Difluoromethane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No specific fertility studies were conducted for difluoromethane. The assessment was made based on the outcome of repeated inhalation studies for HFC-32 where no treatment-related findings were noted up to 50000 ppm v/v and read-across with1,1,1,3,3-pentafluoropropane (HFC-245fa) and 1,1,1,2-tetrafluoroethane (HFC-134a). An overall systemic and reproductive NOAEC 2000 ppm v/v based on the effects seen in F0 animals was determined in a 2-generation reproduction study performed with HFC-245fa. No effects on fertility through a multi-generation study and dominant lethal assay were observed up to 50000 ppm v/v with HFC-134a.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

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

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.

Specific details on test material used for the study:

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

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.

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.

Dose / conc.:

0 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

10 000 ppm

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.

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):

Organ weights were measured from one male and one female per litter on uterus, ovaries, heart, kidneys, liver, lungs and testes. Histopathological examinations were conducted on control and high level animals looking at heart, liver, kidneys, and lung.

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.

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 morbound 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: 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

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.

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)

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

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

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: 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

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.

Key result

Dose descriptor:

NOAEC

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

neuropathology

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

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 begining 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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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. 

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

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

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.

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.

Reference 2

Endpoint:

fertility, other

Remarks:

other: dominant lethal assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guidleline study

Qualifier:

according to guideline

Guideline:

other: Rodent dominant lethal assay, antifertility and germ cell mutation (equivalent or similar to OECD 478)

Principles of method if other than guideline:

Rodent dominant lethal study: antifertility and germ cell cell mutation assay.

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

CD-1 male mice, seven to eight weeks old, were supplied by Charles River UK Ltd, Margate, Kent and housed individually on double-sided mobile mouse racks, each having stainless steel cages of internal measurements: length 28.5 cm, width 11 cm and height 7.5 cm and wire mesh floors. The mice were sequentially numbered by ear punching and were housed in this same order.

Female CD1 mice, eight to nine weeks old, were supplied with the males while further batches of females were supplied at weekly intervals during the experiment. Before mating they were acclimatised to their new environment and were housed 10 per cage on rack similar to that described above with cages of internal measurements: length 27.5 cm. Width 25.5 cm, height 10 cm. After mating they were housed 2 per cage and identified with the number of the male with which they were housed during mating.

The environment was maintained at 21 – 25 °C with relative humidity at 50%. Alternate 12 hr light and dark cycles were controlled with a timer starting at 6 am. The animals received Alderley Park diet supplied by Oaks Ltd, Congleton, Cheshire UK and water (provided by an automatic drinker system) ad libitum except for the males during the exposure period.

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

The HFC 134a treated animals were exposed by inhalation each day for six hours per day on five consecutive days. The negative and positive controls were similarly housed in exposure chambers receiving air. During exposure animals were housed individually without access to food or water in one of twenty compartments in a box made of steel and glass with an internal capacity of approximated 3 litres. Atmospheres of HFC 134a were generated by mixing volumes of the test compounds with air and using rotameters as initial indicators of concentration. The concentrations were monitored by a gas-liquid chromatograph (GLC). The animals of the two positive control groups were maintained in a similar air flow to the negative control animals but were dosed prior to being housed in the exposure boxes. They received either five daily oral doses of ethyl methanesulphonate (EMS) in distilled water or a single intraperitoneal injection of cyclophosphamide in 0.9% saline.

Details on mating procedure:

On the day following the last dose (Monday morning), thirty of the healthiest and most fertile males in the negative control group and fifteen similar males in the HFC 134a and positive control groups were selected. Two ten week old females were place with each male and left for four consecutive nights (until Friday morning). The males were then separated from the females, while the females (identified by the males number) remained. This procedure was repeated each week with females of similar age until eight test matings had been carried out.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmospheric concentrations in the exposure chambers were monitored by gas-liquid chromatography and mean atmospheric concentrations calculated

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours /day for 5 days

Remarks:

Doses / Concentrations:
0, 900, 10200, 50700 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

20 males (15 dosed males chosen for mating)

Control animals:

yes, concurrent no treatment

Details on study design:

After an acclimatisation period of 12 days, a pre-experimental fertility test was carried out on the males. Two females were housed with each male, left for four nights, then re-housed. It was assumed that most matings would take place soon after pairing. The females were therefore not examined for vaginal plugs, but were killed (by cervical dislocation) 15 days after first introducing them to the males. Their uteri were examined for live implantations, early deaths and late deaths. The animals were graded according to fertility and extent of background dominant lethal frequency, choosing where possible those successful in fertilising both females. The selected males were randomly allocated to six experimental groups. Following the males remained housed sequentially on the racks so the groups to which they were allocated were therefore randomly distributed. The experimental outline and groups sizes are shown in Table 1, excess animals being included at this stage to allow for deaths or ill resulting from dosing.

Positive control:

5 oral doses of ethyl methane sulphonate (EMS) or a single i.p. dose of cyclophosphamide (CTX)

Parental animals: Observations and examinations:

All animals were checked daily throughout the study, the males being checked twice daily during exposure. All abnormal observations were recorded. Deaths were recorded, but the animals were not given a post-mortem examination.

Reproductive performance:

no effects observed

Dose descriptor:

NOEL

Effect level:

50 000 ppm

Sex:

male

Basis for effect level:

other: No effects on reproductive performance of male mice

Remarks on result:

other: the fertility was assessed by a Rodent dominant lethal test in which the F1 generation is not produced.

Reproductive effects observed:

not specified

Table 2: Daily atmospheric concentrations (%v/v) - standard deviations are in parenthesis

Day	HFC 134a
	1000 ppm (0.1% v/v)	10 000 ppm (1.0% v/v)	50 000 ppm (5.0% v/v)
1	0.07 (0.03)	1.10 (0.23)	5.24 (0.60)
2	0.08 (0.01)	1.05 (0.25)	5.41 (0.51)
3	0.07 (0.06)	0.98 (0.12)	4.74 (0.21)
4	0.10 (0.01)	0.96 (0.08)	5.00 (0.14)
5	0.12 (0.09)	0.99 (0.12)	4.97 (0.73)
Mean	0.09 (0.02)	1.02 (0.06)	5.07 (0.26)

Fertility (Tables 3 and 4)

a) Percentage of Females pregnant (Table 3). The only significant effect in the HFC 134a treated groups occurred in week 7 where there was a reduction in pregnancy in the low dose group. The positive control groups had significantly lower percentages in weeks 1,2,7 and 8 for EMS and weeks 1,3,4,6 and 7 for CTX.

