2,2-dichloro-1,1,1-trifluoroethane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: High reliability because a scientifically defensible and guideline method was used. Critical study for SIDS endpoint. Only study summary reviewed from secondary source.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dichloro-1,1,1-trifluoroethane

EC Number:

206-190-3

EC Name:

2,2-dichloro-1,1,1-trifluoroethane

Cas Number:

306-83-2

Molecular formula:

C2HCl2F3

IUPAC Name:

2,2-dichloro-1,1,1-trifluoroethane

Constituent 2

Reference substance name:

Ethane,2,2-dichloro-1,1,1-trifluoro

IUPAC Name:

Ethane,2,2-dichloro-1,1,1-trifluoro

Details on test material:

- Name of test material (as cited in study report): Ethane,2,2-dichloro-1,1,1-trifluoro, HCFC-123
- Purity >= 99.8%

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)BR VAF/Plus strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats in the F0 generation were 4 weeks-old upon arrival.

Animal room controls for temperature and humidity were set at 20ºC and 55%, respectively, and lighting was controlled to give 12 hours light and 12 hours dark.

All animals were given free access to pelleted diet and tap water.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

not specified

Vehicle:

not specified

Details on exposure:

During exposure, the animals were held in stainless steel mesh cages. A cage had 10 compartments; each compartment held one rat. Cages were evenly distributed on 5 levels within a chamber and cage positions were altered on a weekly basis. Liquid HCFC-123 was forced from a reservoir (under pressure with nitrogen) through metering values into copper coils where evaporation took place. The vapor was mixed with diluent air at 25 L/min and carried to the air entry points of the inhalation chambers. Information of the environment within each inhalation chamber was collected at 6 minute intervals during each 6-hour exposure. Airflow, temperature, relative humidity, and analyzed concentration were collected.

The inhalation chambers used had a nominal internal volume of approximately 2.43 m3. During operation, filtered, temperature and humidity controlled air was supplied to the chambers by an air handling unit. The volume flow of air to the chambers was monitored and recorded.

Details on mating procedure:

During the premating period, male and females were housed separately, 4 to a cage. During the mating period, animals were housed on the basis of 1 male to 1 female. At the end of the mating period, the males were rehoused with their former cagemates and the females were housed in individual breeding cages.

From presumed day 20 of gestation through to Day 4 post-partum females remained in their breeding cages and were allowed to deliver their young and to establish lactation without exposure to the test substance. Exposures were re-introduced on day 5 postpartum. Vaginal smears were taken daily, prior to exposure, for the 7 days before, and during the 20-day mating period, to enable the number of animals that mated on specific days to bedetermined. This data was used to determine whether or not pregnancy occurred, to detect marked anomalies of the estrus cycle, to determine, where possible, day 20 of pregnancy, and to determine the mean pre-coital time and duration of pregnancy of dams that littered.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: F0 generation: from 6 weeks of age through weaning of all litters (a total of at least 23 weeks of treatment)
F1 generation: from 4 weeks of age through weaning of all their litters (a total of approximately 28 weeks of treatment)

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

Number of generation studies: 2

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

30 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

100 ppm

Doses / concentrations 4

Dose / conc.:

300 ppm

Doses / concentrations 5

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

32 male and 32 female/exposure group

Control animals:

yes

Details on study design:

Due to differences in age at commencement of treatment, F0 animals received 10 weeks of treatment prior to mating. F1 animals received 12 weeks oftreatment prior to mating.

Twenty rats/sex/group of the F0 generation were sacrificed after the weaning of all litters (a total of at least 23 weeks of treatment). The remaining 12 animals/sex/group (designated as geriatrics, F0g) were retained and sacrificed after 39 weeks of treatment. On day 4 post-partum, pups were weighed and where possible, the litter was standardized to a total litter size of 8 pups (ideally 4 male and 4 female), by random selection for each sex. No pups were culled from litters of 8 or less regardless of sex ratio. One male and 1 female pup of each litter in the group was selected by median body weight for organ weight analysis and preservation of tissues. In addition for the 0 and 1000 ppm groups, 1 weanling of each sex was also chosen by random selection. On Day 21 post-partum 1 male and 1 female pup in each of 28 litters per group were retained for further study.

