Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

Exposure at 175 ppm for 6 hours daily produced effects on the mean water consumption for female rats during gestation, longer mean pre-coital interval, longer mean gestation length, and the reduced postnatal survival but no effects on the male rats. Exposure at 10,000 ppm for 5 min daily produced effects on the mean body weight gain and food consumption after 28 days exposure, clinical signs which are resolved one hour after exposure but no effects on reproduction or developmental toxicity.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Exposure Methods: Exposures for Groups 1-4 were conducted in 1.0-m3 stainless steel and glass whole-body inhalation exposure chambers and exposures for Group 5 (10,000 ppm) were conducted in two 0.5-m3 stainless steel and glass whole-body inhalation exposure chambers. All chambers were operated under dynamic conditions, at a slight negative pressure (ca. 0.5 in. of water) with at least 12 to 15 air changes per hour. Oxygen content of the exposure atmosphere was at least 19.0%. Temperature and relative humidity during exposures were measured at least once every hour for each chamber.

Test atmosphere generation methods (50, 100, 175 ppm):
A single vapour generator was used to produce a vapor and a distribution system was used to deliver a controlled amount of test substance to each chamber.

Test atmosphere generation method (10000 ppm):
A single vapor generator was used to produce a vapour atmosphere.
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
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.
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
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: 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
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.

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.
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
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.
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
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
175 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

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. 

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.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 417.18 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study, referred to above, was considered reliable without restriction; the overall database is therefore considered to be of acceptable quality.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

An assessment of the substance for reproductive/developmental toxicity was based upon an analysis of the results of two inhalation screening reproductive/developmental toxicity studies that were conducted in accordance with OECD 421 and in conjunction with assessment of the overall toxicity profile of the substance. The assessment evaluates how findings observed in the OECD 421 studies can be interpreted for assessment of reproductive/developmental toxicity and classification. 


Summary of Justification:


1. Impact of Respiratory Irritancy and CNS Depression


Respiratory Irritancy and CNS Depression in both acute and repeat dose animal experiments point to a general malaise and lethargy in male and female animals. Whilst the CNS depression is not seen clinically at the lower dose levels on one reproductive toxicity study (WIL Research, 2014), it was more obvious in a second reproductive study (Huntingdon Life Sciences, 2013) where an increased incidence of CNS changed was seen alongside increasing effects on reproduction/developmental parameters. A prolonged exposure period of up to 6 hours during each  day of treatment prevents full recovery from the effects that are both clinical and sub-clinical and thereby leaving the animals in a less than optimal condition.


2. Non-Specific Toxicity


Physiological changes experienced in the reproductive/developmental studies indicate non-specific toxicity amongst adult animals. This non-specific toxicity is due to varying sensitivity and susceptibility of individual animals and is not represented in mean test results. Poor maternal health and well-being can be responsible for post-natal mortality (post-implantation loss, reduced live birth index and post-natal survival rate).


3. Mode of Action


The mode of action does not appear to be through Endocrine Disruption involving normal sex hormones (oestrogen, progesterone, and testosterone) since functions regulated by such hormones both increased and decreased in the same dose groups. Some adult animals experienced an increase in reproductive endpoints (e.g., pre-coital interval and oestrus cycle) and others in the same dose group experienced a decrease in the same endpoints, meaning that reproductive endpoint is not the specific target of toxicity.


4. Inter-species Variability


Rats tend to be more susceptible than humans to respiratory irritation because they have more convoluted nasal turbinates and are obligate nose breathers. Heightened respiratory irritation leads to increased water consumption, lack of appetite, and poor general condition of test animals, but would not necessarily occur in humans due to odor and taste discernment.


5. Impact of Toxicokinetics


Although the substance is rapidly cleared from the body, small amounts of chemical may accumulate in fat and slowly be released into the body and some residual toxic effects can take place. Effects seen in rats are potentially the consequence of greater exposure to the test item than you would expect from human exposure.


Detailed Discussion:


Impact of Respiratory Irritancy and CNS Depression:


Acute toxicity testing (inhalation route) has shown evidence of sedation and decreased motor activity along with respiratory tract changes. These were at dose concentrations up to 44 000 ppm for 30 minutes in one study with the rat but in a further study, 4-hour exposure at up to 25 000 ppm resulted in death and with associated bronchiolar lesions 24 hours after exposure. This shows that a shorter exposure period at high concentrations can be tolerated but more prolonged repeated exposures at lower dose concentrations can lead to adverse reactions including death. The fact that mortality was seen up to 24 hours after exposure does suggest that effects of treatment are not necessarily resolved following withdrawal of exposure and regardless of a rapid elimination of the test item from the blood being demonstrated in toxicokinetic studies.


