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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP using methodologies consistent with OECD Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-Bromopropane
- Physical state: Colorless liquid
- Analytical purity: 99.96%
- Lot/batch No.: 60323
- Storage condition of test material: Packed in chemidrum (steel with polyethylene lining) under notrogen. Chemidrum stored at temperature below 20°C.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 8 weeks for males and non-pregnant females, 11 weeks for pregnant females
- Weight at study initiation: 246 - 248 g
- Fasting period before study: Not fasted
- Housing: Stainless, wire-mesh cages
- Diet (e.g. ad libitum): CFR-1 pellet diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 60 +/- 10
- Air changes (per hr): 15-17
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Route and Rationale of Test Substance Administration

Inhalation by whole body exposure was the route of administration, since this is an anticipated route of exposure to humans.

Inhalation Methods

The fresh HEPA filtered dry air was used for bubbling and dilution of test substance at a constant temperature. Test substance vapor was generated from reservoir by bubbling. The vapor was diluted to the target concentrations of each exposure group and supplied into the inhalation chambers. Rats were then exposed to the test substance in the exposure chambers. Chambers were constructed of stainless-steel and glass and were operated under dynamic conditions. Each chamber was dedicated for one exposure level. The control group was exposed to clean filtered air under conditions identical to those for the treated groups exposed to the test substance.

Exposure Period

Male and female rats were exposed to the test substance for six hours per day, seven days per week for 14 days. Pregnant females were exposed from day 6 up to day 19 of gestation for six hours per day.

Target Exposure Concentration Levels

Rats were exposed to the test substance at target concentrations of 0, 125, 250, 500, 1,000 and 2,000 ppm.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual exposure concentrations of the test substance in each chamber were measured every 15 minutes by gas chromatography (GC). The GC samples were constantly drawn by vacuum pump (7000mL/min) through stainless steel lines (15-20m) from each chamber to GC. This constant flow assured the delivery of fresh sample to GC without condensation. The GC was equipped with an automatic stream selector valve, a 0.2mL sample loop, flame ionization detector, and a 3mmx1.5m column packed with 15% chromosorb W on silicon D.C.200 80/100, operated at 80°C. The carrier gas was nitrogen.
Duration of treatment / exposure:
14 days
Frequency of treatment:
6 hr/day for 14 consecutive days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 125, 250, 500, 1000 and 2000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 125, 252, 502, 1003 and 2005 ppm mean
Basis:
analytical conc.
No. of animals per sex per dose:
Control -- 5 male/5 female/3 pregnant female
125 ppm nominal -- 5 male/5 female/3 pregnant female
250 ppm nominal -- 5 male/5 female/3 pregnant female
500 ppm nominal -- 5 male/5 female/3 pregnant female
1000 ppm nominal -- 5 male/5 female/3 pregnant female
2000 ppm nominal -- 5 male/5 female/3 pregnant female
Control animals:
yes, sham-exposed
Details on study design:
OBSERVATIONS AND EXAMINATIONS

Survival and Clinical Observations

Dosing period-- All animals (male/female/pregnant femals) were observed for mortality twice daily, once in the morning and once in the afternoon. Detailed clinical observations were performed twice daily, once before exposure and approximately one hour after completion of exposure.

Body Weight Measurements

Males and females were weighed on 0-0 (administration week-day; first exposed day), 1-1, 1-2, 1-4, 1-7, 2-3 and 2-7. Pregnant females were weighed on days 0, 6, 7, 8, 10, 13, 16 and 20 of gestation.

Food Consumption Measurements

Food consumption for males and females was recorded once a week. Food consumption for pregnant females was recorded on days 6-13 and 13-20 of gestation.

Pathological Examinations

Method of sacrifice-- All animals were sacrificed by exsanguination under ether anesthesia.

Necropsy

All males and females were sacrificed and examined grossly on day 14 of the study. All pregnant females were sacrificed for gross examination and cesarean sectioning on day 20 of gestation. In addition, the number of corpora lutea of all pregnant females were recorded. The uterus of each female within the pregnant group was stained with 10% ammonium sulfide solution and the number of implantation sites was determined.

Observations of Fetuses

Fetuses were removed from the uterus after gross observation and were observed for numbers of dead or live fetuses, body weight, sex, placental weight and external examination. All fetuses were fixed per litter in 10% neutral buffered formalin for possible future examination.