Selection of pups was made using computer-generated random number tables.

Examinations

Parental animals: Observations and examinations:

During the course of the study, food consumption, water consumption, body weight, and reproductive indices (copulation index, fertility index, pregnancy rate, gestation index) were measured. The onset of vaginal opening was monitored in all selected females from 28 days post-partum. The occurrence of cleavage of the balanopreputial skinfold was monitored in all selected males from 35 days post-partum. Clinical pathology investigations occurred at 14 and 15 weeks of age and included 8 parameters for the males and females.

Oestrous cyclicity (parental animals):

Among females, fat determinations of milk were also made during the lactation phase. For the F0 geriatrics, blood samples were obtained at 44 and 45 weeks of age.

Sperm parameters (parental animals):

In addition, male analysis included follicle stimulating hormone, luteinising hormone, estradiol, progesterone, and testosterone.

Postmortem examinations (parental animals):

Organ weight analysis of the reproductive organs and pituitary were performed on adults and selected weanlings. Weight analysis of the liver was performed on F0 and F1 adults and on selected F2 weanlings. Histopathological examinations were confined to F0 and F1 adults and selected F2 weanlings. Approximately 40 organs and/or tissues were preserved for possible analysis; however, histology was restricted to reproductive tract-associated tissues, liver, and pancreas.

Postmortem examinations (offspring):

Shortly after day 21, all excess pups were sacrificed and examined externally and internally for abnormalities. Weight analysis of the liver was performed on F0 and F1 adults and on selected F2 weanlings. Histopathological examinations were confined to F0 and F1 adults and selected F2 weanlings. Approximately 40 organs and/or tissues were preserved for possible analysis; however, histology was restricted to reproductive tract-associated tissues, liver, and pancreas.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

The inhalation exposure systems produced comparable results across the groups for airflow, temperature, and relative humidity. Mean air flow ranged from 652-658 L/min across the groups. Mean chamber temperature ranged from 20.5-21.4ºC and mean relative humidity ranged from 49-53% across the test groups. The chamber mean analyzed concentrations for the F0 generation were 0, 30, 100, 298, and 1008 ppm.

There were no significant differences in mating or fertility indices in either generation.

There were no clinical signs of reaction to treatment during exposure. While loading the animals into the exposure chambers, 1 F0 male at 30 ppm was reported as showing a brief convulsion. These single instances were not considered attributable to treatment.

There were no significant differences in mating or fertility indices in either generation. In terms of reproductive performance, the only adverse finding was of decreased implantation counts among F1 females at 1000 ppm.

There was a total of 7 mortalities during the study, 3 in the F0 generation and 4 in the F1 generation. In the F0 generation, 1 male at 300 ppm was sacrificed during week 14 due to poor condition. Macroscopic findings of note included enlarged and swollen liver, enlarged spleen and adrenals, and enlarged and congested pancreatic lymph nodes. The remaining 2 animals (1 control male and 1 geriatric female at 1000 ppm) died during blood sampling procedures. In the F1 generation, 1 male at 30 ppm was sacrificed as a consequence of a damaged eye following blood sampling procedures, 1 male at 100 ppm was sacrificed at week 22 due to ulceration of a subcutaneous mass, and 2 females at 30 ppm were sacrificed following parturition due to poor condition. Both of these females had dead pups present in the uterus. Of these mortalities, the only one considered possibly associated with treatment was the F0 male at 300 ppm with an enlarged liver.

Retarded weight gains were observed in the F0 and F1 animals which received 1000 ppm, and the F0 males at 100 and 300 ppm. Retarded weight gains were observed during week 3 of pregnancy for the F0 1000 ppm females and throughout pregnancy for the 1000 ppm F1 females. Increased food intake occurred in the 300 and 1000 ppm F0 males and females. Decreased food intake was noted during lactation in the F0 and F1 300 and 1000 ppm females and during days 4-6 of lactation in the 100 ppm F1 females. Impaired food utilization occurred in the 1000 ppm F0 males and females, the 1000 ppm F1 females, and the 300 ppm F0 females. Increased water intake occurred in the 300 and 1000 ppm F0 males and females and in the 100 ppm F0 females. All of these findings were attributed to treatment with HCFC-123.

