1,1,1-trifluoroethane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th June 2004 - 4th April 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Oestrous cycles were not recorded.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Principles of method if other than guideline:

The study described in this report was conducted following the reproductive study guidelines as described in U.S. EPA Health Effects Test Guidelines OPPTS 870.3800 Reproduction and Fertility Effects (August 1998) and OECD Guideline 416 Two-Generation Reproduction Toxicity Study (January 2001) and in compliance with the Principles of Good Laboratory Practice (German Chemicals Law § 19a, Appendix 1, pp. 2119 2129, BGB1. I, June 28, 2002) and German Animal Protection Law (Tierschutzgesetz) of May 25, 1998.

GLP compliance:

yes

Limit test:

no

Justification for study design:

The dose levels were based on the results of a 13-week study conducted with doses of 0, 500, 2000, 10000 and 50000 ppm of the substance. The nose-only inhalation was chosen since a whole-body inhalation exposure was incompatbile with German legislation due to the high amount of substance released.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

419-170-6

EC Name:

-

Cas Number:

460-73-1

Molecular formula:

C3H3F5

IUPAC Name:

1,1,1,3,3-pentafluoropropane

Test material form:

gas

Details on test material:

Purity : 99.8%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: 0050404064004

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Hsd:Sprague Dawley SDr

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sprague Dawley rats approximately 4 weeks old were obtained from Harlan Winkelmann. They were acclimated to the exposure tubes for 3 weeks prior to treatment. Each animal was given a unique number. They were housed individually in polycarbonate cages type III. They were fed 1314 N specially prepared pelleted chow from Altromin International, Lage, Germany, ad libitum. Fresh tap water was provided ad libitum in 300 ml polycarbonate bottles and changed weekly. The temperature in the animal room was maintained at 20-24°C and the rel. humidity 40-70%. The lighting in the animal housing room was maintained on a 12 hr light/dark cycle.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

unchanged (no vehicle)

Details on exposure:

Method of administration or exposure: Nose only administration. The exposures were conducted in four identical oro-nasal exposure chambers of cylindrical shape with four levels, each housing 16 rats. Rats were placed around the chambers in tapered acrylic glass tubes with adjustable back stops. The exposure cylinder was operated at a slightly positive pressure. The exposure atmosphere was generated by injecting a defined mass flow of HFC-245fa into a constant air flow.. The air flow rates were controlled by mass flow meters.

Details on mating procedure:

Animals were mated beginning after 10 weeks of exposure for four consecutive weeks or until successful mating as indicated by the presence of sperm or a vaginal plug. Over night mating was preformed with one male and one female from the same treatment group. The time to successful insemination (precoital time) was recorded.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The HFC-245fa concentration was measured using a flame ionization detector (FID). For the FID measurement, the test and control atmospheres were sampled every 10 min. The FID was calibrated against n-butane so drifts in the detector could easily be detected. The exposure levels of HFC-245fa were determined by comparison to the n-butane signals based on a calabration curve.

Duration of treatment / exposure:

Exposures were 4 hours per day

Frequency of treatment:

Duration of exposure per day: 4 hours
Dosing regime P0: 7 days/week (males)
Dosing regime P0: 7 days/week (females)
Dosing regime F1: 7 days/week (males)
Dosing regime F1: 7 days/week (females)

Details on study schedule:

F0 animals were exposed for 10 weeks prior to mating and during the mating period. After successful mating, females were exposed daily from day 0 to day 20 post-conception (p.c.) as well as from day 5 post partum (p.p.) to day 20 p.p. Daily exposure of animals not successfully mated was continued until sacrifice. After completion of weaning on day 21 p.p., F1 animals were trained to the tubes for 2-3 weeks. Daily exposure of the F1 animals started thereafter and lasted 10 weeks before mating and during mating. Females were exposed daily from day 0 to day 20 p.c. as well as from day 5 to day 20 p.p. Daily treatment of males and females not successfully mated was continued until their sacrifice.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Doses / concentrations 4

Dose / conc.:

50 000 ppm

No. of animals per sex per dose:

30 males and 30 females except for top dose F1 which had 27 males and 27 females.