Organ Weight

The testes, epididymis, seminal vesicle (including the coagulating gland), prostate, ovaries, uterus, lungs, kidneys and liver of all male and female rats were weighed. The ovaries, uterus (after removing the fetuses), lungs, kidneys and liver of all pregnant females were weighed.

Collection of Organ/Tissue

The following tissues were collected from all rats. All tissues except left epididymis and left testis were fixed in 10% neutral buffered formalin. Left epididymis and left testis were fixed in Bouin's fixing fluid.

Integumentary system-- Skin
Respiratory system--Nasal cavity, Nasopharynx, Larynx, Trachea, Lungs
Hematopoietic system -- Bone marrow (with femur bone), Lymph node (inguinal and axillary), Thymus, Spleen
Circulatory system -- Heart
Digestive system-- Tongue, Salivary glands, Esophagus, Stomach, Small intestine (includes duodenum), Large intestine, Liver, Pancreas
Urinary system-- Kidneys, Urinary bladder
Endocrine system-- Pituitary, Thyroid (with parathyroid), Adrenals
Reproductive system-- Males - Testes, Epididymides, Seminal vesicle, Prostate; Females - Ovaries, Uterus (except pregnant females), Vagina (except pregnant females), Mammary glands
Nervous system-- Brain, Spinal cord, Peripheral nerve (sciatic)
Special sense organs/appendage-- Eyes, Harderian glands
Musculoskeletal system-- Muscle (thigh), Bone (femur)
Other-- All gross lesions

Histopathology

The testes, epididymis, seminal vesicle (including the coagulating gland), prostate, ovaries, uterus, lungs, kidneys and liver collected from male and female rats were examined microscopically. The ovaries, lungs, kidneys and liver of preganant females were examined microscopically.

Examinations

Statistics:
All data without the lesion at hisopathology was preliminarily tested by the Bartlett method whether the variance of obtained data between control and treated groups were different or not. When the variance was not different, the one way-layout analysis of variance was made. When difference was significant between the groups, the significance of difference in the group mean was analyzed by the Dunnett's multiple comparison test.

When the variance was different, the Kruskal-Wallis rank sum test was made by arranging all data of control and treated groups in a descending order. When difference was significant between the groups, the non-parametric Dunnett's multiple comparison test by rank was made. Two sided analysis of P-values of 0/05 and 0.01 was made for final ones.

Non-neoplastic lesions at histopathology were analyzed by the Chi-square test by classifying animals without lesions into grade 0. The Chi-square test was made between control and each treated group.

The data of pregnant females and fetuses were not analyzed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Decreased testis weights in males
Gross pathological findings:
no effects observed
Description (incidence and severity):
Slight debris of spermatic elements were noted in four male rats at the 2,000 ppm exposure level
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the kidney, slight hyaline droplet in glomerulus, slight and moderate hyaline cast in Bowman's capsule and urinary tubule were observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
ENVIRONMENTAL CONDITIONS IN EXPOSURE CHAMBERS

In each exposure chamber, mean temperatures ranged between 22.2 to 22.7°C, mean humidity ranged between 53.2 to 59.8%, mean air flow ranged between 211.6 to 213.0 L/min, mean air changes ranged between 12.0 to 12.1 time/h. were similar to the target environmental condition.

CONCENTRATIONS OF TEST SUBSTANCE IN CHAMBERS

Concentrations of the test substance in each chamber were measured at 15-minute intervals by gas chromatography throughout the six hour
exposure period. The overall mean and standard deviation for actual concentrations of the 2-bromopropane in each exposure chamber were 0 (control), 125.2±0.7 (target concentration: 125), 251.6±2.8 (target concentration: 250), 501.8±3.0 (target concentration: 500), 1,002.8±7.1 (target concentration: 1,000) and 2,005.4±9.1 (target concentration: 2,000) ppm.

OBSERVATIONS AND EXAMINATIONS OF MALES AND FEMALES

Survival -- All male and female rats survived to the end of study.

Clinical Observations -- No abnormal clinical sign was noted in any male or female rat during the exposure periods.

Body Weight Measurements -- Mean body weight changes of male and female rats in the exposure groups were similar to those of the control group. However, body weight gain in females at 2,000 ppm was slightly decreased throughout the study period, but not statistically significant.