Table 3: Percentage of mated female mice that became pregnant

		HFC 134a			EMS	CTX
Weeks	Control	1000 ppm	10 000 ppm	50 000 ppm	150 mg/kg orally	200 mg/kg i.p.
Before treatment	98.3 (60)	96.7 (30)	100 (30)	100 (30)	100 (30)	96.7 (30)
1	90.0 (60)	93.3 (30)	90.0 (30)	83.3 (30)	60.0 ** (30)	70.0* (30)
2	93.3 (60)	80.0 (30)	96.7 (30)	90.0 (30)	66.7* (30)	83.3 (30)
3	95.0 (60)	93.3 (30)	93.3 (30)	90.0 (30)	100 (30)	76.7* (30)
4	96.7 (60)	93.3 (30)	100 (30)	93.3 (30)	93.3 (30)	76.7* (30)
5	95.0 (60)	86.7 (30)	93.3 (30)	93.3 (30)	90.0 (30)	92.9 (28)
6	91.7 (60)	90.0 (30)	96.6 (29)	90.0 (30)	100 (30)	57.7*** (26)
7	100 (60)	83.3** (30)	96.7 (30)	93.3 (30)	90.0* (30)	70.0*** (20)
8	98.3 (60)	100 (26)	96.7 (30)	96.7 (30)	83.3* (30)	57.1*** (14)

The number in brackets is the total number of females mated in the experiment at a given time.

Statistically significant differences compared to control: *5%, **1%,

***0.1% level based on the ¿²test.

b) Successful mating per male (Table 4). Both the low and high dose HFC 134a . Both the low and high dose HFC 134a groups has reduced mean numbers of successful matings per male in week 7. The value for the middle dose group was slightly low than for control and the pooled group value was also significantly lower. The analysis of the two positive control groups gave the same significant values as those described for the percentages of females pregnant.

Table 4: Frequency distribution (and mean number†) of successful matings per male mouse

			HFC 134a			EMS	CTX
Weeks	Number of successful matings	Control	1000 ppm	10 000 ppm	50 000 ppm	150 mg/kg orally	200 mg/kg i.p.
Before treatment	0 1 2	0 1 29 (100%)	0 1 14 (100%)	0 0 15 (100%)	0 0 15 (100%)	0 0 15 (100%)	0 1 14 (100%)
1	0 1 2	1 4 25 (96.7%)	0 2 13 (100%)	0 3 12 (100%)	0 5 10 (100%)	1*** 10 4 (93.3%)	1* 7 7 (93.3%)
2	0 1 2	0 4 26 (100%)	2 2 11 (86.7%)	0 1 14 (100%)	0 3 12 (100%)	2** 6 7 (86.7%)	1 3 11 (93.3%)
3	0 1 2	0 3 27 (100%)	0 2 13 (100%)	0 2 13 (100%)	0 3 12 (100%)	0 0 15 (100%)	2* 3 10 (86.7%)
4	0 1 2	0 2 28 (100%)	1 0 14 (93.3%)	0 0 15 (100%)	0 2 13 (100%)	0 2 13 (100%)	2** 3 10 (86.7%)
5	0 1 2	0 3 27 (100%)	1 2 12 (93.3%)	0 2 13 (100%)	0 2 13 (100%)	0 3 12 (100%)	1 0 13 (92.9%)
6	0 1 2	0 5 25 (100%)	1 1 13 (93.3%)	0 1 13 (100%)	1 1 13 (93.3%)	0 0 15 (100%)	5** 1 7 (61.6%)
7	0 1 2	0 0 30 (100%)	1** 3 11 (93.3%)	0 1 14 (100%)	0* 2 13 (100%)	0** 3 12 (100%)	2*** 2 6 (80.0%)
8	0 1 2	0 1 29 (100%)	0 0 13 (100%)	0 1 14 (100%)	0 1 14 (100%)	0** 5 10 (100%)	3*** 0 4 57.1%

Statistically significant differences compared to control: *5%, **1%, ***0.1% level based on Student’s t-test of mean number of successful matings.

†Only males paired with two live healthy females have been included

In week 7, the significant decrease in the number of males mating with both females in the 1000 ppm HFC 134a group can be explained by two factors: unusually high control values (100% of males mated with both females) and by two males which mated with only one or neither of the two females because of health reasons. One of these males was found to have an abscess on the penis and, rectrospectively, it can be seen that from week 4 of mating (abscess first detected week 5) until week 7 when it was killed, the animal failed to mate with and of the females with which it was paired. After both animals had been killed in week 7, 100% of the males in this group mated with both females the following week. It should also be stressed that despite any statistical differences, the fertility in the HFC 134a treated groups remained high throughout the experiment, while the two positive control groups showed definite fertility effects due to treatment in the early weeks of the experiment. The significant effects in the EMS group in weeks 7 and 8 were again probably influenced by the negative control values while those for weeks 6,7 and 8 in the CTX group were probably an indirect effect caused by the delayed toxicity of CTX since several males died during this time and presumably did not mate before they died.

Implantations

Analysis of the numbers of implantations per pregnancy revealed no significant effects in the HFC 134a groups. Both positive controls EMS and CTX produced very significantly reduced mean numbers of implantations per pregnancy in weeks 1 and 2. The effect of CTX was also apparent in weeks 3 and 6.

Early Deaths

Early deaths which are the most sensitive indicator or mutagenicity in the dominant lethal test were statistically significantly increased in the group of male mice exposed to the top dose of HFC 134a in weeks 4 and 8, while in week 7 the pooled value for all three HFC 134a treated groups was significantly higher than that of the negative control. The actual increases, especially when compared to the positive control values in weeks 1 and 2 were very small. On examination of the negative control data, it was seen that at weeks 4, 7 and 8 the numbers of early deaths were particularly low. Furthermore except when the percentage of pregnant females with at least one early death was considered, the values found to be statistically significant for the HFC 134a treated groups fell within the range obtained for the negative control group. That these effects were due to low negative control values was also borne out by the statistically significant increases in the positive control groups in weeks 4,7 and 8 which have not been seen in previous studies which showed a good degree of correlation. A subsequent experiment using cyclophosphamide (CTX) did, however, show a slight increase in week 4. There was no effect on the number of implantations in the HFC 134a treated groups and it is usual for pre- and post-implantation loss to associated with a mutagenic effect, as was demonstrated by the positive controls.

Late deaths

The proportion of females with late deaths showed a significant increase in the 10 000 ppm HFC 134a group. Since this was an isolated finding which did not show a dose response it was unlikely to have been treatment related and was slightly greater than a value observed before treatment in one of the groups.

Conclusion

In conclusion 1,1,1,2-tetrafluoroethane (HFC 134a) had no effect on fertility and is unlikely to cause mutagenic effects as assessed by the mouse dominant lethal assay.

Conclusions:

HFC 134a did not affect male fertility or cause mutagenic effects through sperm.

Executive summary:

In a dominant lethal assay, CD1 male mice were exposed to up to 50000 ppm HFC 134a for 5 days. After the last exposure, each male was housed with 2 virgin females for 4 consecutive nights. Further matings with new females were conducted at weekly intervals for a total of 8 times. The study indicated that HFC 134a did not affect male fertility or cause mutagenic effects through sperm.

Reference 3

Endpoint:

fertility, other

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Equivalent to a GLP guideline study with the restriction that a 1 hour / day exposure was used

Principles of method if other than guideline:

Pre-clinical safety evaluation for use of HFA 134a as a propellant in a MDI (metered dose inhaler). In the fertility study snout only inhalation techniques using a single 1 hour daily exposure regimen were used. HFA 134a was generated from MDI to evaluate the propellant delivered through a metering system similar to that used by patients.