Control animals:

yes, sham-exposed

Details on study design:

Exposure levels were based on a previous 13 week inhalation toxicity study that was conducted at levels of 0, 500, 2000, 10000 and 50000 ppm.The main finding was myocarditis in most animals in the 10000 and 50000 ppm groups. No effects were seen at the lower levels. Also in a perinatal study no effects were seen at levels up to 50000 ppm.

Positive control:

None.

Examinations

Parental animals: Observations and examinations:

Animals were observed daily. Once per week they were removed from their cages and given a detailed assessment. Body weights were recorded weekly. After successful mating, body weights of the females were determined on day 0, 4, 7, 10, 14, and 20 p.c. as well as day 0, 4, 7, 14, and 21 p.p. Food consumption was recorded weekly. After successful mating, food consumption of the females were determined on day 0, 4, 7, 10, 14, and 20 p.c. as well as day 0, 4, 7, 14, and 21 p.p.

Oestrous cyclicity (parental animals):

Since the investigaion of precoital time in the F0 animals did not indicate a substance-induced effect, oestrus stages were not determined in the F1 generation in order to prevent pseudopregnancies.

Sperm parameters (parental animals):

Sperm analysis was preformed using the right cauda epididymis collected immediately after sacrifice. The number of cauda epididymal sperm reserves was determined in a sample of the stock solution using a Makler cell counting chamber. The percent motile sperm was estimated by assessing 20 sperm using a microscope. A morphological evaluation of an epididymal sperm sample was preformed by investigating 200 sperm under the microscope.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of 8 pups/litter (4/sex/litter as close as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, locomotor activity using the ActiMot/Motitest computerised light beam system. Between day 30 and 60 learning and memory was tested using an M water maze.

GROSS EXAMINATION OF DEAD PUPS:
Necropsy was performed in all pups not selected for postweaning investigations or found dead during the study.

Postmortem examinations (parental animals):

Necropsy was preformed on all females after weaning of offspring, in animals found dead or sacrificed in moribund condition and in all males and females on successfully mated. The number of implantation sites was determined using ammonium sulfide staining were there were no visible implantations. Organ weights were determined for: uterus, ovaries, testes, epididymides, seminal vessels, prostrate, liver, kidneys, adrenal glands, spleen, heart and lungs. In females organ weights were measured only in females that had pups surviving until day 21 p.p. Histopathological examination was performed on control and 50000 ppm exposure group animals. The following tissues were examined: vagina, uterus, oviducts, cervix, ovaries, testes one epididymis, seminal vessels, prostate, coagulating glands, brain, heart kidneys, liver, lungs.

Postmortem examinations (offspring):

F1 pups: Gross pathology was performed on organs that appeared to be normal. In addition, the following organs were selected from one male and one female of each litter: heart, kidneys, liver and lungs. The following organs and tissues were investigated histopathologically from one and one female of each litter: heart, kidneys, liver and lungs.

F2 pups: Gross pathology was performed on organs that appeared to be normal. In addition, the following organs were selected from one male and one female of each litter: heart, kidneys, liver, lungs and brain. The following organs and tissues were investigated histopathologically from one and one female of each litter: heart, kidneys, liver and lungs.

Statistics:

All data were recorded by the on-line data aquisition system (Toxicology Analysis System), PROVANTIS 5.0.1, PLACES 2000.1.8 or on special sheets. Statistical evaluation was conducted at p = 0.05. Body weights and food consumption were analyzed using analysis of variance. If differences were noted, the treatment groups were compared to controls using Dunnets modification of the t-test. Kruskall-Wallis ANOVA and Mann-Whitney U-test were applied in the case of non-normal data. Organ weights were compared using the Dunnett's modification of the t-test. Qualitative data were analyzed using the two-tailed FISHER test with Bonferroni correction or Chi-square test.