Food Consumption Measurements -- Mean food consumption of male and female rats in the exposure groups were similar to those of the control group.

Pathological Examinations

Gross examinations --No gross lesion was observed in male or female rats in any of the exposed groups.

Organ weights-- Absolute testis weights of males in the 1,000 and 2,000 ppm group were significantly lower than that of the control group. Relative
testis weights were slightly decreased in the 1,000 and 2,000 ppm groups, but the differences were not statistically significant. No significant difference of organ weight was found in female rats at any exposure level.

Histopathology -- In the epididymis, slight debris of spermatic elements were noted in four male rats at the 2,000 ppm exposure level. There was no histopathological change in female rats in any of the exposed groups.

OBSERVATIONS AND EXAMINATIONS OF PREGNANT FEMALES

Survival -- All pregnant rats survived to the end of the study.

Clinical Observations -- Clinical signs in pregnant females were restricted to the 2,000 ppm group; 2 of 3 pregnant females showed vaginal hemorrhaging after exposure on day 19 of gestation.

Body Weight Measurements -- The mean body weight on day 20 of gestation and body weight gain from day 13 to 20 of gestation of pregnant female rats in the 2,000ppm group were lower than those of control group.

Food Consumption Measurements -- The mean food consumption from day 13 to 20 of gestation of pregnant female rats in the 2,000ppm group was lower than that of control group.

Pathological Examinations

Gross examinations -- No gross lesion was observed in pregnant females at any exposure.

Organ weights -- Absolute ovary weights and absolute and relative uterus weights in pregnant females at the 2,000 ppm group were less than those of the control group.

Histopathology -- In the kidney, slight hyaline droplet in glomerulus, slight and moderate hyaline cast in Bowman's capsule and urinary tubule were
observed in 2 of 3, 1 of 3 and 1 of 3 pregnant females, respectively, at the 2,000 ppm group.

Observations and Examinations of Fetuses at Cesarean Section

Reproductive examinations -- The numbers of fetal deaths and implantation losses were increased in the 1,000 ppm and 2,000 ppm groups. There was no live fetuse at 2,000 ppm. Body weights of male and female fetuses in the 1,000 ppm group were slightly lower than those of the control fetuses. At this exposure level, weights of the placenta were also slightly lower than those of the control group.

External observations of fetuses -- Omphalocele was observed in 1 of 41 fetuses in the 250 ppm group. Ectopic pinna, microphthalmia, agnathia and dwarf was observed in 1 of 48 fetuses, microphthalmia was observed in 1 of 48 fetuses, respectively, in the 500 ppm group. Menigocele was observed in 1 of 40 fetuses in the 1,000 ppm group.

CONCLUSIONS

1. The mean actual concentration of 2-bromopropane in each chamber were
0, 125, 252, 502, 1,003 or 2,005 ppm (The target concentrations were 0, 125,
250, 500, 1,000 and 2,000 ppm, respectively).

2. Effects on male and female
a) No death occurred.
b) No abnormal clinical sign was noted in either male or female rats.
c) No significant difference in mean body weight was indicated between the exposed groups. However, body weight gain in females at
2,000 ppm was slightly decreased throughout the study period, but not statistically significant.
d) No significant difference in food consumptions was indicated between the exposed groups.
e) No gross lesion associated with test substance exposure was observed.
f) Absolute testis weights in the 1,000 and 2,000 ppm groups were significantly decreased. Relative testis weights were slightly
decreased in the 1,000 and 2,000 ppm groups, but the differences were not statistically significant. No histopathological change was
found. In addition, no significant difference on epididymis weight was noted.
g) The slight debris of spermatic elements in the epididymis were found in 2,000 ppm male rats.