There was no obvious difference apparent in the pre-mate vaginal smears for either generation. For both generations, there were no obvious treatment-related effects on mating performance, as assessed by the cell type in the vaginal smear on the day of conception or in the median pre-coital time.Pregnancy rate was high for all treated groups for both generations, at 92%.

Decreased triglycerides occurred in the 300 and 1000 ppm F0g and F1 males and females, in the 100 ppm F0g females, and in the 100 ppm F1 males and females. Decreased very low density lipoprotein (VLDL) was noted in the 1000 ppm F0, F0g, and F1 males and females, in the 300 ppm F0 and F1 males and females, in the 100 ppm F0, F0g, and F1 females, and in the 30 ppm F0g and F1 females. The cholesterol profile of the 300 and 1000 ppm F0, F0g, and F1 females was decreased, as well as in the 100 ppm F0 females. The cholesterol profile of the 1000 ppm F0 and F0g males, the 100 and 300 ppm F0, F0g, and F1 males, and the 30 ppm F0 males appeared increased. All of these findings were attributed to treatment with HCFC-123.

Among the F0 males at 14 weeks of age, LH values were slightly, but significantly, higher than controls at 300 and 1000 ppm. Among the F0 geriatrics, there were no significant differences. Among the F1 males, progesterone levels were slightly, but significantly, lower at 100 ppm and above. As there was neither persistent nor consistent pattern in the hormonal assays, the significant differences in LH and progesterone values were not considered attributable to treatment.

There was no suggestion in the values obtained from the CCK assay of an effect on treatment.

There was no obvious effect of treatment on milk fat content in either generation.

The mean age of occurrence of balanopreputial skinfold cleavage was slightly delayed among males at 300 and 1000 ppm compared with controls. The group mean body weight at attainment of sexual maturity was comparable to controls, suggesting differences in growth rate may have been a factor in the delay of sexual maturation.

There was no obvious effect on the age of attainment of cleavage at 30 and 100 ppm. There was no obvious effect on the mean age of attainment of vaginal opening.

In the F0 generation, there were 2 instances of total litter loss: 1 female at 300 ppm was a total resorption and 1 female at 1000 ppm was a total litter loss in the post-partum phase. In the F1 generation, there were 3 total litter losses all in the post-partum phase: 2 at 30 ppm and 1 at 300 ppm. As there was a low incidence of losses and no evidence of a strict dosage relationship, these losses were considered coincidental.

The 100, 300, and 1000 ppm F0 and F1 males and females, and the 30 ppm F0 males and females had increased liver weights. Macroscopic changes in the liver with associated microscopic changes of centilobular hepatocyte enlargement and vacuolation was found in the 300 and 1000 ppm F0 and F1 males and females. At 100 ppm, microscopic changes limited to centilobular hepatocyte enlargement/swelling occurred in the F1 animals. Liver glycogen content was decreased in the 1000 ppm F0 and F1 animals. All of these findings were attributed to treatment with HCFC-123.

Exposure to HCFC-123 was principally associated with effects on growth and on the liver. Retarded weight gains were observed among directly exposed adults as well as among offspring during the pre-weaning period when direct exposure was confined to the lactating parent female. In terms of effects on the liver, slight changes were seen in some biochemical parameters among animals exposed at 30 ppm. Liver weights were increased at 30 ppm, but only among the F0 animals. Histological changes appeared confined to exposures of 100 ppm and above. It was not possible to identify a no-effect level as effects were seen in some parameters at the lowest exposure level. In terms of reproductive performance, the only adverse finding was of decreased implantation counts among F1 females at 1000 ppm. In terms of development, all exposure levels of HCFC-123 were associated with impaired pup growth in the offspring of the F1 generation.

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

< 30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

Details on results (F1)

The inhalation exposure systems produced comparable results across the groups for airflow, temperature, and relative humidity. Mean air flow ranged from 652-658 L/min across the groups. Mean chamber temperature ranged from 20.5-21.4ºC and mean relative humidity ranged from 49-53% across the test groups. The chamber mean analyzed concentrations for the F1 generation were 0, 30, 100, 299, and 1010 ppm for the 0, 30, 100, 300, and 1000 ppm groups, respectively.