GLP compliance:

yes

Species:

rat

Strain:

other: AHA - a strain of rat having both Sprague-Dawley and Wistar origins suppiled from the Glaxo colony.

Sex:

male/female

Details on test animals or test system and environmental conditions:

For the fertility study, males were within the weight range 177 – 233 g at Week 0 (the first week of treatment) and females were within the weight range 168 – 232 g at Week 6.

Animals were allowed to acclimatise to the experimental exposure conditions for at least two weeks. This included a period of acclimatisation to the exposure procedures. Animals were randomly allocated to treatment groups.

Food (Biosure Laboratory Animal Diet No.1 Special Diet Services Ltd. (Biosure), Manea, Cambridgeshire, UK) and tap water were provided ad libitum except during inhalation exposure and clinical procedures. Each animal was identified by an ear and/or foot tattoo.

The temperature and humidity generally remained within the ranges 21±3°C and 51±10% respectively, throughout the fertility study. A 12 hour light/dark photoperiod was provided.

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

nose only

Vehicle:

unchanged (no vehicle)

Details on exposure:

Each animal was exposed daily for a period of 1 hour in a snout-only exposure system. Animals were placed in individual restraining tubes, which were positioned in tiers radially around a snout-only exposure chamber (ADG Developments, Hitchin, Hertfordshire, UK). The inhalation system was designed to ensure a constant stream of fresh test material of uniform distribution at all chamber levels. The chamber was operated under dynamic airflow conditions of 20/l min. An electronically timed, pneumatically operated device was used to discharge HFA 134a from pressurised MDI into the top of the chamber. Waste vapour was drawn from the base of the chamber. The different exposure concentrations of test material were achieved by varying the number and rate of discharge of the MDI, which were weighed before and after exposure to estimate consumption.

Details on mating procedure:

Animals were mated one-to-one. Within treatment groups, pairing was random, except that the mating of siblings was avoided. During the pre-mating periods four or five animals of the same sex and treatment group were housed per cage. One male was kept with one female during mating. At the end of the mating period, males were replaced with their former cage mates and females were housed individually. Animals were housed in stainless steel cages with mesh floors although during mating, gestation and littering, solid floor polycarbonate breeding cages with sawdust bedding were used.

Oestrous cycles were monitored by means of vaginal lavage with cytology by light microscopy for 14 consecutive days prior to pairing. The stage of the cycle (dioestrous, pro-oestrous or oestrous) was recorded daily. Vaginal smears were examined daily during the mating period and mating was confirmed by the observation of spermatozoa in the smear.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were withdrawn daily from each inhalation chamber to assess the atmospheric concentration of HFA 134a gas, which was determined by specific gas chromatography using a flame ionisation detector. Quantification was by an external standard method.

Duration of treatment / exposure:

10 weeks before pairing for males, 3 weeks before pairing for females, and during mating. F0 males were further exposed until week 18 of exposure and then killed. 14 of the pregnant F0 females from each group were further exposed until day 19 post coitum and terminated on day 20 post coitum for examination of their uterine contents and ovaries. The remaining F0 females were further exposed until day 20 post coitum, at which time exposures were supsended to allow parturition to occur. Exposure of these F0 females recommenced at the same concentrations on day 1 post partum until day 21 post partum, at which time the dams were terminated and the male and female F1 pups seperated.

Frequency of treatment:

1 hour / day

Details on study schedule:

The fertility study comprised four groups, each consisting of 30 males and 30 females. The groups of rats received treatment as shown in Table 1. The dosed animals formed the parental (F0) generation and were treated throughout the periods of gametogenesis (10 weeks before pairing in males and 3 weeks before pairing in females) and mating. Males continued to be treated post mating, were killed in Week 18 and examined post mortem, whilst all mated females continued to be treated until their termination.
Fourteen of the pregnant females from each group were killed on Day 20 post coitum from examination of uterine contents and ovaries. Their treatment continued on Day 19. The remaining females were allowed to litter and rear their young (F1 generation). To allow parturition, dosing of these females was discontinued after dosing on Day 20 of pregnancy and recommenced on Day 1 post partum until Day 21 post partum. The dams were then killed and the male and female pups separated.
The survival, and physical and functional development of the F1 generation was assessed. On approximately Day 21 post partum, one animal of each sex was retained from each litter (12 litters/group) and raised to maturity. The remaining pups, together with their mothers, were killed and examined post mortem. The selected F1 rats were mated at approximately 70 days of age and survival and development of the resulting F2 progeny was monitored for 21 days post partum. F1 males failing to mate were examined post mortem. One F2 animal of each sex was retained from each litter (8 litters/group) and the remaining pups together with their mothers were killed and examined post mortem. Once F2 rats had attained sexual maturity they too were killed and examined post mortem.

Remarks:

Doses / Concentrations:
0, 2595, 10080, 49308 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

30/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

The maximum exposure concentrations were limited by the need to ensure adequate oxygenation (>19%) of the atmosphere and were based on findings from maximum tolerated dose studies in which rats were exposed to HFA 134a concentrations of up to 150 000 ppm. Low exposures were small multiples of the likely human exposure from drug administration from an MDI (33ppm hr/m2 lung surface area/day) and the intermediate exposures were the approximate geometric means of the high and low exposure concentrations.

Animals were randomly allocated to treatment groups.

Parental animals: Observations and examinations:

Animals were inspected daily throughout the studies for any signs of ill health or reaction to treatment. Rats of each generation were weighed regularly through the study.
The quantity of food consumed by each cage of F0 rats was recorded weekly before and after mating. Group mean food consumption g/animal/week) was calculated.
Blood samples for analysis of HFA 134a concentration were collected by tail venepuncture into gas-tight vials during the last 15 minutes of exposure and 15 minutes after the completion of each exposure. Three F0 animals of each sex and treatment group were sampled during Week 15 (relative to the start of treatment) of the fertility study. The concentration of HFA 134a in whole blood samples was determined by gas chromatograph headspace analysis with flame ionisation detection.

Oestrous cyclicity (parental animals):

Oestrous cycles were monitored by means of vaginal lavage with cytology by light microscopy for 14 consecutive days prior to pairing. The stage of the cycle (dioestrous, pro-oestrous or oestrous) was recorded daily.

Sperm parameters (parental animals):

Prostate, seminal vesicles, testes, epididymides, and pituitary were weighed and preserved only if the male in question had apparently failed to mate.

Litter observations:

Physical development of the F1 and F2 generations was assessed by noting the time at which certain events occurred. These events were pinna detachment, upper incisor eruption, eye opening, cleavage of the balanopreputial skinfold in males and vaginal opening (females). Similarly, the age at which certain reflexes (surface righting, startling response, air righting, and pupillary light reflexes) were attained in surviving offspring were recorded. In addition the eyes of all F1 offspring were examined by indirect ophthalmoscopy.
At 4 weeks of age, the locomotor co-ordination of offspring was tested using an accelerating rotarod. The time taken for each pup to lose balance was recorded. Spontaneous exploratory activity in an unfamiliar environment was measured at 5 weeks of age using the Actimat Doppler shift radar monitor. Learning, memory and reverse learning (relearning) were assessed between 7 to 9 weeks of age using the Biel maze.