Repeated-dose, whole body exposure to the substance demonstrated a number of changes in the rat when administered for periods up to 5-6 hours for either 14 or 90 days. In the 14-day range-finding studies , the most obvious signs, both clinically and histopathologically, were those associated with an irritant type effect in the upper respiratory tract. The findings from the sub chronic 13-week inhalation study (Huntingdon Life Sciences, 2013) highlight both the clinical effects associated with respiratory irritation and evidence of CNS depression at 505 ppm and above. There was also a reduced weight gain and food consumption, which is a consequence of post-treatment general malaise. This gives an indication as to the compromised health status of the animals during treatment and was observed at the lowest dose level of 200 ppm.


A major factor in the reactions of animals to treatment was the evidence of irritation effects in the respiratory tract following exposure. Whilst the skin and eye irritation studies gave practically no evidence of irritancy, the single dose and repeated dose inhalation toxicity studies did reveal significant irritancy, particularly in the upper respiratory tract of rats. Prolonged respiratory irritation causes significant effects upon adult animals, which compromises general health and well-being of the animals. The irritant effects were seen clinically in adult rats during gestation, where an increase in water consumption was exhibited in females, plus the observation of nasal discharge in greater proportions in females experiencing deleterious reproductive effects. The histopathological changes in the nasal turbinate of rats also provides an insight into the irritant type effect, contributing to the overall malaise of animals during and after exposure.


The results of the two reproduction/developmental toxicity screening studies identified a number of effects upon male and female reproductive organs/tissues plus effects upon the in utero and post- natal survival of offspring. A clear difference in one study (WIL Research, 2014) was seen between a high dose concentration of 10 000 ppm administered over a short period of the day (5 minutes) when compared with a lower dose concentration up to 175 ppm over a longer daily period of 6 hours. The long duration of exposure prevents recovery from adverse effects during the day and will allow a more cumulative daily effect to occur. This type of daily cumulative exposure means that any CNS /irritant effects will occur over a longer period, thereby magnifying any effects on the reproductive system; albeit the CNS effects were seen clinically only at a high dose concentration of 10,000 ppm for short term exposure duration (WIL Research, 2014) or at dose levels of 198 ppm and above for 6 hours duration (Huntingdon Life Sciences, 2013). This therefore adds weight to the opinion that dose duration has a profound impact as much as dose concentration.


Non-Specific Toxicity:


Additional effects upon tissues such as the heart and pancreas indicate other systemic effects have been the result of substance exposure but where the No Observed Adverse Effect Concentration (NOAEC) was deemed to be 200 ppm (sub-chronic 90-day study). The presence of such systemic changes also contributes to a condition whereby animals are functioning sub optimally but not necessarily causing an effect upon adult survival, nevertheless rendering the animals to be in a compromised health condition.


The reproduction/developmental screening studies were conducted at dose levels between 50 and 3000 ppm for 6 hours per day during 2-week premating, gestation and early lactation. Concentration-related clinical signs in adult animals at 200 ppm and above were linked to CNS depression, such as unresponsiveness and underactivity. Whilst recovery was seen quickly after cessation of treatment, it is likely that physiologic changes to the CNS occurred and persisted after exposure. It is noted that clinically CNS depression in animals from the reproductive studies was restricted to dose levels of 198 ppm and above (Huntingdon Life Sciences, 2013) and only at 10,000 ppm for 5 minute exposure (WIL Research, 2014) but it is apparent that the CNS is a target organ. The lack of clinical signs seen below 198 ppm in one study does not necessarily preclude any adaptive alteration to the CNS below this dose level. Physiological change as a result of non-specific toxicity can explain the variability seen in treated animal responses, due to varying sensitivity or susceptibility of individual animals. This is also the reason why neonatal mortality at 175 ppm was not consistent amongst individuals and is likely to be due to differences in maternal tolerance to the substance. Effects on general body weight gain and a decline in food consumption, signs of non-specific toxicity, were seen in these dose groups. However, a NOAEC for effects on adult animals was set at 175 ppm for males and 100 ppm for females.


