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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Lot No: 56100 (supplied by Mitsui, Japan)
Purity: 99.7%

Test animals

Species:
rat
Strain:
other: CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable animal supplier
- Age at study initiation: not reported
- Weight at study initiation: mean weight: 227.4g control; 228.0g 10 mg/kg/day; 227.0g 100 mg/kg/day; 226.4 400 mg/kg/day

- Fasting period before study: none
- Housing: wire bottom cages
- Diet: certified laboratory rodent feed
- Water: municipal tap water (ad libitum)
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 22°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light:12 hours dark

IN-LIFE DATES: From: To: 29 January to 18 February 1996

Administration / exposure

Route of administration:
dermal
Vehicle:
water
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle: deionised water was selected as the vehicle because DMI is soluble in water, water should not produce any additional skin irritation, and water absorption would not confound the results of the study.
- Concentration in vehicle: such that a dose volume of 1ml/kg will yield the appropriate dose

SUBSTANCE ADMINISTRATION
- An area of approximately 5 cm2 (>10% body surface area) on the back of each rat was clipped free of hair prior to •initiation of dosing. The test material was allowed to be in contact with the skin for approximately 6 hrs/ day. The exposure site was covered with an absorbent gauze pad and non-absorbent cotton, and the animal was wrapped in an elastic bandage to hold the test material, gauze pad and cotton in place. The control rats were dosed with DI water. After completion of each daily 6 hr eposure period, the bandage, gauze and cotton were removed and the exposure site wiped with a water-dampened towel to remove residual test material. The application site was re-clipped as needed during the study.


Dose solutions were mixed once prior to the start of the dosing period. Dosing solutions were analyzed to determine concentrations of DMI. The low- and high-dose solutions were analyzed for homogeneity. Stability of DMI in the dosing solutions was established prior to the start of the study.
Reference samples of all dosing solutions including the control were retained
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details are reported separately in:

Flint G.T. and Putzig, (1995). Analytical Characterization of l,3-Dimethyl-2-Imidazolidinone, Lot 56100, in Support of Toxicology Testing. Analytical Report of the Dow Chemical Company, Midland, Mitchegan. ML-AL 95-004020.

Details on mating procedure:
- Impregnation procedure: sexually mature adult virgin females, weighing approximately 200 grams were naturally mated with one male of the same species
- Verification of same strain and source of both sexes: mating undertaken by the animal supplier
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
days 5-15 of gestation
Duration of test:
21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose level selection was based on the results of preliminary study. The 400 mg/kg/day exposure level was expected to result in decreased body weights and body weight gains compared to the controls. The lower dose levels of 10 and 100 mg/kg/day were selected to establish dose-response relationships for any treatment-related effects which may be observed at the high dose.

- Rationale for animal assignment: Randomization of the time-mated rats into various dose groups was performed using a computerized procedure designed to increase the probability of uniform group mean body weights and standard deviations at the start of the study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (for alterations in behaviour or demeanour)
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 (at the supplier), daily during days 6 through 16, 19 and 21 of gestation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE: not applicable - not a drinking water study


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver, kidneys and gravid uteri

OTHER: site of application of test substance was assessed for irritation immediately prior to dosing on days 6-15. In addition, the site of application was also assessed for exfoliation and fissuring.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Means and standard deviations) were calculated for feed consumption. Maternal body weights, weight gains and mean fetal body weights were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the parametric or nonparametric ANOVA was significant, analysis by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed, respectively. Statistical evaluation of the frequency of preimplantation loss, resorptions and fetal alterations among litters and the fetal population was performed using a censored Wilcoxon test with Bonferroni's correction. The number of corpora lutea, implants, and litter size were evaluated using a nonparametric ANOVA followed by the Wilcoxon RankSum test with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test. Nonpregnant females were excluded from the appropriate analyses. Fetal sex ratios were evaluated using a binomial distribution test. Statistical outliers were identified using a sequential method, but only values for feed consumption were routinely excluded unless justified by sound scientific reasons unrelated to treatment.

Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) is expected to
be much greater than the cited alpha levels suggested. Therefore, the final interpretation of the numerical data took into consideration the statistical
analyses along with other factors such as dose-response relationships and whether the results were significant in light of other biologic and pathologic
findings.
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related changes in behaviour or demeanour were observed at any dose level.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
All rats survived the test period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Exposure to 400mg substance/kg/day resulted in statistically significant decreases in body weights on days 16, 19 and 21 of gestation and body weight gain during gestation days 6-9, 9-12,0-21 and 6-16. Statistically significant decreases in body weight gain were also noted in dams exposed to 100 mg substance/kg/day during days 19-21 and 6-16. The change in body weight during the post-dosing period (days 19-21), in dams given 100 mg substance/kg/day, was not attributed to treatment due to the lack of an effect during this test interval in dams given 400 mg substance/kg/day. The body weight and/or weight gain changes noted during the exposure period at 100 and 400 mg/kg/day were consistent with the decreased feed consumption noted at these exposure levels. No effects on body weights or body weight gains that were considered to be of toxicological significance were observed at 10 mg substance/kg/day.
Terminal body weights were statistically decreased in the high-dose dams. This was consistent with the body weight decreases noted during the in-life phase of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related decrease in feed consumption during days 6 through 21 of gestation in the dams exposed to 100or 400 mg/kg/day substance which was interpreted to be treatment related. Marginal decreases in feed consumpton in the 10 mg/kg/day dose group during the treatment period were not considered to be treatment related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed on absolute or relative liver or kidney weights at any dose levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathologic alterations noted during necropsy that were attributable to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal body weights were significantly decreased at the high dose. The decreases in fetal body weights were concluded to be secondary to the decreases in maternal body weight and were not considered a direct effect of the test substance.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Statistical analysis of the reproductive parameters indicated that fetal body weights and gravid uterine weights were significantly decreased at the high dose. The decreases in fetal body weight and gravid uterine weight were concluded to be secondary to the decreases in maternal body weight and were not considered a direct effect of the test substance. There were no treatment-related effects on the pregnancy rate, number of corpora lutea, number of implantations, preimplantation loss, resorptions, litter size or fetal sex ratio.
The incidence of delayed ossification of cervical centra was increased at the high dose. Although not statistically identified as being significant, the incidence of delayed ossification of the skull was higher in the high-dose rats compared to the controls. Given the significant decreases in maternal weights, uterine weights and fetal body weights at the high dose, a delay in the ossification of cervical centra and skull bones would not be unexpected in these fetuses. The number of malformed fetuses observed within treatment groups was low and fell within the historical control values for our laboratory. The control group had two fetuses in two litters that were malformed. Both of these fetuses exhibited Class II wavy ribs. Class II wavy ribs were also found in one malformed fetus in the 10 mg/kg/day dose group. The 100 mg/kg/day dose group had one malformed pup which had hydronephrosis. The high-dose group had two pups that were malformed, one with agnathia, cleft lip and palate, and the other with a filamentous tail.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Since the maternal NOEL was lower than the developmental NOEL, the substance was not interpreted to be a selective developmental toxicant. The substance is not teratogenic at any dose level tested.
Executive summary:

The developmental toxicity of the substance was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 414.

During the study, groups of 25 time-mated female rats were exposed dermally to test material at levels of 0, 10, 100 or 400 mg/kg bw/day for 6 hours per day on days 6-15 of gestation. rats were evaluated for clinical signs, dermal irritation, feed consumption and body weight gains. All rats were sacrificed on gestation day 21. The ovaries were examined for number of corpora lutea and the uteri were weighed and examined for number and type of implantations and resorptions. Fetuses were weighed and examined for external, visceral and skeletal alterations.

Under the conditions of the study, exposure to 100 ot 400 mg/kg bw/day resulted in dose-related decreases in feed consumption (days 6 through 21 of gestation) and body weight gains. Significant decreases in body weights were also noted in dams exposed to 400 mg/kg bw/day.

A significant decrease in gravid uterine weight and fetal body weight, and increase in the incidence of delayed ossification of skull bones and cervical centra were noted in high dose fetuses. The uterine and fetal effects observed at 400 mg/kg bw/day were interpreted to be secondary to decreased maternal body weight and not a direct effect of the test material.

The NOEL were therefore determined to be 10 mg/kg bw/day for maternal toxicity and 100 mg/kg bw/day for developmental toxicity. Since the maternal NOEL was lower than the developmental NOEL, the substance was not interpreted to be a selective developmental toxicant. The substance is not teratogenic at any dose level tested.