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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2013 - 21 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 2-Bromo-3,3,3-trifluoropropene
- Physical state: Pale yellow liquid
- Analytical purity: >99.4%
- Lot/batch No.: WNCC-BTP111128
- Expiration date of the lot/batch:
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 73 days (55 days on receipt at the test site, followed by an acclimation period of 18 days prior to first treatment)
- Weight at study initiation: (P) Males: 338 - 413 g; Females: 220 - 273 g
- Housing: Stainless steel wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): Ad libitum but withheld during exposure periods.
- Water (e.g. ad libitum): Ad libitum but withheld during exposure periods.
- Acclimation period: 18 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominal - 22 ± 3°C; Actual - 21.3 - 22.5°C
- Humidity (%): Nominal - 50% ±20%; Actual - 38.6 - 49.7% Relative humidity
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours darkness

IN-LIFE DATES: From: 14 October 2013 To: 05 December 2013

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1.0 m3 stainless steel and glass whole-body exposure chambers
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Details on mating procedure:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Vaginal plug or presence of sperm in vaginal lavage referred to as day 0 of gestation
- After successful mating each pregnant female was caged: Plastic maternity cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from the animals' breathing zone for analysis by Gas Chromatography (GC).
Duration of treatment / exposure:
6 hours per day to 3 groups (Groups 2-4)
5 minutes per day to 1 group (Group 5) then filtered air for the remainder of the 6-hour exposure period.

Males exposed to test substance for 14 days prior to mating and throughout the mating period for a total of 28-29 days of exposure.
Females exposed to the test substance for 14 days prior to pairing until gestation day 20 for a total of 35-46 days of exposure.
Females that failed to deliver were dosed through the day prior to euthanasia (post cohabitation day 25) for a total of 52 days of exposure.
Frequency of treatment:
Daily
Details on study schedule:
Age at mating of the mated animals in the study: 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm
Dose / conc.:
100 ppm
Dose / conc.:
175 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random):
The test substance is currently intended for use as a fire extinguishing agent and a special acute exposure group was included in the study to mimic the maximum exposure to the test substance as discharged from a fire extinguisher in all known environments where clean agents are used, including aviation.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

Each male and female was also observed for signs of toxicity approximately 1 hour following the 6-hour exposure period. In addition, each male and female in Group 5 was observed approximately 15 minutes and approximately 1 hour following the 5-minute exposure period.
During the post-natal period, any abnormalities in nesting and nursing behavior were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly
Food intake was not recorded during the breeding period. Once evidence of mating was observed, F0 female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily (not recorded during the mating period)

Oestrous cyclicity (parental animals):
- Time schedule for examinations: daily for 10 days prior to test substance administration and continuing until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, sperm production rate, sperm motility, sperm morphology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. Males were euthanized following completion of the mating period.
- Maternal animals: All surviving animals. Females that delivered were euthanized on lactation day 4. The female that failed to deliver was euthanized on post-cohabitation day 25. The female with total litter loss was euthanized within 24 hours of litter loss.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

MACROSCOPIC EXAMINATION
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Brain, Pituitary gland, Coagulating glands, Prostate gland, Kidneys, Seminal vesicles (2), Liver (sections of 2 lobes), Spleen, Lungs, Testes with epididymides (2) and vas deferens, Mammary glands (females only), Uterus with cervix and vagina, all gross lesions

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain, Lungs, Epididymides (total and caudal), Ovaries (without oviducts), Kidneys, Pituitary gland, Liver, Spleen, Testes

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
Microscopic evaluations were performed on the following tissues for all F0 parental animals from the control, 175, and 10,000 ppm groups.
Cervix, Coagulating gland, Epididymides, Lungs, Mammary glands (female only), Ovaries, Pituitary gland, Prostate gland Seminal vesicles, Spleen
Testis, Uterus, Vagina, Vas deferens, All gross (internal) lesions (all groups)

Postmortem examinations (offspring):
SACRIFICE
- On PND 4, surviving F1 rats were euthanized and discarded without examination.

GROSS NECROPSY
- Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).