Inhalation exposure to rats in two reproduction/developmental toxicity studies  together with findings from acute and subchronic toxicity studies have identified toxicity to adults and an impact upon reproduction and offspring development. Based on the findings of the toxicity studies, it can be concluded that a number of adverse events are seen with exposure to the substance. It is likely that there is no common aetiology for the changes seen. A multifactor-related cause of toxicity to adults and their offspring is seen and it is not possible to identify one single cause that contributes to all the adverse reproductive events.


Mode of Action:


In the studies, adult effects were seen at similar dose levels to effects upon reproduction, which demonstrates an association between these events. The nature of these studies makes it a challenge to prove that effects on reproduction were specifically caused by the test item instead of a non-specific consequence of adult toxicity.  A mode of action does not present itself within the study; however, it is be possible to infer a potential mode of action. Based on the observations of CNS depression especially at higher dose levels on the reproduction study (Huntingdon Life Science, 2013) and respiratory irritancy, it can be concluded that adult health has been affected and the changes are sufficient to influence normal reproductive physiology when compared with animals not exposed to the substance. It stands to good reason that general lack of well-being reduces the capability of adult females to maintain their offspring post-partum and thereby leading to greater neonatal mortality due to lack of maternal care from compromised adults. Other factors that need to be considered are the effects of a respiratory irritant upon reproduction and development plus the findings of systemic toxicity, such as inflammatory alterations in the heart and the splenic changes.


Oestrous cycle lengths did increase in the first reproduction study at higher dose levels (Huntingdon Life Sciences, 2013) but the most prominent feature was the number of irregular cycles amongst treated females. This appears to be an inconsistent effect since a variety of oestrous cycle lengths were recorded for females at higher dose levels. At these higher dose levels, the influence of toxicity to the adult female is a more likely explanation as to the cause of the effect identified as irregular oestrous cycles. For offspring, there was an increased post implantation loss, reduced live birth index and litter survival. Although mostly concentration dependent, not all effects were resolved at a concentration of 200 ppm and ultimately in a later study (WIL Research, 2014), the NOAEC for reproduction/development was established at 100 ppm for post implantation survival. This effect does not necessarily mean an impact upon the offspring since poor maternal care can be equally responsible for post-natal mortality.


The study findings indicate that mode action is unlikely to be through endocrine disruption as there is no consistency for individual animal findings within dose groups. The effect upon oestrous cycle length was not consistent as demonstrated in the second of two reproduction studies (WIL Research, 2014). The group mean oestrous cycle length was similar between the control and high dose group (5.2 days as opposed to 5.0 days). However, there was a significant intergroup difference in individual oestrous cycle lengths for high dose animals ranging from 3.3 days to 7 days. This is of note as variance to the mean value indicates a contradictory finding suggesting not one specific mode of action such as endocrine disruption.


This intergroup variation is less likely to represent a direct effect on oestrous cycles as the material would have to have both an agonistic and antagonistic effect at the same exposure duration and dose. The implication is that the health of the individual animal is compromised such that normal physiological function is impaired by a non-endocrine mechanism.


A comparison can be made to altered nutritional status, such as in the case of vitamin A deficiency, where an impact on parturition and neonatal survival/development can be seen. This is likely due to altered physiology within the parent animal and not attributed to one specific event based on the number of chemical interactions involving vitamin A and the multitude of adverse responses vitamin A deficiency has as a consequence (Mason, K.E. 1935).


A developmental defect in a small number of post-natal offspring deaths (interventricular septal defects) in one study (WIL Research, 2014) was subject to independent review (York, R. 2014).  It was concluded that the true nature of the defects was not fully established and that doubts remain as to the aetiology of this effect. It is not common to identify such malformations in offspring using the method employed in the study as this is usually done in foetal offspring that have been preserved and evaluated under magnification. Since the term interventricular septal defect can be ascribed to more than one developmental process, the observation and reporting of these findings does result in confusion as to the true effect and whether this was actually the cause of mortality in these and other offspring. No such malformations were found in offspring at higher dose concentrations in a separate study (Huntingdon Life Sciences, 2013) and so a true effect on neonatal heart development cannot be confirmed.