Reproductive indices:

Time to mating, number of successul matings, litter size and survival were determined. Oestrus cycle was determined in F0 animals only.

Offspring viability indices:

Survival, body weight and body weight gain, sex ratios, learning and memory were measured.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality of lactating dams in the medium and high dose group was observed begining with day 10 of lactation. The following numbers of animals were found dead or had to be killed in moribound condition - expressed as mortality per number of animals with liveborn offpsring: 0/22 in the control group, 0/21 in the low dose group, 2/21 in the medium dose group and 12/25 in the high dose group. In most cases, animals were within normal limits after the last exposure, but found dead the next morning.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, there was a statistically significant decrease in body weight of the high dose group compared to the control group at the beginning of exposure (day 7 and 14) and during the first weeks of mating (day 77 to 91), accompanied by a decrease in body weight gain between days 0 and 7 as well as day 70 and 77. This was followed by an incrase in body weight gain between days 91 and 98 in the high dose group. The total weight gain between days 0 and 70 was decreased in the high dose group. In females, body weight gain was decreased between days 4 and 7 p.p. as well as during the whole period of lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

A significant increase in histopathological changes were seen in the cerebellum of 9/27 female rats in the high level group. A similar finding occurred in 1/30 female rats in the medium dose. Also 5/28 female rats in the high level group showed lesions in the cerebrum. No brain lesions were seen in male or non-pregnant female animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Myocarditis was seen in both male and female rats: 3/30 (male, air control), 3/30 (male, medium level) and 15/30 (male, high level) as well as 1/30 (female, air control), 0/30 (female, medium level) and 6/30 (female, high level). Thus the findings in the medium level were similar to the air control, while the increase in the high level could be related to the exposure to HFC-245fa. Moderate to severe congestion was reported in the liver (10/30) and kidneys (11/30) in the high level exposure female rats. As the liver and kidney findings occurred only in animals that had died during the study and were present in all animals that died except for one dying on the last day, they appear to be agonal in nature and unrelated to the test compound exposure. All other lesions were judged to be incidental.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility was significantly decreased in the high dose group with the same (statistically not significant) in the medium dose group. There was a trend towards a decrease in F0 sperm count in the medium and high dose group which, however, was not statistically significant in the single group comparisons with the control.

Reproductive performance:

no effects observed

Details on results (P0)

There was an unexpected mortality of lactating dams in the medium and high exposure level groups beginning with day 10 of lactation. In the F0 dams, the mortality pattern was 0/22 (control), 0/21 (low level), 2/21 (medium level), and 12/25 (high level). In addition, 4 of the dams in the high level were noted in poor condition. These animals generally appeared normal at the end of the day and were found dead the following morning. There was no treatment related mortality in the males or non-pregnant females. While there were a few incidents of decreases in body weight gain in the high level group, these tended to be at the beginning of the exposure phase of the study. No effect on mating or pregnancy rates were seen in the F0 generation. There were no gross pathological findings or effects on organ weights in the F0 rats, however there was a decrease in sperm motility in the high level males with a suggestion of a similar effect in the medium level. A significant increase in histopathological changes were seen in the cerebellum of 9/27 female rats in the high level group. A similar finding occurred in 1/30 female rats in the medium dose. Also 5/28 female rats in the high level group showed lesions in the cerebrum. No brain lesions were seen in male or non-pregnant female animals. Myocarditis was seen in both male and female rats: 3/30 (male, air control), 3/30 (male, medium level) and 15/30 (male, high level) as well as 1/30 (female, air control), 0/30 (female, medium level) and 6/30 (female, high level). Thus the findings in the medium level were similar to the air control, while the increase in the high level could be related to the exposure to HFC-245fa. Moderate to severe congestion was reported in the liver (10/30) and kidneys (11/30) in the high level exposure female rats. As the liver and kidney findings occurred only in animals that had died during the study and were present in all animals that died except for one dying on the last day, they appear to be agonal in nature and unrelated to the test compound exposure. All other lesions were judged to be incidental.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

neuropathology

histopathology: non-neoplastic

reproductive function (sperm measures)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

