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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
Study period:
30th November 1998 - 23rd December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3600 (Inhalation Developmental Toxicity Screen)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity as measured by GLC is 99.4 %

Test animals

Species:
rat
Strain:
other: CD (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: 205-259 grams
- Fasting period before study: NDA
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4 - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 ºC
- Humidity (%): 30 - 56 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2nd December 1998 To: 23rd December 1998

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: nitrogen.
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 litre stainless steel and glass chamber
- Method of holding animals in test chamber: Rats held within individual wire-mesh cages.
- Source and rate of air: flow rate of 20 L/minute
- Method of conditioning air: NDA
- System of generating particulates/aerosols: Nitrogen pump
- Temperature, humidity, pressure in air chamber: 20 - 25 ºC, 21 - 60 % relative humidity, under slight negative pressure.
- Method of particle size determination: Particle size samples were drawn once during each exposure at a flow rate of 5.00 Lpm using a TSI Aerodynamic Particle Sizer.
- Treatment of exhaust air: NDA

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber air samples were drawn 4 times during each exposure using a Miran air analyser, with a strip chart recorder. The chamber air samples were drawn through a glass fiber filter in a filter holder and then through ¼" tubing to the inlet of the Miran. A vacuum gauge was connected to the outlet of the Miran. The gauge was connected via ¼" tubing to a flowmeter equipped with a metering valve. The pump was turned off and the absorbance was read off the multimeter, which was attached to the Miran.
- Samples taken from breathing zone: NDA
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
daily
Duration of test:
13 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.5, 2.5 and 5.0 mg/L. Equivalent to 0, 100, 498 and 996 ppm in air.
Basis:
nominal conc.
No. of animals per sex per dose:
25 females per dose.
Control animals:
yes
Details on study design:
- Dose selection rationale: Exposure levels selected by sponsor based on available toxicological data, in order to produce a gradation of toxic effects.
- Rationale for animal assignment (if not random): Females were placed into study groups at receipt using a computerised randomisation procedure, which ranked Gestation Day 0 body weights (provided by the supplier) and randomly assigned each animal within the block into groups. Animals were replaced based on physical examinations and/or body weight changes up to Gestation Day 6. Disposition of all animals not utilised in the study is maintained in the study file.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals checked for mortality, morbidity, and signs of severe toxicity. Animals in extremely poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals on study were examined daily from receipt through terminal euthanasia (Gestation Day 20).
- Examinations: General condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration. During the treatment period these evaluations were performed prior to exposure as animals were placed into the chamber.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Days 0 (provided by the supplier), 4 and 6-20 of gestation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal: Feed consumption was recorded on Gestation Days 4 and 6-20 for each female and reported for the following intervals of Gestation: Days 4-6, 6-9, 9-12, 12-15, 15-18, 18-20 and 6-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross lesions or tissues with significant findings were saved (10% neutral buffered formalin) at the discretion of the Supervising Fetal Pathologist or Study Director. Corresponding tissues from control animals were saved for comparative purposes.
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 implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
The following parameters were analysed statistically and means and standard deviations reported:

• Body weights (all recorded intervals)*
• Body weight change (between all recorded intervals and Gestation Days 6-20, 0-20*
• Feed consumption values (all recorded intervals and Gestation Days 4-6, 6-20, 0-20)*
• Mean number of corpora lutea*
• Uterine implantation data (fetuses*, resorptions**, implantation*)
• Mean Pre-implantation loss**
• Mean Post-implantation loss**
• Number of male and female fetuses*
• Fetal weight distinguished by sex and as a composite for both sexes (litter as experimental unit)*

Continuous Data - Multiple Group Analysis:

Employed when data for more than one test group was compared to control. All statistical tests were conducted at the 5% and 1%, two-sided risk levels.

* Statistical evaluation of equality of means was made by the appropriate one way analysis of variance (ANOVA) technique, followed by a multiple comparison procedure, if needed. If ANOVA showed no difference, no additional comparisons were made. If ANOVA was significant, Dunnett's test was used to determine which data, if any, differed from the control.

