Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Effects of Ethylene Dibromide on Reproduction in Male and Female Rats
Author:
Short, R.D., Winston, J.M., Hong, C.-B., Minor, J.L., Lee, C.-C. & Seifter, J.
Year:
1979
Bibliographic source:
Toxicology and Applied Pharmacology, Vol. 49, pp. 97-105.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
EDB was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin, with a purity of 99% and no further analysis was performed.

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River CD rats (Charles River Breeding Laboratories, North Wilmington, Mass.)
- Age at study initiation: nda
- Weight at study initiation: Mean body weights - Males: 266-267 g; Females: 197-203 g
- Fasting period before study: During the 7-hr inhalation exposure food and water were withheld.
- Housing: Stainless-steel cages with wire-mesh bottoms
- Diet/Water: Rats were given free access to powdered rodent chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill.) in glass jars and tap water except
during the 7-hr inhalation exposure when food and water were withheld.
- Acclimation period: At least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animal rooms were maintained at 22°C
- Humidity (%): 50 ± 10%
- Photoperiod (hrs dark / hrs light): 7:00am to 7:00pm photoperiod.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
EDB vapor generation:
Rochester-type stainless-steel chambers with a volume of about 3.5 m3 were used in this study. Filtered air, at a flow rate of 10 to 12 chamber
air changes/hr, entered at the top of the chamber.
EDB vapor was generated by bubbling nitrogen into a heated glass vessel containing EDB which was maintained at 60°C. The EDB vapor entered the air supply upstream from the chamber. Mixing was initiated in a plenum at the top of the chamber and completed using two squirrel cage fans and a diffusion plate.
The EDB concentration in each chamber was generally monitored three times a day, and a time weighted average concentration was calculated for
each day. The concentration was measured using gas chromatography and a flame ionization detector.
EDB was resolved using a stainless-steel column packed with a 5% dodecyl phthalate on 80/100 chromosorb and nitrogen (80 ml/min) as the carrier
gas. The injection, column, and detector temperatures were 150, 120, and 170°C, respectively. Standards were prepared by serial dilutions of an EDB
stock solution prepared in carbon disulfide.
Details on mating procedure:
Protocol for male reproduction study:
9 to 10 males in each group were housed individually with two virgin females a week for a total of 2 weeks. In this way, each male was given the opportunity to mate with four females.

Protocol for female reproduction study:
20 females in each group were housed with proven male breeders in the ratio of two females to one male. The females were examined in the morning for evidence of mating as demonstrated by the presence of sperm in vaginal smears.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The EDB concentration in each chamber was generally monitored three times a day, and a time weighted average concentration was calculated for
each day.

Males inhaled EDB 7 hr/day, 5 days/week, for 10 weeks. The average ± SE (range of daily values) concentration for each chamber for 50 exposures was 19 ± 1 (16-25), 39 ± 1 (30-48), and 89 ± 2 (65-134) ppm of EDB. Females inhaled EDB 7 hr/day, 7 days/week, for 3 weeks. The average ± SE (range of daily values) concentration for each chamber for 21 exposures was 20 ± 0 (18-22), 39 ± 1 (35-43) and 80 ± 1 (77-89) ppm of EDB.
Duration of treatment / exposure:
Protocol for male reproduction study: 10 weeks

Protocol for female reproduction study: 3 weeks
Frequency of treatment:
Protocol for male reproduction study: 7 hr/day, 5 days/week.

Protocol for female reproduction study: 7 hr/day, 7 days/week.
Details on study schedule:
- Protocol for male reproduction study:
Four groups of male rats were exposed to 0, 19, 39, or 89 ppm of EDB 7 hr/day, 5 days/week, for 10 weeks. Afterwards, 9 to 10 males in each group were killed, testes were weighed, and aortic blood was obtained for the determination of serum testosterone concentrations using commercially prepared assay kits (New England Nuclear, Boston, Mass.). An additional 9 to 10 males in each group were housed individually with two virgin females a week for a total of 2 weeks. In this way, each male was given the opportunity to mate with four females. At the end of the 2-week mating period, males were killed and the reproductive organs (testes, epididymides, seminal vesicle, and prostate glands) were removed and preserved in neutral buffered 10% formalin. The tissues were processed, sectioned, and stained with hematoxylin and eosin for microscopic examination. Females housed with these males were killed at midgestation, which was calculated from the midweek of their presumptive mating. The number of implants, viable implants, and resorptions was determined.