System:

cardiovascular

Organ:

brain

heart

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

A pattern of mortality similar to that seen in the F0 dams was seen in the F1 dams beginning on the 12 day of lactation, 0/18 (controls), 0/23 (low level), 2/23 (medium level) and 6/13 (high level) exposure groups. As noted above, these animals appeared normal at the end of the day and were found dead the following morning.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group there was a decreased body weight at the begining of exposure after weaning in females. This difference disappeared during exposure and a statistically significantly increased body weight gain was seen in females between days 7 and 14 of treatment as well as over the whole premating period. In males, the same trend was observed, with statistically significantly increased body weight gains in the high dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a slight, but statistically significant, decrease in food consumption in females of the high dose group at the begining of exposure (day 0-7 and 14-21).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant substance-related histopathological changes were observed in the cerebellum of 10 females of the high dose group. Very severe multifocal malacia was detected in 5 females. Moderate multifocal neuronal degeneration (in one animal with necrosis) in the cerebellar cortex accompanied by a moderate sponigiosis was diagnosed in 2 other females. In the medium dose group, 1 female with very slight focal pervascular haemorrhages in the cerebellar cortex was seen. No such findings were observed in the control animals.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was a trend towards a decrease in F1 sperm count in the high dose group, which, however, was not statistically significant. The percentage of abnormal sperm was decreased in all substance-treatment groups, but this was not considered an adverse effect.

Reproductive performance:

no effects observed

Details on results (P1)

As with the F0 generation, there was no effect on mating and pregnancy rates in the F1 generation. A similar pattern of mortality was seen in the F1 dams beginning on the 12 day of lactation, 0/18 (controls), 0/23 (low level), 2/23 (medium level) and 6/13 (high level) exposure groups. As noted above, these animals appeared normal at the end f the day and were found dead the following morning. In both male and female pups in the high level group and the female pups of the medium level group, there were depressions in many organ weights. These were judged to be a consequence of the lower terminal body weights. Histopathological examination of these pups did not show abnormalities in the lungs.  Findings in the heart and kidney were equally divided in the control and high level groups. There were no other reported findings.

Effect levels (P1)

Key result

Dose descriptor:

NOAEC

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

neuropathology

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 000 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in average litter size in the medium and high dose group in the late lactation period. Some of this decrease in the high dose group could be attributed to maternal mortality in that group and therefore not attributed to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a significant decrease in pup weight in the high dose group compared to controls beginning with day 14 p.p.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group, all organ weights were decreased compared to the control group. This reached statistical significance in males for heart and lung, and in females for ovaries, kidneys, liver, heart and lungs. This also occurred in the heart of females of the medium dose group. This decrease could be a result of the lower terminal body weight of the pups in the high dose group.

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings in the heart and kidney were equally divided in the control and high level groups. There were no other reported findings.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

In the F1 pups, clinical observations were generally unremarkable. Sexual maturation was comparable across all groups. Body weight, body weight gains and food consumption, while showing some variations, tended to be unremarkable. Evaluations of locomotor activity, learning and memory were unaffected. 

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a statistically significant increase in the number of still born pups in the low and high level groups, but not the medium level. When the incidence of stillborn pups is combined with the incidence of pups not surviving until day 4, the incidence in the control, low and medium level groups is comparable. Thus it would appear that the increase in still born pups and pups not surviving until day 4 was only associated with the high exposure level group.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the high level group, the right (but not left) testes was increased in weight in the males and the uterus and heart weight was increased in females. All other organ weights were normal.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadically occurring findings in F2 weanling gross pathology included dark red areas in the lung, spongy condition of the lung, and ovarial cysts. There were no statistically significant differences to the control group. Consequently, none of these findings were considered substance-related.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Very slight and slight chronic progressive nephropathy was diagnosed in rats of the control, medium dose and high dose group.