Inter-species Variability:


It is important to note the differences in respiration systems between rat and humans.  Rats are obligate nasal breathers in contrast to humans, who breathe through both their nose and mouth. In addition, laboratory rats have much more convoluted nasal turbinate systems than humans and the length of the nasopharynx in relation to the entire length of the nasal passage also differs between species. This greater complexity of the nasal passages, coupled with the obligate nasal breathing of rodents, is generally thought to result in greater deposition of chemicals in the upper respiratory tract of rats than in humans breathing orally or even nasally. The respiratory irritancy is displayed by increased incidence of red nasal discharge at dose levels of 50 to 175 ppm substance and supported by an increase in daily water consumption.  It is therefore likely that the choice of the rat as animal model highlighted the contribution of respiratory irritation and its subsequent contribution to a deterioration in health of adult animals when exposed to the substance. 


Such toxic/irritant effects are expected to also contribute to the impact upon maternal aspects of reproduction such as the prolongation of gestation and ability to maintain offspring during early lactation due to a direct effect upon the health of adult females. It has been demonstrated, for example, that the introduction of infectious agents such as trypanosoma may affect animal health and also affect gestation length at the same time. It is not unreasonable to assume that females displaying adverse health effects in general as a consequence of adverse toxicity and, as in the case of infection, will show prolonged gestation. Under these circumstances of ill health, females will not maintain a pregnancy/litter to the same ability as those that are not affected by toxic insult or other forms of health effects.


Impact of Toxicokinetics:


The nature of the toxicity to adults is such that all effects may not be completely resolved after treatment is concluded each day. This is despite the fact that clearance of the test item was quite rapid on the basis of measured arterial blood concentrations. However, the high body fat: air partition coefficient suggests some chemical accumulation in fat, which may have an impact during the off-dose periods where slow release of the substance from body fat may occur. This may have exacerbated any toxic effects seen during the dosing period and whilst clinical signs of toxicity rapidly diminish, some residual toxic effects can take place and influence events such as food consumption and mating performance.  In general, the NOAEC for findings were set in the region of 175-200 ppm. For reproductive effects, the effects upon male and female reproductive organs were seen at 200 ppm and above in a concentration related manner. Effects on pre-coital interval and gestation length were seen at 175 ppm with a NOAEC as 100 ppm. Effects on offspring post-implantation survival and post-partum survival were also seen at 175 ppm with a NOAEC of 100 ppm. In addition, the appearance of offspring with abnormal hearts added to the conclusion of offspring NOAEC at 100 ppm.

Effects on developmental toxicity

Description of key information

See fertility section for discussion.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study, referred to above, was considered reliable without restriction; the overall database is therefore considered to be of acceptable quality.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

It is assessed that the reproductive endpoint is not the specific target of toxicity - see full discussion in fertility section. 

Justification for classification or non-classification

Based on the observed effects upon adults and their subsequent reproductive performance, a relationship between adult effects and reproductive effects has been demonstrated. It is likely that this centers around the effects upon the CNS initially plus contributions from respiratory irritation, which contribute to adverse health responses in adult animals. These effects are significantly affected by duration of exposure. The results of two repeated dose screening reproduction studies have shown that both adult and reproductive toxicity effects are related to treatment duration and dosage, and the NOAEC for effects on adult and reproductive toxicity are essentially in the same dose range. The likely daily accumulation of 2-BTP in the rat will result in a magnification of clinical effects as a consequence of exposure . It is clear that a broad range of circumstances (including dietary conditions and infectious agents) can affect the health of the adult animal and as a consequence can impact the reproductive performance. This can include maintenance of a litter during gestation and care for pups during lactation. It is the assertion, in this case that cumulative adverse health effects as a consequence of exposure over long daily periods to 2-BTP, have contributed to a decline in reproductive capacity and therefore presentation of adverse reproductive effects.  As outlined by the ECHA document related to Guidance for the application of the CLP criteria, the evidence with 2-BTP provides a link between adult toxicity and reproductive effects. It is not, however, completely clear as to whether the effects upon offspring can be completely attributed to effects via the adult, although a plausible argument can be made to demonstrate that toxic effects to the adult do compromise maternal (and hence, reproductive) performance. As such, since there is not clear evidence of reproductive/development effects in the absence of other non-specific consequences and there is interspecies and mechanistic information that raises doubt about the human relevancy, the most appropriate self-classification for 2-BTP would be Category 2 H361: Suspected of damaging fertility or the unborn child.


 

Additional information