Postmortem examinations (parental animals):

Animals were killed by carbon dioxide asphyxiation. Routine post mortem examinations were carried out on all animals killed or found dead. They consisted of general macroscopic examination and preservation of samples of any apparently abnormal tissues.
In addition to routine post mortem examination, the uterus and ovaries of females killed on Day 20 of pregnancy (F0 and F1 generations) were removed entire, and the number of corpora lutea in each ovary was recorded. Upon removal of each uterus, the number of implantation sites, the positions of any foetuses, and of any apparent early or late intra-uterine deaths were recorded.
Uteri of apparently non-pregnant females were examined for evidence of implantation using a modified Salewski technique. Prostate, seminal vesicles, testes, epididymides, and pituitary were weighed and preserved only if the male in question had apparently failed to mate.

Postmortem examinations (offspring):

All live foetuses were weighed, examined externally, and the sex and any abnormalities recorded. For the fertility study, half of the pups in each litter were fixed in 85% industrial methylated spirit for subsequent macroscopic examination and evisceration. They were later stained with Alizarin red S for skeletal examination (modified Dawson technique). The remaining pups of each litter were fixed in Bouins fluid for sectioning and visceral examination.

Statistics:

The group mean food consumption and body weight change of the adults and the litters were calculated for each day/occasion they were weighed. The change in group mean body weight of pregnant F0 females for each day of weighing was calculated with respect to the first day of treatment. Values for the F0 females during lactation, F0 and F1 females during pregnancy, were calculated with respect to the initial body weight recorded at the start of each relevant period.
Depending on the heterogeneity of variance between treatment groups, parametric (analysis of variance followed by Williams’ or t tests) or non-parametric (Kruskal-Wallis test) tests were used to analyse data.
Mean litter data and foetal abnormalities were analysed by the Kruskal-Wallis test. Intergroup comparisons were made by the non-parametric equivalent of the t test together with the Jonckheere test for an ordered series of treatments. Where 75% or more of the values for a given variable were the same, a Fischer’s exact test was used, with the Mantel-Haenszel test applied for analysing trend.
The rotarod data were analysed using an analysis of variance and the Actimat and Biel maze data by the Kruskal-Wallis test. Intergroup comparisons were made using the Williams test or the non-parametric equivalent, Shirley’s test.
Statistical analysis is declared at the 5% level and refers to differences between mean values for Group 1 and Groups 2,3 and 4.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Concentrations
Exposure concentrations were close to target values (Table 1)

Pharmacokinetics
HFA 134a was absorbed into the systemic circulation. Concentrations increased with increasing dose. Elimination was rapid and there was no apparent accumulation on repeat dosing (Table 2). The mean half-life of HFA 134a was 5.8 and 7 minutes for the study.

Clinical observation
Mortality: no mortality was related to treatment.

One F0 male dosed at 50 000 ppm was killed for human reasons in Week 5 due to noisy respiration and red staining around the nose and eyes. There were no findings at autopsy to account for its condition.

Clinical signs: there were no clinical effects of treatment observed.

Food consumption: There was no effect of treatment on food consumption.

Body weights
There was a slight but statistically significant reduction in body weight gain of F0 males dosed at 10 000 and 50 000 ppm after 2 weeks of exposure. Cumulative (Weeks 0 – 5) and overall (Weeks 0 – 10) weight gains of males were reduced also at 50 000 ppm. However, there were no effects on weight gain in F0 females.
Amongst F1 rats, there were no significant effects upon the mean body weights of pups, nor on the growth rate of litters, when compared with the control group. This continued to be the case with all retained F1 animals during pre-mating and pregnancy. Towards the end of lactation, F2 litters in the 50 000 ppm group showed a slight decrease in weight to those from control animals which was slight and considered not to be attributable to treatment of the F0 generation. Weight gains of the F2 animals were comparable between the groups.

Breeding performance
There was no evidence of any effect on oestrous cycles, mating, pre-coital times, conception or on the length of gestation in the F0 and F1 generations (Tables 3 and 5).

Post mortem observations
No abnormalities attributable to HFA 134a were detected in F0 and F1 males, in F0 and F1 Females at termination of pregnancy or at the end of lactation

Findings in F0 females.

The number of corpora lutea. Implants, embryonic deaths, live you, sex ratio, litter weights and F1 foetal body weights did not differ significantly between treated and control groups.

Dose descriptor:

NOEL

Effect level:

>= 50 000 ppm

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Gross pathological findings:

no effects observed

Post natal observations

Litter data of F0 dams rearing young. The numbers of live born pups, their sex ratio and their survival post partum were unaffected by treatment (Table 4). There were two litter losses, one due to total embryonic death (2500 ppm), the other to unsuccessful weaning (10 000 ppm). Neither were attributed to treatment.

Post natal development of the F1 generation. There was no evidence of a treatment related effect on the mean day of eruption of upper incisors nor in the appearance of surface, air righting and pupillary reflexes (Table 6). Vaginal opening and cleavage of the balanopreputial skinfold were likewise unaffected, and differences between animals in the treated groups and controls in the rotarod, activity and learning tests were considered to be on no importance. There were no treatment related ocular effects in F1 animals. There were no effect of treatment on the mean day on which pinnae detachment, eye opening and the startle response occurred (Table 6).

Litter data of F1 dams rearing young. The numbers of live born pups, their sex and their survival post partum were unaffected by treatment (Table 5). Physical and functional development of the F2 generation was considered to be unaltered as, despite the occasional statistically significant difference in certain developmental markers (pinna detachment, startle response, and air righting reflex), non of the group means exceeded one day’s difference from control values and there was no dose relationship (Table 7).

No abnormalities attributable to HFA 134a were detected in the sexually mature F2 animals. In addition, macroscopic examination of F1 and F2 generation rats at weaning did not reveal any abnormalities of significance.

There were no effects of treatment upon the incidence, type and distribution of visceral or skeletal abnormalities in foetuses from the F0 generation females killed on Day 20 post coitum (Table 8).

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 50 000 ppm

Sex:

male/female

Dose descriptor:

NOEL

Generation:

F2

Effect level:

>= 50 000 ppm

Sex:

male/female

Reproductive effects observed:

no

Table 1. Treatment of the dose groups

Group	Target Conc. (ppm)	Fertility Study Measured mean ppm (SD)
1.Control	0
2.Low	2500	2595 (170)
3.Intermediate	10000	10080 (452)
4.High	50000	49308 (3281)

Table 2. Mean (standard deviation) blood HFA 134a concentration (Cmax ug/ml)

Group	During exposure	After exposure
2.Low	2.9 (0.5)	<1
3.Intermediate	11.3 (3.6)	2.7 (1.2)
4.High	68.2 (14.6)	5.2 (2.1)

Table 3. Breeding performance of F0 females.