- Protocol for biochemical and distribution study in males:
Three groups, each consisting of four male rats, received by oral intubation 0, 10, or lOOmg/kg of EDB dissolved in corn oil, and aortic blood was obtained 24 hr later. Serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), and blood urea nitrogen (BUN) were determined according to the methods described by Amador and Wacker (1962), Henry et a/. (1960), and Chaney and Manback (1962), respectively. Additional groups, each consisting of four males/ dose at each time point, were similarly treated, and the non-protein sulfhydryl concentration was determined in the liver and right testis 2 and 4 hr after treatment. Two groups each consisting of three male rats were treated orally with 10 mg/kg (5.4&i/mg) or 100 mg/kg (0.54 pCi/mg) of [1,2-14C]EDB (New England Nuclear, Boston, Mass.) and killed 4 hr later. Samples of liver, right testis, right kidney, and stomach, which was washed of its contents, were obtained. Portions of these tissues, which weighed about lOOmg, were combusted in a Biological Material Oxidizer (R. J. Harvey Instruments, Hillsdale, N.J.) and radioactivity was captured as Y02 An additional portion of each tissue, which weighed about 1 g, was homogenized in 5 ml of cold water, and an equal volume of 1 N perchloric acid (PCA) was added. The precipitate was washed with successive 5-ml portions of 0.5 N PCA, 1 M sodium acetate in absolute methanol, absolute methanol : chloroform (1 :l), absolute ethanol, and then heated for 1 hr at 37°C in 5 ml of 0.5 N sodium hydroxide. Afterwards, 5 ml of 1 N PCA was added and the sample was cooled on ice. The supernatant, which is referred to as the RNA fraction, was saved; and the precipitate was washed twice in 5 ml of 0.5 N PCA. The supernatants from these washings were added to the RNA fraction. The precipitate was heated for 15 min in 5 ml of 0.5 N PCA. The supernatant, which is referred to as the DNA fraction, was saved; and the precipitate was heated a second time in a similar fashion. The supernatant was added to the DNA fraction. The precipitate was washed in 5 ml of 0.5 N PCA, and the resulting supernatant was added to the DNA fraction. The precipitate was dissolved in 10 ml of sodium hydroxide and is referred to as the protein fraction. Radioactivity in whole tissue and aliquots of the RNA, DNA, and protein fractions was determined by liquid scintillation counting. The results were initially expressed as disintergrations per minute per milligram tissue and then converted to nanogram equivalents of EDB per milligram of tissue using the specific activity of the dose.

- Protocol for female reproduction study:
Four groups of female rats were exposed to 0, 20, 39, or 80 ppm of EDB 7 hr/day, 7 days/week, for 3 weeks. Exposure was not continued after mating because we were interested in evaluating the effect of EDB on conception rather than early development. Afterward, 20 females in each group were housed with proven male breeders in the ratio of two females to one male. The females were examined in the morning for evidence of mating as demonstrated by the presence of sperm in vaginal smears. Females were killed at midgestation during the third week following exposure and examined for the number of implants, viable implants, and resorptions. In addition, the uterus and ovaries were prepared for histopathological evaluation as previously described.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Males: 19, 39 & 89 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Females: 20, 39 & 80 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
2 females to 1 male. 20 females per dose and 10 males.
Control animals:
yes
Positive control:
not applicable.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
Oestrous cyclicity (parental animals):
nda
Sperm parameters (parental animals):
At the end of the 2-week mating period, males were killed and the reproductive organs (testes, epididymides, seminal vesicle, and prostate glands) were removed and preserved in neutral buffered 10% formalin. The tissues were processed, sectioned, and stained with hematoxylin and eosin for microscopic examination.
Litter observations:
Exposure was not continued after mating because the interes was in evaluating the effect of EDB on conception rather than early development.
Postmortem examinations (parental animals):
At the end of the 2-week mating period, males were killed and the reproductive organs (testes, epididymides, seminal vesicle, and prostate glands) were removed and preserved in neutral buffered 10% formalin. The tissues were processed, sectioned, and stained with hematoxylin and eosin for microscopic examination.
Females were killed at midgestation during the third week following exposure and examined for the number of implants, viable implants, and resorptions.
In addition, the uterus and ovaries were prepared for histopathological evaluation.
Postmortem examinations (offspring):
Exposure was not continued after mating because the interest was in evaluating the effect of EDB on conception rather than early development.
Statistics:
Data are reported as the mean or mean ± SE. Comparisons were made by analysis of variance techniques (Steel and Torrie, 1960), two sample
rank test (Goldstein, 1969) or Fisher exact probability test (Siegel, 1956). The level of significance was selected as p < 0.05.
Reproductive indices:
not reported.
Offspring viability indices:
not reported.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

