistical analyses of maternal body weight and body weight gain were performed using data recorded on gestation days 6, 9, 12, 16 and 21. Food and water consumption were recorded at 3 day intervals during days 6 through 21 of gestation.

Adult animals which died spontaneously were submitted for gross pathologic examination and details of the condition of the uterus and contents were recorded. At the termination of the study, all maternal animals and neonates were sacrificed with no further examination.
Ovaries and uterine content:
On day 21 of gestation, all pregnant females were transferred to plastic shoe box cages provided with corn cob nesting material and allowed to deliver their litters. All litters were examined as soon as possible after delivery for the presence of external anomalies, with the day of delivery considered day 1 post partum. The following parameters were recorded for each litter: ( 1 ) the date of parturition, (2) litter size on day 1 and day 3 postpartum, (3) the weight of the litter and lactating mother on day 1 and day 3 postpartum, ( 4 ) the sex of each pup; and (5) the number of live pups on day 1 and day 3 postpartum which had milk in their stomachs.

Animals which did not deliver a litter within three days afte the expected date of parturition were sacrificed and the uterus examined for the presence of resorbed implantations. Uteri which did not show visible evidence of implantations were stained with a 10% solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of implantation sites. Adult animals which died spontaneously were submitted for gross pathologic examination and details of the condition of the uterus and contents were recorded.
Fetal examinations:
Pups which were found dead during the course of the study were examined for anomalies to the extent allowed by the condition of the pup. At the termination of the study, all maternal animals and neonates were sacrificed with no further examination.
Statistics:
Descriptive statistics (means and standard deviations) were reported for food and water consumption. Body weights and body weight gains were evaluated by Bartlett's test (Winer, 1971) for equality of variance. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis was performed by Dunnett's test (Winer, 1971) or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966). Pup body weights were analyzed by Bartlett's test for equality of variances using the number of live pups on days 1 and 3 as a covariate to correct for differences in weights due to litter size. Statistical outliers were identified by a sequential outlier test (Grubbs, 1969) but, only outliers for food and water consumption values were excluded from analysis unless justified by documented, scientifically sound reasons, unrelated to treatment. (Statistics cont'd below)

Indices:
The pregnancy rate was analyzed by the Fisher exact probability test (Siege, 1956). Survival indices and other incidence data among neonates were analyzed by the Wilcoxon test as modified by Haseman and Hoel (1974), using the litter as the experimental unit.
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
During the course of dosing, the majority of animals in the 1000 mg/kg/day dose group exhibited perineal staining and/or loose and watery stools. The majority of these observations were made during the initial 3-4 days of dosing, but occasionally animals would exhibit these signs during the later half of the dosing period. Two deaths occurred during the course of the study, both among animals at 1000 mg/kg/day. One death was considered to be associated with an intubation error; however, the cause of death of the other animal was not determined.

There were no statistically identified differences in the overall body weights in any dose group when compared to controls, though the body weights in the 1000 mg/kg/day dose group were frequently slightly lower than in the controls. Evaluation of the maternal body weight gains during gestation, however, revealed significant treatment-related decreases in weight gain in both treatment groups during the dosing period when compared to controls. Weight gain during the dosing period among pregnant rats given 1000 mg/kg/day was approximately 24% lower than in the controls, while weight gain ,among the 300 mglkglday dose group was approximately 26% lower than the control value. This decrease in weight gain was still evident on day 21 of gestation in the 1000 mg/kg/day dose group, while animals given 300 mg/kg/day gained slightly more weight than the controls after dosing had been completed and overall weight gain (days 6 through 21) was not significantly different from the controls.

Comparison of mean food and water consumption data for these animals revealed no appreciable differences in food consumption among the various groups. However, water consumption among the 1000 mg/kg/day animals was increased during the period of dosing when compared to controls, a finding consistent with data generated in a preliinary study (Hanley, et al., 1985).

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOEL
Effect level:
< 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The pregnancy rate among rats given 1000 mg/kg/day was slightly lower than the control rate; however, there were no totally resorbed litters in any of the animals which failed to deliver a litter detected after staining the uteri with sodium sulfide. Since treatment of animals in this study did not begin until day 6 of gestation, the low pregnancy rates observed in all groups were not considered to be related to treatment. The mean litter size among rats given the high dose level was slightly lower than the control value but this difference was not statistically significant. There were no fetuses born dead among the litters given 1000 mg/kg/day during gestation. Examination of the fetuses on the day of delivery (day 1) revealed significant percentages of fetuses at 1000 and 300 mg/kg/day which did not have milk in their stomachs. However, there were no differences in the incidence of milk in the stomachs of pups examined on day 3 postpartum and neonatal survival was not affected at either dose level when compared to controls. Evaluation of pup body weights on days 1 and 3 postpartum using litter size as a covariate did not reveal any significant differences in either of the treated groups when compared to controls. No malformations were noted in any pups in any of the treatment groups. Examination of the sex ratio revealed a significantly larger proportion of males among litters given 1000 mg/kg/day than would be expected. Maternal body weights during the post-partum period were also unaffected when compared to the controls.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
In conclusion, oral administration of DCA on days 6 through 15 of gestation at dose levels of 300 and 1000 mg/kg/day produced significant depressions in maternal weight gain during the treatment period, with no significant adverse effects on reproductive parameters in either group. Thus, DCA does not appear to be a selective developmental toxicant even at dose levels producing significant maternal toxicity.
Executive summary:

A Chernoff test was conducted using DOWFAX* C-6 A1kylated Sodium Sulfonate (DCA) (an alkylated diphenyl oxide compound) in which pregnant female Fischer 344 rats were administered dose levels of 0, 300 or 1000 mg/kg/day of DCA in deionized water by gavage on days 6 through 15 of gestation. These animals were then allowed to deliver their litters and the litters were evaluated for size, neonatal growth and survival.

Administration of oral doses of 300 or 1000 mg/kg/day of DCA during gestation produced significant depression in maternal weight gain at both dose levels. Evaluation of the litters for mean litter size, neonatal growth and survival through the first 3 days post-partum did not reveal any significant adverse effects at either dose level. Thus, DCA did not appear to have a selective developmental toxicity even at dose 1evels producing significant maternal toxicity.