	Group 1	Group 2	Group 3	Group 4
Females paired	30	30	30	30
Females mated	30	30	30	30
Females pregnant	30	30*	30**	30
Median precoital time (days)***	3.0	2.0	2.0	3.0
Females examined	14	14	14	14
Corpora lutea	15.6	16.6	15.8	16.2
Implantations	14.2	15.5	15.4	14.4
Intra uterine deaths	1.0	0.6	1.1	1.4
Live young/litter	13.2	14.9	14.3	13.1
Litter weight (g)	45.22	50.30	47.91	44.18
Foetal weight (g)	3.44	3.38	3.36	3.37

* one dam with total embryonic death; ** one dam with total litter loss post partum; *** day by which half the number of pair females had mated.

Table 4. Group mean findings from F0 females allowed to litter and rear their offspring

	Group 1	Group 2	Group 3	Group 4
Females examined	16	16	16	16
Females pregnant	16	15*	16**	16
Gestation length (days)	21.8	21.9	22.0	21.8
Implantation sites	14.9	14.3	14.6***	14.8
Number in litter	13.8	13.4	14.0	13.3
% males/litter at birth	56.2	54.1	51.4	51.1
% males/litter on Day 21 post partum	55.7	54.5	51.6	52.1
Live young/litter at birth	13.8	13.4	13.7	13.1
Live young/litter Day 21 post partum	13.6	13.1	13.5	12.8
Litter weight at birth (g)	77.8	78.3	79.3	74.8
Litter weight Day 21 post partum (g)	537.9	527.9	526.3	496.1
Pup body weight at birth (g)	5.7	5.9	5.8	5.8
Pup body weight Day 21 post part (g)	40.0	41.0	40.2	40.2

* one dam with total embryonic death; ** one dam with total litter loss post partum; *** excludes one litter where implantation sites were not recorded.

Table 5. Breeding performance of F1 generation

	Group 1	Group 2	Group 3	Group 4
Females paired	12	12	12	12
Females mated	12	12	12	12
Females pregnant	12	11	12*	12
Median pre-coital time (days)	3.0	3.0	2.0	3.0
Mean gestation length (days)	21.6	21.8	22.1	21.6
Females weaning young	12	11	11	12
Implantation sites	15.8	15.0	15.9	15.8
Number in litter	14.5	14.4	15.5	15.4
% Males/litter at birth	48.6	44.6	51.9	53.4
% Males/litter Day 21 post partum	48.4	44.4	51.2	53.5
Live young/litter at birth	14.5	14.4	15.0	15.3
Live young/litter Day 21 post partum	14.3	14.4	14.9	15.3
Litter weight (g) at birth	86.2	87.3	90.5	91.1
Litter weight (g) Day 21 post partum	580.2	580.1	581.9	558.8
Pup body weight (g) at birth	5.9	6.1	6.1	6.0
Pup body weight (g) Day 21 post part	40.7	40.7	40.4	37.1**

* one dam with total litter loss post partum; **statistically significant decrease from control (P<0.05)

Table 6. Development of the F1 generation

	Mean age (day post-coitum) of appearance
	Group 1	Group 2	Group 3	Group 4
Number of animals	12	12	12	12
Pinna detachment	24.9	25.1	24.9	24.8
Upper incisor eruption	30.3	30.0	30.0	30.1
Eye opening	37.0	37.0	37.1	36.8
Reflex development:
Surface righting	24.2	24.6	24.7	24.6
Startle response	34.7	34.6	34.3	34.3
Air righting	37.8	37.5	37.5	37.5
Pupil reflex (% success on Day 20 post partum)	100	100	100	100
Cleavage of balanopreputial skinfold	43.9	44.7	43.5	44.5
Vaginal opening	32.3	33.4	33.3	33.6

Table 7. Development of the F2 generation

	Mean age (day post-coitum) of appearance
	Group 1	Group 2	Group 3	Group 4
Number of animals	8	8	8	8
Pinna detachment	23.7	23.8	24.4**	23.7
Upper incisor eruption	30.2	30.4	30.7	30.3
Eye opening	36.6	36.7	37.0	36.7
Reflex development:
Surface righting	23.5	23.5	24.0	23.4
Startle response	33.8	33.9	34.3**	34.3*
Air righting	36.6	37.2*	37.6*	37.1
Pupil reflex (% success on Day 20 post partum)	100	99	100	100
Cleavage of balanopreputial skinfold	44.6	45.0	43.8	45.4
Vaginal opening	31.6	32.5	32.6	32.4

* statistically significant increases from control group (P<0.05); **(P<0.01)

Table 8 Abnormalities in foetuses from F0 generation killed on Day 20 post coitum.

	Group 1	Group 2	Group 3	Group 4
Major abnormalities
Foetuses examined	185	209	200	183
Foetuses with major defects	1	2	0	0
Anury	1
Retroesophageal aortic arch		1
Cranial meningocoele		1
Minor abnormalities*
Foetuses examined for visceral defects	91	102	103	93
Foetuses with visceral defects	12	9	15	9
Absent innominate artery	2	1
Variation in origin of arteries from aortic arch			1
Abnormal lobulation of the liver	1	3	2	6
Dilatation of renal pelvis +/- ureter	10	3	6	3
Displacement of one testis		2	4
Reduction in size of thyroid			1
Small intraventricular septal defect			1
Minimal dilatation of orbital sinus				1
Foetuses examined for skeletal defects	93	105	97	90
Foetuses with skeletal defects	7	9	13	11
One/two bipartite thoracic centra	2	3	7	4
One/two butterfly-shaped thoracic centra	4	3	2	3
Butterfly-shaped lumbar centrum			1
Shortened rib	1
Right cervical rib		1
Extra thoraco-lumbar vertebrae		2
Asymmetrical alignment of costal cartilage elements		1	1
Reduced ossification of cranium				1
Reduced ossific’n of vertebral arches		1		1
Reduced ossification of rib				1
Unossified 5^thmetatarsals		1	3	1

Conclusions:

There were no treatment-related effects on fertility, reproductive performance of rats treated with 1,1,1,2-tetrafluoroethane (HFA 134a) or on the development, maturation or reproductive performance of up to two sucessive generations. HFA 134a is judged not to be toxic to reproduction (fertility).

Executive summary:

1,1,1,2 -tetrafluoroethane (HFA 134a) was administered to AHA rats by snout only inhalation to assess the effects on reproduction and development. In the fertility study (detailed above), rats were exposed to atmospheres of 2500, 10 000 or 50 000 ppm HFA 134a throughout gametogenesis, mating, pregnancy and lactation.

The only treatment related effect was a slight reduction in body weight gain of the treated parental generation at 50 000 ppm.

There were no adverse effects of HFA 134a on the fertility and reproductive performance of treated animals or on the development, maturation or reproductive performance of up to two successive generations.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

208 000 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good. GLP reliables studies allowing to assess the potential effects of the substance on reproduction.

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Additional information

No specific fertility studies were conducted for difluoromethane. In two repeated-dose inhalation studies, rats were exposed until 50,000 ppm (105000 mg/m3) HFC-32 for 28 or 90 days. In both studies, several reproductive organs of males (testes, epididymis and prostate) and females (uterus, ovaries and vagina) were evaluated both macro- and microscopically. No treatment-related findings were observed at any of the administered doses.