** The Kruskal-Wallis test (2 X n) was used to test equality of means. If no differences were seen, no additional analyses were performed. If differences were indicated, a 2 X 2 Kruskal-Wallis test was used to determine which treatments differed from control.
Indices:
Incidence data:

• Mortality rate
• Pregnancy rates
• Incidence of females with only resorptions (no live fetuses)
• Incidence of litters containing fetuses with malformations (external, soft tissue and skeletal - total affected and by finding)

Incidence Data Analysis:

A Fisher Exact Test with Bonferonni correction was performed to identify differences between the groups.
Historical control data:
Visceral malformations have been observed previously in this strain of rat in historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All female rats in the control, 100, 496 and 996 ppm test groups survived to study termination, with the exception of one female sacrificed on Gestation Day 13 in the 996 ppm group.

The sacrificed female (no. 992) in the 996 ppm treated group was euthanised for humane reasons due to pale eyes, porphyrin staining and swollen paws that hindered her hindlimb posture and mobility. This female rat also had red staining on the snout immediately post-dose and/or during clinical observation assessment from Gestation Days 9 through 12.

Postmortem examination revealed discolored liver foci and an internal firm abdominal mass attached to the left peritoneal wall (2.2 cm x 1.1 cm.). The liver and abdominal mass was examined microscopically. Examination of the liver revealed foci of hepatocellular necrosis, acute/subacute inflammation, scattered microgranulomas, scattered foci of extramedulary hematopoiesis and periportal cell infiltrates. Microscopic finding of the "mass" was determined to be the spleen, which had extensive lymphoid cell hyperplasia (moderate) and necrosis, chronic purulent inflammation/abscesses (severe). Granulation tissue and fibrosis (both severe) on the surface of the spleen resulted in adhesions between the visceral and parietal peritoneum.

The findings in the liver and spleen were not considered to be related to 1-Bromopropane exposure as similar findings were not observed in otherexposure levels.

Pregnancy rates were unaffected by I-Bromopropane exposure since both control and test groups were comparable. These rates were 92% for the control and 100 ppm exposure groups and 100% for the 498 and 996 ppm exposure groups.

No effect of exposure to 1-Bromopropane at 100 ppm was evident from body weight or body weight gain data during gestation.
The body weights for the 498 ppm 1-Bromopropane exposure group were slightly but not statistically reduced when compared to the control group until Gestation Day 17. Thereafter until sacrifice the body weights for this group remained significantly decreased. However, body weight gains for this 498 ppm exposure group were significantly decreased, when examined by periods, for Gestation Days 6 through 9, 15 through 18, and overall for Gestation Days 6 through 20 and 0 through 20. Body weights in the 996 ppm exposure group were statistically decreased beginning on Gestation Day 8 and remained suppressed throughout the gestation period. Body weight gains for this exposure group were also significantly decreased on Gestation Days 6 through 7, 7 through 8, 16 through 17, 17 through 18, 6 through 9, 15 through 18, and overall for Gestation Days 6 through 20 and 0 through 20.

There were no 1-Bromopropane exposure-related effects noted for feed consumption (grams/animal/day and grams/kg/day) at the 100 ppm dose level when compared to the control group. A single statistically significant increase and decrease in feed consumption was observed at 100 ppm during gestation. Feed consumption for the 498 ppm exposure group expressed as grams/animal/day and grams/kg/day was initially decreased statistically during Gestation Days 6 through 7 (g/kg/day), 7 through 8 and 6 through 9. Thereafter, feed consumption was only slightly decreased but not statistically throughout the remainder of gestation. The decrease in feed consumption in the 498 ppm exposure group corresponded with the slight decreases in body weight and body weight gains.

Feed consumption (grams/animal/day) in the 996 ppm exposure group was significantly decreased during Gestation Days 6 through 12 and overall for Gestation Days 6 through 20. Feed consumption (grams/kg/day) in this group was also significantly decreased on Gestation Days 6 through 9 (daily and for the entire interval), 9 through 10 and overall for Gestation Days 6 through 20. Decreased feed consumption paralleled the decreases in body weight for this group.

No effect of exposure to 1-Bromopropane was indicated from the maternal macroscopic postmortem examinations. Sporadically occurring postmortem findings were observed in all treatment groups including the control group.

