Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2012 to 11 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official test guidelines and in compliance with GLP; on this basis the study is considered reliable without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
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 (as cited in study report): 2-Bromo-3,3,3-trifluoropropene(stabilized)
- Physical state: Liquid
- Analytical purity: Greater than 99%, Excluding stabilisers; >99.6% including stabilisers
- Lot/batch No.: WNCC-BTP111128
- Reassay date: 12 January 2013
- Storage condition of test material: Ambient temperature

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:47 to 54 days
- Weight at study initiation: in the range of 216 to 263 g for males and 160 to 224 g for females.
- Housing: housed five of one sex per cage made of a polycarbonate body with a stainless steel mesh lid.
- Diet:free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 to 23°C
- Humidity (%):40 to 70%
- Photoperiod (hrs dark / hrs light): 12h light/12 h dark

IN-LIFE DATES: From: 10 July 2012 to 11 January 2013

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system consisted of a Glass vaporiser and a whole body exposure chamber a 0.75 m3 chamber of stainless steel and glass construction
- Method of holding animals in test chamber: Wire mesh modular caging
- Source and rate of air: From in-house compressed air system – breathing quality
- System of generating particulates/aerosols:Aerosol Generator: Sintered glass vaporiser for Groups 2 to 4 all in a water bath set at 40°C; Syringe driver, polypropylene syringe for Group 2; Liquid reservoir and feed rate used to alter concentration Groups 3 and 4; Test groups - 30 L/min
- Temperature, humidity in air chamber: within expected parameters for this type of exposure system.
- Air flow rate: Group 1 - 149 L/min, Group 2 to 4 - 119 L/min





Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test atmosphere sample (chamber) directly analysed using the IR spectrophotometer

Vapour samples collected as follows:
Sample frequency – minimum of 3 samples/group/exposure
Sample location – representative animal level
Sample position remained constant

Sample analysis by Infra-red spectrophotometer
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day for 5 days each week (Monday to Friday)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 199, 505, 2876 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a previous 2-week dose range finding study (Huntingdon Life Sciences Study Number: WAG0024), 3 groups of rats were exposed to 2-bromo-3,3,3-trifluoropropene for 6 hours per day, 5 days per week at achieved aerosol concentrations of 533, 1167 and 2980 ppm.
There were no unscheduled deaths at doses up to 2980 ppm. In-life findings, consisting of transient clinical signs, evident during and after exposure for all treated animals, and reduced bodyweight gain and food consumption, seen for all treated males, were not considered adverse.
Histopathological changes related to treatment were seen mainly in the nasal turbinates of rats exposed to 1167 or 2980 ppm and were consistent with a localised toxic effect of the test material. No histopathological changes were evident at 533 ppm and this was considered to be the no observed adverse effect level (NOAEL).
Although adverse histopathological findings were evident at 1167 ppm it was considered that these may not progress over 13 weeks and may be readily reversible during a 4 week off-exposure recovery period. A target high exposure level of 3000 ppm was thus selected for this study, as it was considered that exposure for 13 weeks at this level would have adverse histopathological effects on rats which may or may not be reversible.
The target intermediate exposure level of 500 ppm was selected as it was shown to have no adverse effects after 2 weeks exposure.
Since the 500 and 3000 ppm target exposure levels were used on the 2-week study, their use on the 13-week study would also enable the relationship of changes over time to be explored.
The target low exposure level of 200 ppm was expected to have no adverse effects and would offer an acceptable worker safety margin.

- Post-exposure recovery period in satellite groups:
Ten male and ten female rats were assigned to each of the Control and high dose groups. These animals were treated for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS : Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment and recovery periods, and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment and during week 13
- Dose groups that were examined: Groups 1 (Control) and 4 (2876 ppm)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 of treatment and week 4 of recovery
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:during week 13 of treatment and week 4 of recovery (same animals as for peripheral haematology: further blood samples collected)
- Animals fasted: Yes

URINALYSIS: Yes
- Time schedule for collection of urine:During Week 13 of treatment, overnight urine samples were collected from all animals.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes (ARENA OBSERVATIONS)
- Time schedule for examinations: weekly


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals killed during the study, animal killed after 13 weeks of treatment or 4 weeks of recovery were subjected to a detailed necroscopy