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

In the F2 litters, there was a statistically significant increase in the number of still borne pups in the low and high level groups, but not the medium level. When the incidence of stillborn pups is combined with the incidence of pups not surviving until day 4, the incidence in the control, low and medium level groups is comparable. Thus it would appear that the increase in still borne pups and pups not surviving until day 4 was only associated with the high exposure level group. While there was a decrease in average litter size in the medium and high level groups, this was associated with the loss of complete litters as a consequence of maternal mortality (when the dams died, the pups in that litter was sacrificed) and not a direct effect of the exposure to the test article. Clinical observations and gross pathological observations did not show evidence for a treatment related effect. In the high level group, the right (but not left) testes was increased in weight in the males and the uterus and heart weight was increased in females. All other organ weights were normal. No abnormalities were detected in the cerebrum, cerebellum or heart by histopathological examination. In the liver, extramedullary haematopoesis was seen in all groups, including the controls. This was considered normal. While seen in the controls, in the high level group there was an increase in dilated tubules and interstitial fibrosis in the kidneys of the females.

Effect levels (F2)

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

In-life clinical observations were generally unremarkable. However, there was an unexpected mortality of lactating dams in the medium and high exposure level groups beginning with day 10 of lactation. In the F0 dams, the mortality pattern was 0/22 (control), 0/21 (low level), 2/21 (medium level), and 12/25 (high level). In addition, 4 of the dams in the high level were noted in poor condition. These animals generally appeared normal at the end of the day and were found dead the following morning. There was no treatment related mortality in the males or non-pregnant females. While there were a few incidents of decreases in body weight gain in the high level group, these tended to be at the beginning of the exposure phase of the study. No effect on mating or pregnancy rates were seen in the F0 generation. There were no gross pathological findings or effects on organ weights in the F0 rats, however there was a decrease in sperm motility in the high level males with a suggestion of a similar effect in the medium level. A significant increase in histopathological changes were seen in the cerebellum of 9/27 female rats in the high level group. A similar finding occurred in 1/30 female rats in the medium dose. Also 5/28 female rats in the high level group showed lesions in the cerebrum. No brain lesions were seen in male or non-pregnant female animals. Myocarditis was seen in both male and female rats: 3/30 (male, air control), 3/30 (male, medium level) and 15/30 (male, high level) as well as 1/30 (female, air control), 0/30 (female, medium level) and 6/30 (female, high level). Thus the findings in the medium level were similar to the air control, while the increase in the high level could be related to the exposure to HFC-245fa.  Moderate to severe congestion was reported in the liver (10/30) and kidneys (11/30) in the high level exposure female rats. As the liver and kidney findings occurred only in animals that had died during the study and were present in all animals that died except for one dying on the last day, they appear to be agonal in nature and unrelated to the test compound exposure. All other lesions were judged to be incidental.

 

In the F1 pups, clinical observations were generally unremarkable. Sexual maturation was comparable across all groups. Body weight, body weight gains and food consumption, while showing some variations, tended to be unremarkable. Evaluations of locomotor activity, learning and memory were unaffected. As with the F0 generation, there was no effect on mating and pregnancy rates in the F1 generation. A similar pattern of mortality was seen in the F1 dams beginning on the 12 day of lactation, 0/18 (controls), 0/23 (low level), 2/23 (medium level) and 6/13 (high level) exposure groups. As noted above, these animals appeared normal at the end f the day and were found dead the following morning. In both male and female pups in the high level group and the female pups of the medium level group, there were depressions in many organ weights. These were judged to be a consequence of the lower terminal body weights. Histopathological examination of these pups did not show abnormalities in the lungs.  Findings in the heart and kidney were equally divided in the control and high level groups. There were no other reported findings.

