Norflurane

Toxicity to reproduction

Administrative data

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

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

Test material information

Constituent 1

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:

1,1,1,2-tetrafluoroethane (HFC 134a) was obtained from Imperial Chemical Industries Limited, Mond Division, Runcorn UK with a purity of >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 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.

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:

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

Doses / concentrations

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)

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. Deaths were recorded, but the animals were not given a post-mortem examination.

Results and discussion

Results: P0 (first parental generation)

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

50 000 ppm

Sex:

male

Basis for effect level:

other: No effects on reproductive performance of male mice

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 50 000 ppm

Sex:

male/female

Basis for effect level:

viability

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	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 χ²test.

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.

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 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.