ORGAN WEIGHTS: Yes

HISTOPATHOLOGY: Yes
All tissues preserved for examination were examined for all animals of Groups 1 (Control) and 4 (2876 ppm) sacrificed on completion of the scheduled treatment period and for all animals killed or dying during the study.
Nasal turbinates, larynx, lungs, trachea (including bifurcation), heart, liver, pancreas, spleen, thymus, teeth and abnormalities preserved for examination were examined for all animals of Group 2 (199 ppm), Group 3 (505 ppm) and all recovery animals sacrificed on completion of the scheduled treatment period.
Tissues reported at macroscopic examination as being grossly abnormal were examined for Main and Recovery animals

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Treatment-related, but transient, clinical signs, related to inhalation of an irritant material (shallow breathing, piloerection, grinding teeth and hunched posture) or possible CNS effects (unresponsiveness to external stimuli, underactivity, and partially closed eyelids), were evident during and after exposure at all exposure levels. Reductions in grip strength and motor activity were evident for animals exposed to 505 or 2876 ppm and were only partially reversible for animals previously exposed to 2876 ppm after a 4 week recovery period.

BODY WEIGHT AND WEIGHT GAIN AND FOOD CONSUMPTION
Lower bodyweight gain was evident, over the 13 week treatment period, for treated males and females exposed to 505 ppm or higher, in a dosage related manner. Total food consumption was similarly lower for treated males and females exposed to 2876 ppm.
Effects on bodyweight and food consumption showed evidence of recovery for animals
previously exposed to 2876 ppm after a 4 week recovery period.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examination revealed no treatment-related effects.

HAEMATOLOGY
Week 13 group mean total white blood cell counts were lower than control for males and females exposed to 2876 ppm (0.78 and 0.68X control respectively), statistically significantly so for females. This was mainly as a result of statistically significantly lower lymphocytes counts (0.68 and 0.66X control for males and females exposed to 2876 ppm, respectively). During Week 4 of recovery, differences from control for total white blood cell and lymphocyte counts remained similar to Week 13 values for both sexes previously exposed to 2876 ppm.
Eosinophil counts were statistically significantly lower than control for all treated animals, with evidence of a dose-related response (0.26 to 0.47X and 0.27 to 0.64X control for males and females exposed to 199 and 2876 ppm respectively). However, during Week 4 of recovery eosinophil counts showed evidence of some recovery to control values.
Monocyte counts were statistically significantly higher than control for males and females exposed to 505 and 2876 ppm (1.74 and 1.84X control for males, and 2.16 and 1.68X control for females respectively). However, during Week 4 of recovery monocyte counts were lower than control values for both sexes previously exposed to 2876 ppm.

CLINICAL CHEMISTRY
Week 13 mean alkaline phosphatase (ALP) values for males and females exposed to 2876 ppm were statistically significantly higher (1.32 and 1.43x control, respectively). During Week 4 of recovery, values for males previously exposed to 2876 ppm were unchanged. However, for females of the same group, values had lowered and were similar to Week 13 control values.
Group mean values for aspartate amino transferase (AST) were statistically significantly higher than control for treated males (2.31, 3.00 and 2.88X with ascending dose), and females exposed to 505 ppm (2.72X control). There was considerable intragroup variation for this parameter. However, the group mean changes seen were outside the range of available background data. During Week 4 of recovery, values for both sexes previously exposed to 2876 ppm were similar to controls.
Group mean values for urea were statistically significantly higher than control for treated females (1.18, 1.24 and 1.54X control with ascending dose), and males exposed to 505 and 2876 ppm (1.25 and 1.64X control, respectively).Group mean phosphate values were statistically significantly higher for treated females (1.42, 1.75 and 2.38X control, with ascending dose) and males exposed to 2876 ppm (1.30X control). During Week 4 of recovery, urea values for both sexes previously exposed to 2876 ppm had returned to control values.

URINALYSIS: no treatment-related effects

NEUROBEHAVIOUR
Sensory reactivity observations for males and females were unaffected.
During Week 13 of treatment grip strength values were statistically significantly reduced for animals exposed to 2876 ppm (forelimbs and hindlimbs) and in males exposed to 505 ppm (hindlimbs only). Grip strength for animals exposed to 199 ppm and for females exposed to 505 ppm was unaffected. Forelimb grip strength values for animals previously exposed to 2876 ppm were similar to those of controls during Week 4 of recovery but hindlimb values were still low, although to a lesser extent than during Week 13 of treatment.
High beam and low beam motor activity scores (rearing and cage floor activity respectively) were consistently reduced in animals exposed to 2876 ppm throughout most of the 1-hour recording period during Week 13 of treatment. High beam scores for animals exposed to 505 ppm were also reduced, as, to a lesser extent, were low beam scores for females exposed to 505 ppm, with a dose-relationship being evident. Motor activity for animals exposed to 199 ppm was unaffected. Low beam scores for females previously exposed to 2876 ppm were again low compared with control scores during Week 4 of recovery but the differences were less marked than during Week 13 of treatment. High and low beam scores for males previously exposed to 2876 ppm were lower than control at a few of the 6-minute intervals (p<0.05 or 0.01) but the total activity scores over the 1-hour recording period did not attain statistical significance.