In a dominant lethal assay carried out with the structural analogue HFC-134a, CD1 male mice were exposed up to 50000 ppm (208000 mg/m3) HFC 134a for 5 days. After the last exposure, each male were housed with 2 virgin females for 4 consecutive nights. Futher matings with new females were conducted at weekly intervals for a total of 8 times. The study indicates that HFC 134a did not affect the male fertility or cause mutagenic effects through sperm.

In addition, no effects on fertility were observed in a 2-generation study carried out with HFC-134a (maximal concentration tested: 50000 ppm or 208000 mg/m3).

 

In a 2-generation, inhalation, reproduction study conducted in Sprague-Dawley rats at exposure levels of 0, 2,000, 10,000 and 50,000 ppm with another structural analogue HFC-245fa, the systemic and reproductive 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 only. 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.

Effects on developmental toxicity

Description of key information

In three developmental toxicity studies, difluoromethane did not induce teratogenic or significant embryo-foetal toxic effects in rats and rabbits exposed up to 50000 ppm (105000 mg/m3) during gestation days 7 to 16 and 6 to 18 respectively. Furthermore, no obvious maternotoxic effects were observed. Thus, the NOAEC for developmental and maternal toxicity were higher than 50000 ppm(105000 mg/m³).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Interfauna UK (Huntingdon, Cambridgeshire, UK)
- Age: 16-24 weeks 
- Weight at study initiation: 3030-4020 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 50 +/- 15
- Photoperiod (hrs dark / hrs light): 14 / 10

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Atmosphere generation: The pressurised gas passed from the cylinder  supplied through a manifold to 3 feedlines each one connected to an  exposure chamber. The rate of gas flow to each chamber was controlled by  an in-line needle valve and monitored by an in-line flow tube (Meterate  Glass Precision Engineering Ltd). The gas entered the base of an  aluminium and glass elutriation column where it was mixed with diluent  air. Different chamber concentrations of HFC 32 were achieved by varying  the rate at which the gas was introduced into the diluent air stream. The  total gas/air mixture flow to each chamber was maintained at 200 l/minute.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with flame ionisation detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of chamber air were withdrawn at  approximately hourly intervals during exposure. Samples were withdrawn  into gas tight syringes and injected into a gas chromatograph via a gas  sample loop (Pye Unicam Series 204 chromatograph, inluding a 1m x 3mm  Porapak Q 80-100 mesh column and coupled to a flame ionisation.

Details on mating procedure:

Females were mated with males of proven fertility; once cairns had been  observed, each female was allowed to remain with the male for at least  one hour. On successfully completing coitus, each doe had been injected  intravenously with 25 IU of Chorulon (luteinizing hormone) to ensure that  ovulation had taken place. The day of mating was considered as Day 0 of  pregnancy.

Duration of treatment / exposure:

from gestation days 6 to 18

Frequency of treatment:

6 hours/day

Duration of test:

29 days post coitum

No. of animals per sex per dose:

24 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

The study was conducted in two phases because of the constraints  of the exposure chambers in relation to the number of animals used. Both  phases consisted of 48 animaIs supplied in three consecutive batches: the  first batch of animals for the second phase of the study was delivered 4  days after termination of the first phase.
Statistical analyses of bodyweight and litter data showed that there were no interaction between exposure and phase allowing presentation of the combined data from the two phases.

Maternal examinations:

PARAMETERS ASSESSED DURING STUDY:
- Clinical observations: at least once daily
- Maternal mortality: at least once daily
- Maternal body weight: on gestation days 0, 2, 6, 8, 10, 14, 19, 23 and  29
- Food consumption: recorded on gestation days 0, 2, 6, 8, 10, 14, 19, 23  and 29

Ovaries and uterine content:

On day 29 of pregnancy, the females were killed by cervical dislocation and the fetuses removed by Caesarean section. Uteri and their content were examined: number of corpora lutea; number and position of  implantation sites, classified as live fetuses, early intra-uterine  deaths (presence of decidual or placental tissue only) or late  intra-uterine deaths (presence of embryonic/fetal tissue plus placental  tissue).

Calculated parameters:
Pre-implantation loss (%) = [(number of corpora lutea - number of  implantations) / number of corpora lutea] x 100 . 
Post-implantation loss (%) = [(number of implantation sites - number of  live fetuses) / number of implantation sites] x 100.

Fetal examinations:

Number of live and dead fetuses, weight of  individual fetuses, sex ratio, external examination, head examination, skeletal examination (modified Dawson technique).
Morphological abnormalities were classified as malformations (rare and/or probably lethal), anomalies (minor differences from "normal" but  relatively frequent) or variants (differences regularly observed in the  control group).

Statistics:

Analysis of variance followed by a William's test or Kruskal-Wallis test followed by a Shirley's test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS:
No exposure-related clinical signs were observed.

MATERNAL MORTALITY: 
There were no mortalities at 15000 and 50000 ppm.  At 5000 ppm there was one mortality post commencement of exposure and  this was considered to be unrelated to treatment. 

MATERNAL BODY WEIGHT:
At 50000 ppm, there was no obvious effect on bodyweight during the first  2 days of exposure (Days 6 to 8 of pregnancy). During Days 8 to 10,  however, there was a slight but statistically significant effect on  bodyweight; a group mean weight loss of 22 g was recorded for treated  rabbits during this period (9/20 animals showed weight loss) compared  with a mean weight gain of 28 g among controls (5/22 animals showed  weight loss). From Day 10, recovery was recorded, with weight gains being  generally comparable with controls. There was no obvious or statistically significant effect on bodyweight at  5000 and 15000 ppm.

FOOD CONSUMPTION: 
There was no obvious effect on food intake.

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER PARAMETERS:
The number of corpora lutea, implants and litter sizes from treated  groups did not significantly differ from control values. Pre- and  post-implantation loss values were comparable in all groups. The only statistically significant change was the mean for total  embryonic deaths that was lower in the high-dose group (0.7) when  compared to control (1.3) but this finding has no toxicological  significance. Fetal weight was unaffected by treatment and no significant changes were observed on sex ratio.

EXAMINATION OF FOETUSES:
- Malformations: The number of malformed foetuses/litters observed in  control, low, mid and high-dose groups were 4 (4), 6 (6), 2 (2) and 8 (5)  respectively, the differences being not statistically significant.  Although the incidence of malformed foetuses at 50000 ppm is higher than  in controls, as there is no consistent pattern to the type of structural  defects observed among foetuses in this group, this finding is considered  likely to be coincidental. This increased incidence of malformations in  the high-dose group was essentially due to 4 foetuses with microphthalmia  compared to 0 in the control group, confined in only 2 litters out of 20  and from study phase 2. Since microphthalmia was not observed among phase  1 litters and overall only 2/20 litters were affected, the higher  incidence in this group is considered to be coincidental and unrelated to  maternaI exposure. The mean percentage incidence of malformed foetuses at 5000 and 15000 ppm  was comparable with controls. No foetuses in these groups showed  microphthalmia.
- Minor anomalies: There was no obvious adverse effect of exposure on the  incidence of foetuses showing visceral anomalies or keletal anomalies.
- Skeletal variants: The percentage incidence of foetuses displaying  variant sternebrae and 13 ribs were comparable in all groups and did not  indicate any adverse effects of exposure.