There were no significant differences in mating or fertility indices in either generation. From Day 14, a dose-related reduction in both litter and meanpup weights occurred at > 100 ppm in the F1 litters. In the F2 litters, a clear, significant dose-related effect on mean pup weight was observed from Day 7 onwards, with pup weights lower than controls at all exposure levels.

There were no clinical signs of reaction to treatment during exposure. While loading the animals into the exposure chambers, 1 F0 male at 30 ppm and 1 F1 male at 1000 ppm were reported as showing a brief convulsion. These single instances were not considered attributable to treatment.

There was a total of 7 mortalities during the study, 3 in the F0 generation and 4 in the F1 generation. In the F0 generation, 1 male at 300 ppm was sacrificed during week 14 due to poor condition. Macroscopic findings of note included enlarged and swollen liver, enlarged spleen and adrenals, and enlarged and congested pancreatic lymph nodes. The remaining 2 animals (1 control male and 1 geriatric female at 1000 ppm) died during blood sampling procedures. In the F1 generation, 1 male at 30 ppm was sacrificed as a consequence of a damaged eye following blood sampling procedures, 1 male at 100 ppm was sacrificed at week 22 due to ulceration of a subcutaneous mass, and 2 females at 30 ppm were sacrificed following parturition due to poor condition. Both of these females had dead pups present in the uterus. Of these mortalities, the only one considered possibly associated with treatment was the F0 male at 300 ppm with an enlarged liver.

Retarded weight gains were observed in the F0 and F1 animals which received 1000 ppm, and the F0 males at 100 and 300 ppm. Retarded weight gains were observed during week 3 of pregnancy for the F0 1000 ppm females and throughout pregnancy for the 1000 ppm F1 females. Increased food intake occurred in the 300 and 1000 ppm F0 males and females. Decreased food intake was noted during lactation in the F0 and F1 300 and 1000 ppm females and during days 4-6 of lactation in the 100 ppm F1 females. Impaired food utilization occurred in the 1000 ppm F0 males and females, the 1000 ppm F1 females, and the 300 ppm F0 females. Increased water intake occurred in the 300 and 1000 ppm F0 males and females and in the 100 ppm F0 females. All of these findings were attributed to treatment with HCFC-123.

There was no obvious difference apparent in the pre-mate vaginal smears for either generation. For both generations, there were no obvious treatment-related effects on mating performance, as assessed by the cell type in the vaginal smear on the day of conception or in the median pre-coital time. Pregnancy rate was high for all treated groups for both generations, at 92%.

Decreased triglycerides occurred in the 300 and 1000 ppm F0g and F1 males and females, in the 100 ppm F0g females, and in the 100 ppm F1 males and females. Decreased very low density lipoprotein (VLDL) was noted in the 1000 ppm F0, F0g, and F1 males and females, in the 300 ppm F0 and F1 males and females, in the 100 ppm F0, F0g, and F1 females, and in the 30 ppm F0g and F1 females. The cholesterol profile of the 300 and 1000 ppm F0, F0g, and F1 females was decreased, as well as in the 100 ppm F0 females. The cholesterol profile of the 1000 ppm F0 and F0g males, the 100 and 300 ppm F0, F0g, and F1 males, and the 30 ppm F0 males appeared increased. All of these findings were attributed to treatment with HCFC-123.

Among the F0 males at 14 weeks of age, LH values were slightly, but significantly, higher than controls at 300 and 1000 ppm. Among the F0 geriatrics, there were no significant differences. Among the F1 males, progesterone levels were slightly, but significantly, lower at 100 ppm and above. As there was neither persistent nor consistent pattern in the hormonal assays, the significant differences in LH and progesterone values were not considered attributable to treatment.

There was no suggestion in the values obtained from the CCK assay of an effect on treatment.

There was no obvious effect of treatment on milk fat content in either generation.