 

Gross pathological examination of the adult F1 rats did not reveal any treatment related findings. There were no treatment related findings on examination of sperm. Organ weights were comparable across all groups. Clinical chemical measurements of LDH, CK, and AST were unremarkable. The CK determinations were used to look for myocardial muscle damage. There was no evidence of myocardial damage. Blood samples were also analyzed for the presence of metabolites (trifluoroacetic acid and trifluoropropanoic acid). Only minimal levels of trifluoroacetic acid were found. Trifluoropropanoic acid levels were below the limits of detection. A significant increase in histopathological changes were seen in the cerebellum of 10/27 female rats in the high level group. A similar finding occurred in 1/30 female rats in the medium dose. Also 1/27 female rats in the high level group showed lesions in the cerebrum. No brain lesions were seen in male or non-pregnant female animals. Myocardial fibrosis was seen only in the female rats: 3/30 (female, air control), 0/30 (female, medium level) and 9/27 (female, high level). This increase in the high level could be related to the exposure to HFC-245fa. All other lesions were judged to be incidental.

 

In the F2 litters, there was a statistically significant increase in the number of still borne pups in the low and high level groups, but not the medium level. When the incidence of stillborn pups is combined with the incidence of pups not surviving until day 4, the incidence in the control, low and medium level groups is comparable. Thus it would appear that the increase in still borne pups and pups not surviving until day 4 was only associated with the high exposure level group. While there was a decrease in average litter size in the medium and high level groups, this was associated with the loss of complete litters as a consequence of maternal mortality (when the dams died, the pups in that litter was sacrificed) and not a direct effect of the exposure to the test article. Clinical observations and gross pathological observations did not show evidence for a treatment related effect. In the high level group, the right (but not left) testes was increased in weight in the males and the uterus and heart weight was increased in females. All other organ weights were normal. No abnormalities were detected in the cerebrum, cerebellum or heart by histopathological examination. In the liver, extramedullary haematopoesis was seen in all groups, including the controls. This was considered normal. While seen in the controls, in the high level group there was an increase in dilated tubules and interstitial fibrosis in the kidneys of the females.

Applicant's summary and conclusion

Conclusions:

In a 2-generation, inhalation, reproduction study conducted in Sprague-Dawley rats the NOAEC was identified to be 2000 ppm based on mortality in several dams in the medium and high level exposure group of the P0 and F1 generation. There was an increase in abnormal findings in the brain and heart that appear to be related to exposure to HFC-245fa seen predominately in the high level exposure group. Brain lesions were not seen in males or non-pregnant females. There was an increase in the number of still-born pups combined with pups not surviving until Day 4 pp in the high level exposure group of the F2 generation. A decrease in sperm motility was found in the high dose males of the F0 generation.

Executive summary:

A 2-generation, inhalation, reproduction study was conducted in Sprague-Dawley rats at exposure levels of 0, 2,000, 10,000 and 50,000 ppm. Several of the dams in the high level exposure group died during the late period of lactation. There were also a few similar deaths in the medium level exposure group, but not in the controls or low level groups. There was an increase in abnormal findings in the brain and heart that appear to be related to exposure to HFC-245fa. And while seen predominately in the high level exposure group, 1 rat in the F0 and one rat in the F1 generation also had similar brain lesions. Brain lesions were not seen in males or non-pregnant females. Finally, in the F2 generation, there was an increase in the number of still-borne pups combined with pups not surviving until Day 4 p.p. in the high level exposure group. While there was a decrease in sperm motility in the high level males of the F0 generation, this effect was not seen in the F1 generation and thus may be spurious. Exposure at 2000 ppm did not appear to result in any adverse effects and while the exposure at 10,000 ppm did result in some effects, they were far less numerous than seen at 50,000 ppm, suggesting that this was close to the threshold.