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

At 50000 ppm, the maternal response to exposure was minimal and confined to a slight and transient loss of body weight during gestation days 8 to 10. There were no obvious adverse effects on fetuses. So the NOAEL for maternal toxicity and for foetus development were both considered to be equal or greater than 50000 ppm.

Executive summary:

Groups 24 mated female New-zealand rabbits were exposed to 5000, 15000 and 50000 ppm HFC-32 in the day 6-18 of pregnancy for 6 hrs/day. Animals were housed individually in metal cages and exposed whole body in chambers. HFC-32 concentrations were monitored at 1 hour interval during the exposure periods.

 All females were subjected to daily examination for clinical signs of toxicity. Body weight gain and food consumption were measured regularly during exposure. On day 29 of pregnancy the animals were killed and examined for pathological changes. Developmental and teratogenic potential of HFC-125 was assessed in the litter and fetuses by examination of the typical parameters as the number of corpora lutea, number and distribution of live young, number and distribution of embryofoetal deaths, individual and litter foetal weight and foetal abnormalities.

At 50000 ppm, the maternal response to exposure was minimal and confined to a slight and transient loss of body weight during gestation days 8 to 10. There were no obvious adverse effects on fetuses. So the NOAEL for maternal toxicity and for foetus development were both considered to be equal or greater than 50000 ppm.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alderley Park (Alpk:APfSD, Wistar-derived)

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Barriered Animal Breeding Unit (Biological Services Section,  Alderley Park, Macclesfield, Cheshire, UK)
- Age: approximately 11 weeks 
- Weight at study initiation: 200-271 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20.5
- Humidity (%): 54-71
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure generation: The test atmospheres were generated by passing  liquid HFC 32 into a copper coil placed in a Techne TE-7 water bath maintained at approximately 45°C. The resultant vapour was then passed  through a copper equilibrium coil to flowmeters via a copper distribution  plenum. From these flowmeters, the HFC 32 vapour was metered to  individual exposure chambers via copper transfer lines and then diluted  by addition of clean dried air (dried and filtered using equipment  supplied by Atlas-Copco, Sweden) into the top of each chamber at a flow  rate of 45 l/min.
- Exposure chamber: ICI-designed PERSPEX chambers, 210 l
- System of generating particulates/aerosols:
- Temperature, humidity: 18.4-19.6°C, 29-55%.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with flame ionisation detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres of HFC 32 were sampled using an  automatic air sampling system and analysed automatically using a gas chromatograph (HP5890 Series II, Hewlett Packard) equipped with a gas sampling valve (Hewlett Packard), a Porapak P-S (80-100 mesh) 1.8m x 4mm  ID stainless steel column (Waters) and flame ionisation detector. The resultant peak area attributable to HFC 32 was used to calculate the  atmospheric concentration in parts-per-million (ppm v/v). Control  atmospheres and room air were also sampled and analysed.

Details on mating procedure:

Females were mated at the supplier's facilities. Female rats were paired overnight with untreated males of the same strain. On the following morning, vaginal smears from these females were examined for the presence of sperm. The day when spermatozoa were detected was designated day 1 of gestation.

Duration of treatment / exposure:

gestation days 7-16

Frequency of treatment:

6 hours/day

Duration of test:

until day 22 of gestation

No. of animals per sex per dose:

24 time-mated female rats

Control animals:

yes, concurrent vehicle

Details on study design:

The highest concentration of 49,800 ppm is considered to be a maximum practicable exposure level to minimise secondary effects due to oxygen depletion.

Maternal examinations:

PARAMETERS ASSESSED DURING STUDY:
- Clinical observations: twice daily
- Maternal mortality: twice daily
- Maternal body weight: on gestation days 4, 7 to 16 (inclusive), 19 and  22
- Food consumption: recorded on gestation days 4, 7, 10, 13, 16, 19 and 22

Ovaries and uterine content:

NECROPSY:
- Organ weights: gravid uteri
- Examination of uterine content: on day 22 of pregnancy, the females  were killed by over-exposure to halothane and the fetuses removed by Caesarean section. Uteri and their content were examined: number of corpora lutea; number and position of implantation sites, classified as  live fetuses, early intra-uterine deaths (presence of decidual or  placental tissue only) or late intra-uterine deaths (presence of  embryonic/fetal tissue plus placental tissue).
- Calculated parameters: Pre-implantation loss (%) = [(number of corpora  lutea - number of implantations) / number of corpora lutea] x  100Post-implantation loss (%) = [(number of implantation sites - number  of live fetuses) / number of implantation sites] x 100.

Fetal examinations:

Examination of fetuses: number of live and dead fetuses, weight of  individual fetuses, sex ratio, external examination (including cleft  palate), brain examination, skeletal examination (alizarin red S).

Statistics:

Fischer's exact test or Student's t-test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS:
None of the animals showed any change in clinical condition during exposure or during the course of the study.

MATERNAL MORTALITY:
No maternal deaths occurred during the study.

MATERNAL BODY WEIGHT:
There was no evidence for an adverse effect of HFC 32 on maternal bodyweight or bodyweight gain. The bodyweights of the HFC 32 groups were marginally lower than the control group but intergroup differences showed no consistent relationship with exposure concentration.

FOOD CONSUMPTION:
There was a reduced food consumption between days 7-10 and 10-13 and to a lesser extent between days 13-16 for animals exposed to 49800 ppm HFC 32 in comparison with the control group. Food consumption values thereafter were similar to controls. There was no effect of 5000 or 15000 ppm HFC 32 on maternal food consumption.

NECROPSY:
- Macroscopic findings in dams:
None of the findings noted were considered indicative of an adverse effect due to exposure to HFC 32.

Dose descriptor:

NOAEL

Effect level:

49 800 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was a non statistically significant reduction in the mean number of live foetuses in the 49800 ppm HFC 32 group reflecting the increase in the total resorptions percentage. In this group there was a statistically significant increase in pre-implantation loss (32.8% vs 21.1% in control) and hence a reduction in the number of implantations (9.8 vs 11.3 in control). There was no significant change in the percentage of post-implantation loss. As implantation occurs prior to day 7 in that strain of rats, thus prior to the initiation of exposure to HFC 32, the occurence of total resorptions in the 49800 exposed group was considered to be not related to exposure.

There was no effect of HFC 32 on foetal weight.

- Examination of fetuses:
No major defects were observed in any dose group.
Minor defects:
In the 49800 ppm group there was a slight increase in the proportion of foetuses with very minor external/visceral defects. This increase was due to the number of foetuses with slightly dilated ureters, with mottled livers or with cysts attached to the liver. However, these findings were of low incidence, affecting a very few litters, and when considered individually were not statistically significantly different from controls. Therefore, this finding was not considered related to treatment.
The percentage of foetuses with minor skeletal defects was similar for all groups.
Variants:
The percentage of foetuses with skeletal variants was similar for all groups. There was a statistically significant increase in the percentage of foetuses with external/visceral variants that was due to an increased percentage of foetuses with kinked ureters. As usually admitted for common transient variants, this finding was not considered of any toxicological significance.