General Observations:
Males exposed to 89 ppm gained less weight and consumed less food than control males (Table 1). In addition, mortality was observed only at the high concentration, at which 7 of 33 (21%) males died. A reduced weight gain also occurred with males exposed to 39 ppm, and their body weight was
significantly less than that of the controls at the end of exposure. In contrast, the weight gain and food consumption of males exposed to 19 ppm of EDB were generally similar to control values.
Females exposed to 80 ppm lost weight and consumed less food than control females (Table 1). In addition, mortality was observed only at the high concentration, at which 10 of 50 (20 %) females died. Although there were some exceptions at a few time points, the weight gain and food consumption
of females exposed to 20 and 39 ppm were similar to control values.

Male Reproduction Study:
At the end of a 10-week exposure to 0, 19, 39, or 89 ppm EDB, the average testes weight of rats in each group was 3.4, 3.3, 3.5, and 0.5 g, respectively. In addition, the serum testosterone concentrations in these rats were 1.04, 1.21, 1.20, and 0.48 µg/100 ml, respectively.
Additional males were mated with unexposed females (Table 2). At least 90 % of all males from the groups exposed to 0, 19, or 39 ppm EDB impregnated at least one female.
In contrast, none of the males exposed to 89 ppm impregnated any females. Females that mated with males exposed to 19 and 39 ppm produced litters that were normal in terms of total implants, viable implants, and resorptions (Table 3).
After a 10-week exposure period and a 2- week mating period, the reproductive organs of these males were examined for histopathological
changes. No treatment-related effects were observed in these tissues from males exposed to 19 or 39 ppm.
In contrast, males exposed to 89 ppm had atrophy of the testis, epididymis, prostate, and seminal vesicles.

Female Reproduction Study:
At the end of 3-week inhalation exposure, vaginal smears from females exposed to 20 or 39 ppm of EDB were normal. In contrast, females exposed to 80 ppm were in diestrus and did not begin to cycle normally until 3 to 4 days after exposures ended. As a result, fewer females mated in this group than other groups during a 10-day mating period. In all groups exposed to EDB, all mated females were pregnant when killed at midgestation and had uterine contents that were normal in terms of total implants, viable implants, and resorptions.
In the females exposed to 0, 20, 39, or 80 ppm of EDB, the incidence (females affected/females inspected) of mild vacuolated degeneration of epithelium of the uterus with/without necrosis was 3/20, 0/20, 0/20, and 6/20, respectively, and ovarian cyst was 0/20, 0/20, 0/20, and 3/20, respectively. No other lesions were observed.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
39 ppm
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Not examined.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

In the present study, both sexes of rats were exposed to one of three concentrations of EDB. Adverse effects on survival, body weight, and food consumption were consistently observed at the highest concentration. In addition, adverse effects on reproduction were also observed at this concentration. Males exposed to 89 ppm had reduced testicular weights and serum testosterone concentrations, atrophy of the reproductive organs, and failed to impregnate any female. Females exposed to 80 ppm had abnormal estrous cycles at the end of exposure; however, this change was reversible and some females successfully mated and produced normal litters. In contrast, the various parameters of reproduction examined were normal in both males and females exposed to the low and middle ccncentrations of EDB.