HISTOPATHOLOGY / ORGAN WEIGTHS
- Pups with suspected skeletal anomalies were eviscerated and stained (Dawson, 1926) for subsequent skeletal evaluation.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group by sex.Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food and water consumption, offspring body weights and body weight changes, gestation length, estrous cycle length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, pre coital intervals, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance exposed groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival, percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-exposed groups to the control group. Histopathological findings in the test substance exposed groups were compared to the control group using a two tailed Fisher’s Exact test (Steel and Torrie, 1980).
Reproductive indices:
Male (Female) Mating Index (%) = [No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/Total No. of Males (Females) Used for Mating] x 100

Male Fertility Index (%) = [No. of Males Siring a Litter/Total No. of Males Used for Mating]x 100

Male Copulation Index (%) = [No. of Males Siring a Litter/No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)] x100

Female Fertility Index (%) = [No. of Females with Confirmed Pregnancy/Total No. of Females Used for Mating] x 100

Female Conception Index (%) = [No. of Females with Confirmed Pregnancy/No. of Females with Evidence of Mating (or Confirmed Pregnancy)] x 100

Offspring viability indices:
Mean Live Litter Size = [Total No. of Viable Pups on PND 0)/(No. of Litters with Viable Pups PND 0)]

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = [Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/No. of Litters Per Group] x 100

Postnatal Survival for All Other Intervals (% Per Litter) = [Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group] x 100

Where N = PND 0-1 and 1-4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only in the 10,000 ppm group at 15 minutes, resolved by 1 h pos exposure
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
only in the 10,000 ppm group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
only in the 10,000 ppm group
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related, adverse, higher mean pre-coital interval and longer mean gestation length were noted in the 175 ppm group F0 females compared to the control group

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no effects on F0 parental survival at any exposure concentration. Test substance related clinical findings noted at 15 minutes following exposure for males and females in the 10,000 ppm group included hypoactivity, decreased respiration, completely shut eyelids, and lacrimation. However, these findings were only noted on the first day of exposure (study day 0) and were resolved by 1 hour following exposure. In addition, salivation and red and/or clear material around the mouth and/or nose were noted for males and females in the 10,000 ppm group throughout the respective exposure periods at 15 minutes and/or 1 hour following exposure. No other test substance-related clinical findings were noted for males or females in the 50, 100, 175, and 10,000 ppm groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the 10,000 ppm group F0 males, lower mean body weight gains were noted throughout the exposure period and resulted in a lower mean body weight on study day 28. In addition, corresponding lower mean food consumption was noted for the 10,000 ppm group males compared to the control group during the pre-mating period (study days 0-13). These body weight and food consumption effects for F0 males were considered test substance-related and adverse
Mean body weights, body weight gains, and food consumption in the 10,000 ppm group F0 females were generally similar to the control group during the pre-mating period, gestation, and lactation. In addition, mean water consumption for F0 males and females at 10,000 ppm was unaffected by test substance exposure.
Mean body weight gains for males in the 100 and 175 ppm groups were similar to the control group during study days 0-21 and when the pre-mating period (study days 0-13) was evaluated. Lower mean body weight gains were noted in these groups compared to the control group during study days 21-28 and resulted in slightly lower mean body weight gains in the 100 and 175 ppm groups when the entire exposure period (study days 0-28) was evaluated; the differences were generally significant (p<0.05), but were not dose-responsive. The changes in mean body weight gain noted in the 100 and 175 ppm groups during the latter half of the exposure period were not of sufficient magnitude to affect mean body weights in these groups, and therefore were considered likely test substance-related but non-adverse.
Mean body weights and body weight gains in the 50 ppm group males were unaffected by test substance exposure throughout the study. None of the differences from the control group were statistically significant.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Unaffected by test substance exposure at all exposure levels.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Unaffected by test substance exposure at all exposure levels.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
A test substance-related, adverse, higher mean pre-coital interval and longer mean gestation length were noted in the 175 ppm group F0 females compared to the control group. Although a longer mean gestation length was also noted at 100 ppm and considered test substance-related, the value was within the range of values in the WIL Research historical control data, and therefore was not considered adverse.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Unaffected by test substance exposure at all exposure levels.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the scheduled necropsy, no noteworthy macroscopic findings were noted for F0 males and females at any exposure concentration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
no test substance-related microscopic changes at any exposure concentration.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increase in mean water consumption for females during gestation, longer mean pre-coital interval, longer mean gestation length, and the reduced postnatal survival noted in the 175 ppm group
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 175 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lack of adverse effects on systemic toxicity for males at any 6-hour exposure regimen or at the 10,000 ppm 5-minute exposure.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
lower postnatal survival was noted during PND 0 1, 1-4, and from birth to PND 4 in the 100 and 175 ppm groups
The postnatal survival values in the 100 ppm group were within the range of values in the WIL Research historical control data, and therefore were considered within normal limits and not adverse
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
increased incidence of interventricular septal defect was noted in the 175 ppm group
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
In the 100 and 175 ppm groups, test substance related, lower postnatal survival was noted during PND 0 1, 1-4, and from birth to PND 4 compared to the control group; the values in the 175 ppm group were below the minimum mean values in the WIL Research historical control data, and therefore were considered adverse. The postnatal survival values in the 100 ppm group were within the range of values in the WIL Research historical control data, and therefore were considered nonadverse. Postnatal survival in the 50 and 10,000 ppm groups was similar to the control group. The mean numbers of F1 pups born, the pup sex ratio, and live litter size in the 50, 100, 175, and 10,000 ppm groups were unaffected by parental test substance exposure.