The mean age of occurrence of balanopreputial skinfold cleavage was slightly delayed among males at 300 and 1000 ppm compared with controls. The group mean body weight at attainment of sexual maturity was comparable to controls, suggesting differences in growth rate may have been a factor in the delay of sexual maturation.

There was no obvious effect on the age of attainment of cleavage at 30 and 100 ppm. There was no obvious effect on the mean age of attainment of vaginal opening.

In the F0 generation, there were 2 instances of total litter loss: 1 female at 300 ppm was a total resorption and 1 female at 1000 ppm was a total litter loss in the post-partum phase. In the F1 generation, there were 3 total litter losses all in the post-partum phase: 2 at 30 ppm and 1 at 300 ppm. As there was a low incidence of losses and no evidence of a strict dosage relationship, these losses were considered coincidental.

The 100, 300, and 1000 ppm F0 and F1 males and females, and the 30 ppm F0 males and females had increased liver weights. Macroscopic changes in the liver with associated microscopic changes of centilobular hepatocyte enlargement and vacuolation was found in the 300 and 1000 ppm F0 and F1 males and females. At 100 ppm, microscopic changes limited to centilobular hepatocyte enlargement/swelling occurred in the F1 animals. Liver glycogen content was decreased in the 1000 ppm F0 and F1 animals. All of these findings were attributed to treatment with HCFC-123.

Exposure to HCFC-123 was principally associated with effects on growth and on the liver. Retarded weight gains were observed among directly exposed adults as well as among offspring during the pre-weaning period when direct exposure was confined to the lactating parent female. In terms of effects on the liver, slight changes were seen in some biochemical parameters among animals exposed at 30 ppm. Liver weights were increased at 30 ppm, but only among the F0 animals. Histological changes appeared confined to exposures of 100 ppm and above. It was not possible to identify a no-effect level as effects were seen in some parameters at the lowest exposure level. In terms of reproductive performance, the only adverse finding was of decreased implantation counts among F1 females at 1000 ppm. In terms of development, all exposure levels of HCFC-123 were associated with impaired pup growth in the offspring of the F1 generation.

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

< 30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Summaries of reproductive outcomes for the F0 and F1 generations are provided in the tables below:

F0 Generation
Concentration (ppm):    0     30     100     300     1000
Copulation Index (%):	93.8  96.9    96.9  100.0    96.9
Fertility Index (%):  	96.7  96.8   100.0   96.9    96.8
Gestation Length 
  (days):	      	22.2  22.3    22.2   22.4    22.5
Implantation sites:   	15.3  16.8    15.6   15.5    14.4
Pregnancy Rate (%):   	90.6  93.8    96.9   93.8    93.8
Gestation Index (%): 	100.0  100.0  100.0   96.8   100.0
Mean % Born Alive:    	99.2  98.7    97.4   96.6    99.1
0-4 Day Viability (%):	99.0  97.4    98.0   98.9    98.8
Weaning Viability 
   Index (%):	97.9  98.3   100.0   99.6   100.0
Sex Ratio (% males):  	54.0  49.1    50.8   48.1    46.9

F1 Generation
Concentration (ppm):  	0     30     100     300     1000
Copulation Index (%): 	89.3  96.4   100.0   100.0   100.0
Fertility Index (%):  	96.0 100.0    96.4    96.4    92.9
Gestation Length 
   (days):            	22.3  22.1    22.4    22.2    22.1
Implantation sites:   	15.7  15.3    15.1    14.3    13.1
Pregnancy Rate (%):   	85.7  96.4    96.4    96.4    92.9
Gestation Index (%): 	100.0 100.0   100.0   100.0   100.0
Mean % Born Alive:    	97.3  98.8    98.4    99.5    98.6
0-4 Day Viability (%):	99.0  95.4    96.9    99.2    98.1
Weaning Viability 
   Index (%):	96.9  99.0    99.1    99.5   100.0
Sex Ratio (% males):  	51.0  46.8    51.7    51.0    56.1

Applicant's summary and conclusion

Conclusions:

In terms of reproductive performance, the only adverse finding was of decreased implantation counts among F1 females at 1000 ppm. In terms of development, all exposure levels of HCFC-123 were associated with impaired pup growth in the offspring of the F1 generation.