3.Effects on pregnant female
a) No death occurred.
b) Vaginal hemorrhage was observed at 2,000 ppm in 2 of 3 pregnant females on day 19 of gestation.
c) Body weight gain was affected at the 2,000 ppm exposure concentration.
d) Food consumption in the 2,000 ppm exposed group was lower than that of control.
e) Absolute ovary weight, absolute and relative uterus weights were decreased in 2,000 ppm. However, no gross lesion or histopathological
change associated with exposure to the test substance on these organs were observed.
f) The slight hyaline droplet in glomerulus and slight to moderate hyaline cast in Bowman's capsule and urinary tubule of the kidney
were observed in the 2,000 ppm exposed group.
g) The numbers of fetal death and implantation loss were increased in the 1,000 ppm and 2,000 ppm groups. Additionally, there were no live
fetuse at 2,000 ppm exposure level.
h) Body weights of male and female fetuses in the 1,000 ppm group were lower than those of the controls. In addition, at this exposure
level, weights of the placenta were also lower than that of control group.
i) External malformations of fetuses were observed sporadically at 250, 500 and 1,000 ppm. However, there were no clear concentration-related
findings in the type and incidence external malformations.
It was concluded that the vaginal hemorrhaging, decreases in body weight and food consumption, and decreased ovary and uterus weights
were related to the increases in the numbers of fetal deaths and implantation losses.

OVERALL CONCLUSIONS

The results of the repeated inhalation toxicity of 2-bromopropane were decreases in testis weight in 1,000 and 2,000 ppm male rats and histopathological changes of epididymis in 2,000 ppm male rats. No toxic effect in females was found at 2,000 ppm.

In terms of developmental toxicity, increases in the numbers of fetal deaths and implantation losses, i.e. embryotoxicity at 1,000 and 2,000 ppm, and
decreases in fetal body weight and weight of placenta at 1,000 ppm were found. In addition, histopathological changes in the kidney were observed in the 2,000 ppm pregnant rats.

Based on repeated inhalation toxicity in male rats at concentrations of 1,000 ppm and 2,000 ppm, no toxic effect in females at 2,000 ppm, effects on pregnant female rats at 2,000 ppm, embryotoxicity noted in pregnant rats at 1,000 and 2,000 ppm, the test concentrations selected for the subsequent combined repeated dose toxicity study with a reproduction / developmental toxicity screening test (OECD 422) in rats were 0, 125, 250, 500 and 1,000 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The results of the repeated inhalation toxicity of 2-bromopropane were decreases in testis weight in 1,000 and 2,000 ppm male rats and histopathological changes of epididymis in 2,000 ppm male rats. No toxic effect in females was found at 2,000 ppm.

In terms of developmental toxicity, increases in the numbers of fetal deaths and implantation losses, i.e. embryotoxicity at 1,000 and 2,000 ppm, and decreases in fetal body weight and weight of placenta at 1,000 ppm were found. In addition, histopathological changes in the kidney were observed in the 2,000 ppm pregnant rats.

Applicant's summary and conclusion

Conclusions:
The results of the repeated inhalation toxicity of 2-bromopropane were decreases in testis weight in 1,000 and 2,000 ppm male rats and histopathological changes of epididymis in 2,000 ppm male rats. No toxic effect in females was found at 2,000 ppm.

In terms of developmental toxicity, increases in the numbers of fetal deaths and implantation losses, i.e. embryotoxicity at 1,000 and 2,000 ppm, and
decreases in fetal body weight and weight of placenta at 1,000 ppm were found. In addition, histopathological changes in the kidney were observed in the 2,000 ppm pregnant rats.

Based on 14-day repeated inhalation toxicity in male rats at concentrations of 1,000 ppm and 2,000 ppm, no toxic effect in females at 2,000 ppm, effects on pregnant female rats at 2,000 ppm, embryotoxicity noted in pregnant rats at 1,000 and 2,000 ppm, the test concentrations selected for the subsequent combined repeated dose toxicity study with a reproduction / developmental toxicity screening test (OECD 422) in rats were 0, 125, 250, 500 and 1,000 ppm.
Executive summary:

In a CoR 1 subchronic inhalation toxicity study, 2 -bromopropane was administered to groups of Crj: CD(SD) rats (5 males/5 females/3 pregnant females per exposure group) by whole body exposure at concentrations of 0, 125, 252, 502, 1003 and 2005 ppm (analytically verified mean) for 6 hours per day, 7 days/week for a total of 14 days. This was done as a limit test. The results of the repeated inhalation toxicity of 2-bromopropane were decreases in testis weight in 1,000 and 2,000 ppm male rats and histopathological changes of epididymis in 2,000 ppm male rats. No toxic effect in females was found at 2,000 ppm.