Table 1. Body weight and food consumption of male and female rats exposed to EDB.

Exposure week Bodyweight (g) Food consumption (g/day)
EDB (ppm) EDB (ppm)
0 19 39 89 0 19 39 89
Male exposed
0 267 a 266 266 267 ND b ND ND ND
2 342 320 c 315 c 268 c ND ND ND ND
4 407 404 386 c 319 c 28.3 39.8 27.8 22.4 c
6 446 443 428 329 c 27.8 28.4 27.8 19.3 c
8 477 466 448 c 333 c 28 28.3 28.5 15.7 c
10 484 463 443 c 317 c 35.2 41.9 41.2 19.5 c
Exposure week Bodyweight (g) Food consumption (g/day)
EDB (ppm) EDB (ppm)
0 19 39 80 0 19 39 80
0 203 d 197 200 200 ND ND ND ND
1 215 200 c 203 c 180 c 18.3 16.0 c 16.9 11.2 c
2 230 218 217 167 c 19.6 18.9 20.4 13.3 c
3 245 234 229 171 c 19.8 20.7 20.7 10.5 c

a=Mean value for 30 -33 animals/concentration (one to two/cage).

b=Not determined

c=Significantly different from control (Dunnett's test).

d=Mean values for 20 animals/concentration (one to two/cage).

Table 2. Fertility of Male rats exposed to EDB for 10 weeks and then housed with unexposed Females.

  EDB (ppm)
0 19 39 89
No. exposed to EDB 9 10 10 9
No. impregnating females(s) a
During week 11 9 9 10 0 b
During week 12 9 9 10 0 b
No. impregnating
4 females 5 6 3 0
3 females  2 2 5 0
2 females  2 1 2 0
1 female 0 0 0 0
0 females 0 1 0 9 b

a=Number of males impregnating at least one female. One males housed with two females each week.

b=Significantly different from control (Fisher exact probability test).

Table 3. Reproductive performance of unexposed female rats mated with males exposed to EDB.

  EDB (ppm)
0 19 39 89
No pregnant rats
Week 11 16 15 14 0
Week 12 14 17 17 0
Total implants/dam
Week 11 13.1±1.1 a 14.2±1.0 11.1±1.2 NP b
Week 12 12.4±0.5 13.4±0.5 11.9±0.5 NP
Viable implants/dam
Week 11 11.8±1.2 13.1±1.0 10.1±1.1 NP
Week 12 11.8±0.5 12.3±0.6 11.3±0.6 NP
Resorptions/dam
Week 11 1.2±0.4 1.1±0.2 1.1±0.2 NP
Week 12 0.6±0.2 1.1±0.3 0.7±0.2 NP

a=Mean±SE

b=None of the females were pregnant.

Applicant's summary and conclusion

Conclusions:
The NOAEC can be determined as 39 ppm for both males and females as no effects were seen at this concentration.
Executive summary:

The effects of ethylene dibromide (EDB) on reproduction were studied in both sexes of rats. Males inhaled average daily concentrations of 19, 39, and 89 ppm of EDB 7 hr/day, 5 days/week, for 10 weeks. Mortality and morbidity occurred in the group exposed to the high concentration. In addition, males in this group had reduced testicular weights, reduced serum testosterone concentrations, and failed to impregnate any females during a 2-week mating period. Atrophy of the testes, epididymis, prostate, and seminal vesicles was observed in these males. The reproductive performance of males exposed to 19 or 39 ppm of EDB was not impaired.

Females inhaled average daily concentrations of 20, 39, or 80 ppm of EDB 7 hr/day, 7 days/week, for 3 weeks. Mortality and morbidity occurred in the group exposed to the high concentration. Females in this group did not cycle normally until several days after the termination of exposure. The reproductive performance of females exposed to 20 or 39 ppm of EDB was normal. Pregnant females in all groups produced normal litters, and histopathological examination of the ovary and uterus did not reveal lesions that would impair reproductive performance. Thus, adverse effects on reproduction were observed in both sexes of rats only at concentrations of EDB that also produced mortality and morbidity. Therefore, it was not possible to attribute the reproductive effects directly to EDB.