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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented publication which meets basic scientific principles.

Data source

Reference Type:

Materials and methods

Principles of method if other than guideline:
Rats were bred 1 male to 1 female, and 25 plug-positive females were assigned to each of 4 experimental groups (0, 100, 500 or 1200 ppm). Pregnant rats were exposed for 6 hr/day by inhalation from gestation days (GD) 6-15. Rats were observed daily for clinical signs of toxicity and food consumption daily. Rats were sacrificed on GD 21 and gravid uterus, ovaries (including corpora lutea), cervix, vagina, abdominal and thoracic cavities and upper and lower respiratory tracts examined grossly. All live and dead fetuses and resorption sites (early and late) were recorded. Live fetuses were weighed and examined for gender, external malformations (including cleft palate) and variations.
GLP compliance:
EPA TSCA Good Laboratory Practice Standards (US EPA, 1983)

Test material

Details on test material:
Test material (Isopropyl benzene, CAS No. 98-82-8) was >99.9% pure and remained stable during the study, as determined by gas chromatographic analysis.

Test animals

Crj: CD(SD)
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 10-11 weeks
- Housing: Inidividually housed in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Prolab Certified Rodent Food (Agway, St. Marys, OH, USA) ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
- Exposure apparatus: 4320-L rectangular glass and stainless steel chambers (Wahmann Manufacturing, Timonium, MD, USA).
- Method of holding animals in test chamber: cages
- Source and rate of air: Air was provided by a separate HVAC system.
- Method of conditioning air: Air was filtered for particulates and temperature and humidity controlled.
- System of generating vapor: Liquid cumene/isopropyl benzene was metered from a piston pump into one or two heated glass evaporators similar in design to that described by Carpenter et al. (1975). The temperature in the evaporators was maintained at the lowest level sufficient to vaporize the liquid (35-63 C). The resulting vapor was carried into the chamber by passing conditioned air through the evaporators.
- Temperature, humidity, pressure in air chamber: Air flow rate, temperature and relative humidity were monitored every half-hour during exposure.

- Brief description of analytical method used: Measurements made hourly using Perkin-Elmer Sigma 3902B gas chromatograph equiped with a flame ionization detector.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Concentration of the test material in the test chambers was determined by GC analysis.
Details on mating procedure:
- Proof of pregnancy: vaginal plug
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
gestation day (GD) 6 - 15
Duration of test:
Through GD 21.
No. of animals per sex per dose:
25 pregnant females per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Rationale for animal assignment: random


Maternal examinations:
- Time schedule: Observed daily for clinical signs of toxicity. Body weights and food consumption recorded on GD 0, 6, 9, 12, 15, 18, and 21.

- Time schedule: GD 6-15 and GD18

- Time schedule for examinations: GD 0 and GD 6-18

- Sacrifice on gestation day #: 18
- Organs examined: Lung, liver, uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: uterine content was examined
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of viable and non-viable fetuses
Fetal examinations:
- External examinations: Yes: all per litter were examined for weight, sex, malformations, and variations
- Soft tissue examinations: All rats examined for visceral abnormalities were decapitated and heads fixed in Bouin's solution for examination of craniofacial structures. All live fetuses in each litter were eviscerated, air-dried and then processed for skeletal staining with alizaring red S. Intact fetuses were examined for skeletal malformations and variations.
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: Heads of half the fetuses were used for soft tissue examination.
The unit of comparison was the pregnant animal or the litter (Weil, 1970). Results of the quantitative continuous variables (maternal body weights, liver weights, etc.) were intercom pared for the three exposure groups and the control group by use of Levene's test for equal variances (Levene, 1960), analysis of variance (ANOV A), and t tests with Bonferroni probabilities. The t tests were used when the F value from the ANOV A was significant. When Levene's test indicated homogeneous variances, and the ANOV A was significant, the pooled t test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 197 4) followed, when necessary, by the separate variance t test for pairwise comparisons. Non parametric data obtained following laparohysterectomy were statistically evaluated using the Kruskal-Wallis test (Sakal and Rohlf, 1969), followed by the Mann-Whitney U test (Sakal and Rohlf, 1969) when appropriate. Incidence data were compared using Fisher's exact test (Sakal and Rohlf, 1969). For all statistical tests, the probability limit of 0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
1200 ppm dams showed significant reductions in body weight gain during the exposure period (GD 6-9 and GD 6-15), although there were no significant differences in maternal body weight in any group on the specific days the dams were weighed.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant differences in the incidences of any individual malformation, of malformations by category (external, visceral, or skeletal), or of total malformations. The most commonly observed malformation, dilated cerebral ventricles, were spontaneous in nature and occurred at similar incidences in treated and control groups. There were also no significant increases in the incidences of individual external variations or external variations by category. While several skeletal and visceral variations had a significantly higher incidence, they were not exposure-related.

Effect levels (fetuses)

Dose descriptor:
Effect level:
1 200 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects at any test concentration

Fetal abnormalities

no effects observed

Overall developmental toxicity

Developmental effects observed:

Applicant's summary and conclusion

The maternal NOAEC was 500 ppm. No statistically significant effects were reported for gestational parameters and/or developmental effects. NOAEC for developmental toxicity was >1200 ppm.
Executive summary:

This study was conducted to determine the developmental toxicity of isopropyl benzene, a C9 aromatic substance and a component of complex C9 aromatic hydrocarbon solvents. Groups of 25 pregnant female rats were exposed via inhalation to 100, 500, or 1200 ppm of isopropyl benzene 6 hrs per day during gestation days 6 -15. Rats were sacrificed on gestation day 21 and both dams and fetuses were examined for adverse effects. Aside from a statistically significant reduction in maternal body weight gain (1200 ppm) and food consumption (500 and 1200 ppm), no other adverse treatment-related effects were found. Maternal NOAEC was determined to be 500 ppm on the basis of reduced maternal body weight gain. Fetal NOAEC was determined to be greater than 1200 ppm.