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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: dominant lethal assay
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report date:
1979

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Norflurane
EC Number:
212-377-0
EC Name:
Norflurane
Cas Number:
811-97-2
Molecular formula:
C2H2F4
IUPAC Name:
1,1,1,2-tetrafluoroethane
Details on test material:
Purity: >99.5%.

Test animals

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 for 12 days and were housed 10 per cage on rack similar to that described for males 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 approximately 50%. Alternate 12 hr light and dark cycles were controlled with a timer starting at 6 am. The animals received Alderley Park diet supplied and water (provided by an automatic drinker system) ad libitum except for the males during the exposure period.

Administration / exposure

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:
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. Test substance atmospheres were generated by mixing volumes of the test compound 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.
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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 10000, 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 900, 10200, 50700 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30 males for mating from negative control
15 males for mating from test substance and positive controls
Control animals:
yes, sham-exposed
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 but excluding those with a high dominant lethal frequency. The selected males were randomly allocated to six experimental groups. Following the allocation process 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)

Examinations

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.
Postmortem examinations (parental animals):
Deaths were recorded, but the animals were not given a post-mortem examination.
Statistics:
Data from the test substance exposed groups and positive control groups were compared statistically with those from the negative control groups.
To assess fertility, two methods were used:
i) the percentage of pregnant females was analysed using the Chi-squared test
ii) the number of successful matings per male was analysed using Student’s t-test
Pre-implantation egg losses, whilst representing some of the mutagenic effect, are not as important as the post-implantational losses since such losses could be due to other than genetic factors. Pre-implantation egg loss in the present study has thus been measured by comparing values of total implantations in females mated with treated males and control males instead of counting corpora lutea. The total number of implantations per pregnancy was considered by analysis of variance and group means were compared using a one-sided Student’s t-test based on this analysis.
Reproductive indices:
The number of early deaths, late deaths and live implantations observed during the uterine examination were recorded and the resultant data processed and analysed statistically.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Dose descriptor:
NOEC
Effect level:
50 000 ppm
Sex:
male
Basis for effect level:
other: No effects on reproductive performance of male mice at highest concentration tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOEC
Generation:
F1
Effect level:
50 000 ppm
Sex:
male/female
Basis for effect level:
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Day

Test substance

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):

 

Percentage of Females pregnant (Table 3): The only significant effect in the test substance 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

Test substance

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 χ2 test.

 

Successful mating per male (Table 4): Both the low and high dose test substance groups had reduced mean numbers of successful matings per male in week 7. The value for the middle dose group was slightly lower 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

Test substance

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 test substance 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, retrospectively, 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 any 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 test substance 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 test substance 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 the test substance in weeks 4 and 8, while in week 7 the pooled value for all three test substance 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 test substance 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 test substance treated groups and it is usual for pre- and post-implantation loss to be 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 10000 ppm test substance 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: The test substance had no effect on fertility and is unlikely to cause mutagenic effects as assessed by the mouse dominant lethal assay.

Applicant's summary and conclusion

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 0, 1000, 10000 or 50000 ppm test substance 6 hours a day for 5 days. Positive control groups received either 5 daily oral doses of ethyl methanesulphonate or a single intraperitoneal dose of cyclophosphamide. 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.

Despite occasional statistical differences between the test substance and the negative control groups, this study indicated that the test substance does not did not affect male fertility or cause mutagenic effects through sperm. This lack of effect was not due to a lack of sensitivity of the test system since the expected positive responses were demonstrated by ethyl mehtanesulphonate and cyclophosphamide. The NOAEC for this study was >50000 ppm.