Difluoromethane

Developmental toxicity / teratogenicity

Administrative data

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment (SIDS score: 1b).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Difluoromethane

EC Number:

200-839-4

EC Name:

Difluoromethane

Cas Number:

75-10-5

Molecular formula:

CH2F2

IUPAC Name:

difluoromethane

Details on test material:

Name of test material: difluoromethane
Source: ICI Chemicals and Polymers
Batch number: A108 and A110
Purity: 99.9% v/v

Test animals

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

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: maternal toxicity

Results (fetuses)

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.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

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.