Dose descriptor:

NOAEL

Effect level:

49 800 ppm

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

49 800 ppm

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Groups 24 mated female Alpk:APfSD, Wistar-derived rats were exposed to 5000, 15000 and 50000 ppm HFC-32 in the day 7-16 of pregnancy for 6 hrs/day. Animals were housed individually in metal cages and exposed whole body in chambers. HFC-32 concentrations were monitored at 1 hour interval during the exposure periods.

 All females were subjected to daily examination for clinical signs of toxicity. Body weight gain and food consumption were measured regularly during exposure. On day 22 of pregnancy the animals were killed and examined for pathological changes. Developmental and teratogenic potential of HFC-125 was assessed in the litter and fetuses by examination of the typical parameters as the number of corpora lutea, number and distribution of live young, number and distribution of embryofoetal deaths, individual and litter foetal weight and foetal abnormalities.

No treatment-related adverse effects were recorded in dams. There was a non statistically significant reduction in the mean number of live foetuses in the 49800 ppm HFC 32 group reflecting the increase in the total resorptions percentage. However, in this group there was a statistically significant increase in pre-implantation loss (32.8% vs 21.1% in control) and hence a reduction in the number of implantations (9.8 vs 11.3 in control). There was no significant change in the percentage of post-implantation loss. As implantation occurs prior to day 7 in that strain of rats, thus prior to the initiation of exposure to HFC 32, the occurence of total resorptions in the 49800 exposed group was considered to be not related to exposure.There was no effect of HFC 32 on foetal weight.

Consequently, HFC32 did not show any teratogenic effect. There was no conclusive evidence for any adverse effect on foetus development at 49800 ppm, maximal practicable exposure level of HFC32.

Conclusions:

HFC32 did not show any teratogenic effect. There was no conclusive evidence for any adverse effect on foetus development at 49800 ppm, maximal practicable exposure level of HFC32.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Principles of method if other than guideline:

modified Chernoff-Kavlock assay (complying with TSCA Health Effects Testing Guidelines part 798.4420).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alderley Park (Alpk:APfSD, Wistar-derived)

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: ICI Pharmaceuticals (Alderley Park, Macclesfield, Cheshire, UK)
- Age: approximately 11 weeks 
- Weight at study initiation: 231-276
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 34-86
- Air changes (per hr): 20-30
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Atmosphere generation: The test atmospheres were generated by passing  liquid HFC 32 into a copper co il placed in a Techne TE-7 water bath  maintained at 45°C. The resultant vapour was then passed through a copper  equilibrium coil to flowmeters via a copper distribution plenum. From  these flowmeters, the HFC 32 vapour was metered to individual exposure  chambers via copper transfer lines and then diluted by addition of clean  dried air (dried and filtered using equipment supplied by Atlas-Copco,  Sweden) into the top of each chamber at a flow rate of 25 l/min.
- Exposure chamber: ICI-designed PERSPEX chambers, 100 l
- Temperature, humidity: 18-22 °C, 17-44%

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatograhy with flame ionisation detector

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres of HFC 32 were sampled using an  automatic air sampling system and analysed automatically using a gas chromatograph equipped with a gas sampling valve and flame ionisation detector. The resultant peak area attributable to HFC 32 was used to calculate the atmospheric concentration in parts-per-million (ppm v/v). Control atmospheres and room air were also sampled and analysed. Daily concentrations were found within 10% of the target value.

Details on mating procedure:

Each virgin female was paired overnight with an unrelated male of the  same strain. On the following morning, vaginal smears were examined and  the day when spermatozoa were detected was considered as the day 1 of gestation.

Duration of treatment / exposure:

gestation days 7-16

Frequency of treatment:

6 hours/day

Duration of test:

up to day 5 post partum

No. of animals per sex per dose:

10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

EVALUATION CRITERIA:
- Negative for foetotoxicity and teratogenicity potential: if no effects on litter size, survival or pup weight gain were observed
- Potentially teratogenic: if reduced litter size (mean < 8) or reduced pup survival (< 80%) were observed
- Potentially foetotoxic: if reduced pup weight gain (< 30%) with no reduction in pup survival were observed.

Maternal examinations:

- Clinical observations: at least once daily
- Maternal body weight: on gestation days 1, 7 to 16 and 21

Ovaries and uterine content:

not examined

Fetal examinations:

Litter data (number of litters, litter size, mortality and litter  weight): on post-partum days 1 and 5

Statistics:

Two-tailed Student's test

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- CLINICAL OBSERVATIONS:
Only one animal (in the 9930 ppm group) showed signs of urinary incontinence on days 8-10 of gestation.

- MATERNAL BODY WEIGHT:
There was no evidence for an adverse effect of HFC 32 on maternal bodyweight gain. Intergroup differences were small and not statistically significant.

Dose descriptor:

NOAEL

Effect level:

49 600 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of litters:
10 litters were obtained in each dose group.

Litter size:
The mean number of pups per litter was 12.4, 9.3 and 10.7 in control, 9930 ppm and 49600 ppm treated groups respectively. There was no dose relationship effect.

Mortality:
All pups were alive on day 1 post-partum.
However, one litter of the 9930 ppm group did not survive until day 5 post-partum. One control female and two females exposed to 49600 ppm showed an unusually high incidence of pup mortality to day 5 post-partum in comparison with the other females. These pup mortalities are reflected in the reduced values for the percentage of pup survival on day 5 post-partum: 90.3, 93.5 and 84.1 in control, 9930 ppm and 49600 ppm treated groups respectively. As none of the other litters in the 49600 ppm group was affected, the pup mortalities in this group were considered not related to HFC 32 exposure.
Mean pup weight at birth was comparable for all groups although there was a slight reduction in mean pup weight gain to day 5 in the 49600 ppm HFC 32 group.
Percentages of pup weight gain to day 5 were 40.36, 44.1 and 30.4 in control, 9930 ppm and 49600 ppm treated groups respectively.

Dose descriptor:

NOAEL

Effect level:

49 600 ppm

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

49 600 ppm

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Negative (no teratogenic and no foetotoxic potential) according to the criteria for assessing this type of study.

Executive summary:

Groups 10 mated Alderley Park female rats were exposed to 15000 and 50000 ppm HFC-32 in the day 7-16 of pregnancy for 6 hrs/day up to day 5 post partum. Animals were housed individually in metal cages and exposed whole body in chambers. HFC-32 concentrations were monitored during the exposure periods.

 All females were subjected to daily examination for clinical signs of toxicity. Body weight gain and food consumption were measured regularly during exposure. Days 1 and 5 after the delivery, the foetotoxicty and teratogenicity potential of the substance was assessed by evaluating the litter size, pup survival and body weight.

There was no evidence for an adverse effect of HFC 32 on maternal clinical signs or bodyweight gain

Compared to controls, the mean number of pups per litter was not statistically different in the treated groups. Pup mortalities occurred among control and treated-groups without dose-relationship. Under these conditions, the NOAEL for maternal toxicity and fetus development were both considered to be equal or greater than 50000 ppm.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

105 000 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Good. Several reliable GLP studies available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data on fertility and development in the available animal studies, the substance does not need to be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.