Gravid uterine weights for the 498 ppm and the 996 ppm group were slightly lower than the control, but the decrease was not statistically significant. The net body weight change and net body weight minus the gravid uterine weight were both statistically significantly decreased in the 498 and 996 ppm exposure groups.

No effect of treatment with 1-Bromopropane on uterine implantation data was observed. The mean number of corpora lutea, pre- and post¬implantation loss, mean number of live fetuses per female rat, and the mean number of early and late resorptions for the 1-Bromopropane exposure groups were comparable to the control group.

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were statistically significant reductions in fetal weights for all three 1-Bromopropane exposed groups when compared to the control group. The difference between the mean fetal weights of 100 ppm exposure group and the control group was 0.2 g for the combined sexes and 0.1 g and 0.2 g for the individual male and female fetuses, respectively. The difference between the mean fetal weights of 498 ppm exposure group and the control group was 0.2 g for the combined sexes and for the individual male and female fetuses. The reduction in fetal body weight for the combined and individual male and female fetuses in the 996 ppm exposure group was 0.3 g compared to the control group.

A newly implemented laboratory procedure conducted during this study created a control group of fetuses with statistically heavier body weights than is usually seen in Gestation Day 20 rat fetuses. This procedure involved saving 1 or 2 control rats until the end the daily cesarean section period to allow for comparable tissues to be save from the control group in the event of observed findings in the treated groups. This produced many control group fetuses weighing approximately 0.2 grams heavier in body weight verses the laboratories usual 3.9 gram fetuses for combined sexes.

Taking this non-random order in the time of cesarean sections into account, the fetuses in the 498 ppm group may have had marginally reduced fetal body weights. The reduced fetal body weights in the 996 ppm group are considered a result of maternal toxicity from the exposure to 1-Bromopropane.

There were no 1-Bromopropane exposure-related external, visceral, or skeletal fetal malformations. One litter in the control group (dam #607) contained a visceral malformation in fetus #11, kidney(s) agenesis. No fetal malformations occurred in either the 100 or 498 ppm treated groups. Three litters exposed to 996 ppm of 1-Bromopropane had a total of 4 fetuses each with a different visceral malformations: hydrocephaly, misshapen hemisphere(s), heart and/or great vessel anomaly, and testicular agenesis. These visceral malformations have been observed previously in this strain of rat, and are considered to be of spontaneous origin, and are not attributed to exposure to 1-Bromopropane.

There were no external fetal variations noted in any exposure groups, including the control group. There were four commonly observed visceral variations, including dilation of ventricle(s), dextrocardia, great vessel alteration, and renal papilla not developed (Grade 0). These visceral variations appeared at a low incidence rate, or the incidence of occurrence was comparable to that of the control group, and thus are considered to be of spontaneous origin, and are not attributed to exposure to 1-Bromopropane.

There were no 1-Bromopropane-related skeletal variations observed in fetuses exposed to 100 ppm, when compared to the control group. Common skeletal variations were: thoracic centrum(s) bipartite, lumbar arch(es) reduced ossification, sternebra(e) 5 and/or 6 unossified, and rib(s) 14th rudimentary. These skeletal variations appeared at a low incidence rate, or the incidence of occurrence was comparable to that of the control group, and thus are considered to be of spontaneous origin, and are not attributed to exposure to 1 -Bromopropane.