CLINICAL SIGNS (OFFSPRING)
No remarkable findings regarding the general physical condition of the F1 pups were noted at any exposure concentration.

BODY WEIGHT (OFFSPRING)
No adverse effects on mean F1 male and female pup body weights and body weight gains were noted during PND 1-4 at 50, 100, 175, and 10,000 ppm.

GROSS PATHOLOGY (OFFSPRING)
At the necropsy for F1 pups that were found dead, an increased incidence of interventricular septal defect was noted in the 175 ppm group and was considered test substance-related and adverse.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
gross pathology
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
175 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
175 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

DISCUSSION:

Adverse clinical signs in males and females and lower mean F0 male body weights, body weight gains, and food consumption after 28 days exposure were noted in the 10,000 ppm group. However that exposure level was intended to mimic and assess the effects of a single maximum exposure in humans, the changes noted in mean body weight gain and food consumption that led to a reduction in mean body weight only after 28 days of exposure would not be relevant to a single exposure scenario at the same exposure level. No effects on F0 reproductive performance and F1 offspring were noted at 10,000 ppm when 2-Bromo-3,3,3-Trifluoropropene was administered as a daily 5-minute whole-body inhalation exposure.

Test substance-related, higher mean maternal water consumption was noted in the 175 ppm group females compared to the control group throughout gestation and was considered adverse. 

In the 175 ppm group females, a higher mean pre-coital interval and longer mean gestation length were noted compared to the control group; these differences were considered test substance-related and adverse. In addition, test substance-related, lower postnatal survival was noted in the 175 ppm group compared to the control group during PND 0-1, 1-4, and from birth to PND 4 and was considered adverse.

Higher mean male and female pup birth weights (PND 1) were noted for males and females in the 50, 100, and 10,000 ppm groups compared to the control group. This was likely due to the slight increases in mean gestation length noted in these groups compared to the control group. Fetal weight gain occurs rapidly during late gestation and immediately prior to delivery. Although not adverse and within WIL Research historical control data, there was a 0.4 to 0.6 day increase in the gestation length noted in these groups compared to the control group, which may have attributed to the increased pup weights noted. However, mean body weights on PND 4 and mean body weight gains during PND 1-4 in these groups were similar to the control group. Therefore, the higher mean male and female pup birth weights on PND 1 may have been secondary to the slightly increased gestation length and were not considered adverse. In the 175 ppm group, mean pup birth weights (PND 1) were similar to the control group. It would be expected that the longer gestation length would produce higher mean birth weights in pups, as mentioned previously. An increased gestation length that was outside of the WIL Research historical control values and considered adverse was noted at 175 ppm. The lack of a statistical increase in the mean pup body weights at 175 ppm was likely due to a potential developmental delay or a reduction in mean pup body weights that was masked by the increased gestation period. 

At the necropsy for F1pups that were found dead, an increased incidence of interventricular septal defect was noted in the 175 ppm group. Experimentally induced interventricular defects have been shown to close during postnatal development and can be considered a developmental delay in the cardiac system that may or may not be considered adverse depending on the size of the opening and the impact on viability and growth of the pups (Fleeman,et al., 2004). The interventricular defects in this study were noted in pups that were found dead. Therefore, while these observations may have been related to the apparent developmental delay noted in the 175 ppm group, they were considered test substance-related and adverse when coupled with the reduction in postnatal survival noted in this group. 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this reproduction/developmental toxicity screening study in rats, an exposure level of 100 ppm 2-Bromo-3,3,3-Trifluoropropene was considered to be the no-observed-adverse-effect level (NOAEL) for female systemic toxicity, reproductive toxicity, and neonatal toxicity based on the increase in mean water consumption for females during gestation, longer mean pre-coital interval, longer mean gestation length, and the reduced postnatal survival noted in the 175 ppm group. The NOAEL for F0 male systemic toxicity was considered to be 175 ppm, the highest exposure concentration tested, based on the lack of adverse effects on systemic toxicity for males at any 6-hour exposure regimen.
No effects on reproduction/developmental toxicity were noted at 10,000 ppm when 2-Bromo-3,3,3-Trifluoropropene was administered as a daily 5-minute whole-body inhalation exposure.