ORGAN WEIGHTS
After 13 weeks of treatment, thymus weights were statistically significantly lower for males and females exposed to 2876 ppm (0.63 and 0.54X control, respectively). However, after 4 weeks recovery, thymus weights appeared to have fully recovered to control values.
Lung and bronchi weights were slightly elevated for males exposed to 505 or 2876 ppm (1.07 and 1.12X control respectively), with similar values after 4 weeks of treatment for males previously exposed to 2876 ppm.
Salivary gland weights were statistically significantly lower than control values for all treated females (0.81, 0.82 and 0.84X control, with ascending dose). For males, a reduction in salivary gland weights was also evident (between 0.88 and 0.89X control for all treated males), however, these did not achieve statistical significance. However, after 4 weeks recovery these have recovered to control values.

GROSS PATHOLOGY
Teeth pallor was noted in all animals exposed to 2876 ppm, the majority of animals exposed to 505 ppm and a few animals exposed to 199 ppm. Thickening was seen in the majority of females and one male exposed to 2876 ppm.
Spleen capsular thickening was observed in the majority of males and some females exposed to 2876 ppm and a few animals exposed to 505 ppm. Adhesions were noted in some females exposed to 505 or 2876 ppm.
The macroscopic examination performed after 4 weeks of recovery revealed the following changes in teeth and spleen. Teeth pallor was noted in all animals exposed to 2876 ppm. Thickening was seen in occasional females and one male previously exposed to 2876 ppm.
Spleen capsular thickening was observed in one male previously exposed to 2876 ppm. Adhesions were noted in some males and one female previously exposed to 2876 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes related to treatment were seen in the liver (centrilobular hepatocyte hypertrophy), pancreas (acinar cell degranulation), heart (chronic inflammation), spleen (capsular inflammation and/or thickening and adhesion), thymus (involution/atrophy), nasal turbinates (findings related to minor local irritants), larynx (ventral squamous metaplasia) and teeth (pulp cavity necrosis). The majority of these findings were seen with animals at 505 and 2876 ppm. Chronic inflammation in the heart and acinar cell degranulation in the pancreas were evident for animals exposed to 199 ppm, however, these changes are not considered to be adverse. The slight and moderate degrees of chronic inflammation in the heart observed at 505 and 2876 ppm are considered adverse.
Following the 4 week recovery period, there was complete recovery of test material related histopathological changes seen in the liver, pancreas, heart, thymus, larynx and teeth and partial recovery for the findings see in the spleen (capsular inflammation and/or thickening) and nasal turbinates (atrophy/disorganisation/vacuolation of the olfactory epithelium and nasolacrimal duct inflammation).

Effect levels

Dose descriptor:
NOAEC
Effect level:
199 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In the heart, chronic inflammation was seen in males exposed to 199 ppm and both sexes exposed to 505 or 2876 ppm. The heart tissue samples from all males (main study and recovery) were independently peer reviewed by an external consultant pathologist and the findings agreed between the consultant and the study pathologist. The minimal degree observed at 199 ppm is considered unlikely to be associated with any alteration of heart function and is considered not adverse. The slight and moderate degrees observed at 505 and 2876 ppm are considered adverse. This change may be related to the elevated alkaline phosphatase and aspartate amino transferase levels measured during Week 13 of exposures, however the toxicological importance of this is unclear. Complete recovery of the chronic inflammation was seen following the 4 week recovery period.

Applicant's summary and conclusion

Conclusions:
The test article, 2-bromo-3,3,3-trifluoropropene, was administered to 3 groups of rats by inhalation administration, using a whole-body exposure system, for 6 hours a day, 5 days a week, for 13 week at achieved aerosol concentrations of 199, 505 and 2876 ppm. Based on adverse chronic inflammation in the heart, transient clinical signs, lower bodyweight gain, food consumption, grip strength and motor activity and histopathological changes in the liver, pancreas, heart, spleen, thymus, nasal turbinates, larynx and teeth seen with animals exposed to 505 or 2876 ppm, the no observed adverse effect level (NOAEL) was considered to be 199 ppm.