There was a significant increase in the litter incidence of reduced ossification of the skull in 498 ppm and 996 ppm exposure groups. There was a statistically significant increase in the 996 ppm exposure group in the litter incidence of bent rib(s) and a slight, but not statistically significant, increase in the litter incidence of hyoid body/arch(es) unossified, sternebra(e) 5 and/or 6 unossified and reduced ossification of ribs. The bent ribs are considered 1-Bromopropane exposure-related; however, the literature supports that this condition is reversible and frequently observed in untreated control rats (Nishimura et al., 1982; Kast, 1994). The slight but statistical significant reductions in ossification in the 498 and 996 ppm exposure groups are probably a result maternal toxicity and associated with the reduction in fetal weight.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOEL
Effect level:
996 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No adverse effects of 1-Bromopropane exposure on mortality, or macroscopic postmortem evaluations on day 20 of gestation were observed. The 996 ppm 1-Bromopropane exposure group exhibited a higher incidence of red stains on the snout or head, bilateral or unilateral lacrimation, and excessive salivation immediately post-exposure. Exposure-related decreases in maternal body weight, body weight gain and feed consumption were observed in rats exposed to 498 and 996 ppm of 1-Bromopropane. No effect of 1-Bromopropane exposure at 100, 498 or 996 ppm on pregnancy rates, implantation data, sex distribution or fetal external, skeletal or visceral malformations were observed. There was a significant and dose related increase in the litter incidence of reduced ossification of the fetus in the 498 ppm and 996 ppm exposure groups. This reduction in fetal ossification is probably associated with maternal toxicity and reduced fetal body weights in these two groups. There was a significant increase in the 996 ppm exposure group in the litter incidence of bent rib(s) and a slight, but not statistically significant, increase in the litter incidence of hyoid body/arch(es) unossified, sternebra(e) 5 and/or 6 unossified and reduced ossification of ribs. The bent ribs are considered 1-Bromopropane exposure-related; however, the literature supports that this condition is reversible and not uncommonly observed in untreated control rats (Nishimura et al., 1982; Kast, 1994).

The results of this developmental toxicity study revealed no maternal or fetal toxicity at 100 ppm exposure level. Slight maternal toxicity and slight fetal toxicity were observed in the 498 ppm group. Moderate to severe maternal toxicity and slight to moderate fetal toxicity was observed in the 996-ppm exposure group. Therefore, the No-Observable-Effect-Level (NOEL) for maternal toxicity and fetal toxicity is 100 ppm and for teratogenicity is 996 ppm.
Executive summary:

This developmental toxicity study, conducted for the Brominated Solvents Consortium (BSOC), was designed to assess the potential maternal and/or embryo-fetal toxicity of inhaled 1-Bromopropane in the pregnant rat. Seventy-five pregnant rats (25 per group) were exposed for six hours daily from Gestation Day (GD) 6 through the period of organogenesis (GD 19) at levels of 0.5, 2.5, and 5.0 mg/L (100, 498, and 996 ppm, respectively). The control group was comprised of twenty-five pregnant rats that received the filtered air, using the same exposure regimen as the treated animals. A cesarean section was performed on all rats on GD 20. The experiment was performed according to OECD 414 and EPA OPPTS 870.3600 and to GLP standard.

No adverse effects of 1-Bromopropane exposure on mortality, or macroscopic postmortem evaluations on day 20 of gestation were observed. The 996 ppm 1-Bromopropane exposure group exhibited a higher incidence of red stains on the snout or head, bilateral or unilateral lacrimation, and excessive salivation immediately post-exposure. Exposure-related decreases in maternal body weight, body weight gain and feed consumption were observed in rats exposed to 498 and 996 ppm of 1-Bromopropane. No effect of 1-Bromopropane exposure at 100, 498 or 996 ppm on pregnancy rates, implantation data, sex distribution or fetal external, skeletal or visceral malformations were observed. There was a significant and dose related increase in the litter incidence of reduced ossification of the fetus in the 498 ppm and 996 ppm exposure groups. This reduction in fetal ossification is probably associated with maternal toxicity and reduced fetal body weights in these two groups. There was a significant increase in the 996 ppm exposure group in the litter incidence of bent rib(s) and a slight, but not statistically significant, increase in the litter incidence of hyoid body/arch(es) unossified, sternebra(e) 5 and/or 6 unossified and reduced ossification of ribs. The bent ribs are considered 1-Bromopropane exposure-related; however, the literature supports that this condition is reversible and not uncommonly observed in untreated control rats (Nishimura et al., 1982; Kast, 1994).

The results of this developmental toxicity study revealed no maternal or fetal toxicity at 100 ppm exposure level. Slight maternal toxicity and slight fetal toxicity were observed in the 498 ppm group. Moderate to severe maternal toxicity and slight to moderate fetal toxicity was observed in the 996-ppm exposure group. Therefore, the No-Observable-Effect-Level (NOEL) for maternal toxicity and fetal toxicity is 100 ppm and for teratogenicity is 996 ppm.