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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 1993 to 17 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Analytical purity: 94.6 %
- Purity test date: 19 June 1992
- Lot/batch No.: 52000726
- Expiration date of the lot/batch: not stated
- CAS: 78-85-3

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD (SD) BR VAF/Plus strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Manston Road, Margate, Kent.
- Age at study initiation: 8 - 10 weeks.
- Weight at study initiation: 173 - 239 g
-Batches of animals: The animals were devided into 2 batches. The first batch (A) consisted of 63 animals followed by an second batch (B) of 42 animals mated one day later. Both batches were treated identically.
- Fasting period before study: No.
- Housing: Animals were housed five to a cage (except during exposure periods) in suspended stainless steel cages (North Kent Plastic Cages Ltd) equipped with solid sides and wire grid front, back and floor. Each group of rats was kept in a separate ventilated cabinet to prevent any possible cross-contamination between groups.
- Diet: ad libitum. SDS Laboratory Animal Diet No. 1.
- Water: ad libitum.
- Acclimation period: no data.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 plus/minus 4 °C.
- Humidity (%): 46 plus/minus 15 %.
- Air changes (per hr): no data.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: 19 May 1993 To: 8 June 1993

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chambers were constructed from stainless steel and glass and were of approximately 5000 litres internal volume. The chambers were of square cross-section fitted with a pyramidal base and top. An extraction plenum was fitted in the base of each chamber.
The chamber atmosphere was extracted by mean of individual air handling units, each fitted with activated charcoal filters. Agate valve fitted in each extract line was used to adjust the pressure within each chamber to approx. 5 mm of water below that of the room.

- Method of holding animals in test chamber: The rats were held within individual compartments of stainless steel wire mesh cages during exposure.

- Source and rate of air: no data.

-Atmosphere generation: Each test atmosphere was produced by metering the liquid from a gas-tight syringe mounted on a Precidor type 5003 syringe pump to a sintered glass disc contained in a glass vessel. Air was passed through the glass sinter at a rate of 100 litres per minute. The air supply was heated prior to entering the vaporiser by passing it through a copper coil submerged in a water bath maintained at 80 °C. The vapour air mixture entered the exposure chamber via the inlet duct.

- System of generating particulates/aerosols: not relevant.

- Temperature, humidity, pressure in air chamber (means):
Group 1 (Air control, Batch A): 23.0 °C/52 %
Group 2 (Low dose, Batch A): 25.0 ° C/41 %
Group 3 (Intermediate dose, Batch A): 25.2 °C/37 %
Group 4 (High dose, Batch A): 24.4 °C/48 %
pressure in air chamber: approx. 5 mm of water below that of the room.

Group 1 (Air control, Batch B): 22.9 °C/53 %
Group 2 (Low dose, Batch B): 24.7 ° C/42 %
Group 3 (Intermediate dose, Batch B): 25.1 °C/36 %
Group 4 (High dose, Batch B): 24.2 °C/49 %
pressure in air chamber: approx. 5 mm of water below that of the room.


- Air flow rate: 100 L/min.

- Method of particle size determination: not relevant.

- Treatment of exhaust air: Each chamber was fitted with ports for withdrawal of chamber air samples for analytical purposes. Routinely a port in the upper centre of the chamber side wall was used.


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography.
- Samples taken from breathing zone: yes

PROCEDURE
-The rats were placed within the exposure cages, the cages loaded into the exposure chambers and the chamber doors sealed.
The air supply to the vaporisers was switched on and the flow rate set to 100 litres per minute. Chamber pressure was checked and adjusted if necessary to 5 mm of water below ambient.
Exposure commenced when the syringe pumps were switched on. The concentration of methacrolein within each chamber was cotrolled by the rate at which the test substance was supplied to each vaporiser. The feed rates required to achieve the target concentrations were determinded during preliminary investigations.
Following 6 hours of exposure the syringe pumps were switched off. After approx. 20 minutes the rats were unloaded from the chambers and returned to their holding cages.
Group 1 (Air control) rats were treated in similar fashion except that no methacrolein was introduced into the chamber.



VEHICLE (if applicable)

-air
- Justification for use and choice of vehicle: no data.
- Composition of vehicle: no data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Gas chromatography.
- For further details see Appendix A1.
Details on mating procedure:
- Cohoused:
- M/F ratio per cage: no data.
- Length of cohabitation: no data.
- Verification of same strain and source of both sexes: yes.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: no.
Duration of treatment / exposure:
6 hours per day, gestation day 6-15 of pregnancy. A total of 10 exposures.
Frequency of treatment:
daily
Duration of test:
20 Days.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 10, 20 ppm nominal (= 0, 0.014, 0.028 and 0.056 mg/L)
Basis:
nominal conc.
No. of animals per sex per dose:
Batch A: 15 female animals per sex and dose.
Batch B: 10 female animals per sex and dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The exposure levels employed were agreed between HRC and the Sponsor and based on the results of a preliminary study in pregnant rats (HRC Report No. BGH 42/921663) performed at these laboratories in which levels of 10, 20 and 30 ppm produced clear evidence of a dose-related maternal response, principally manifested as reduced bodyweight gain and food intake, although embryofoetal development appeared unaffected at all exposure levels. In a previous two-week study in male and non-pregnant female rats (HRC Report No. BGH 40/920648) performed at these laboratories, 5 ppm was identified as a "No Adverse Effect Level".

Group 1 (control, Batch A and B): 0 ppm.
Group 2 (low dose, Batch A and B): 5 ppm.
Group 3 (intermediate dose, Batch A and B): 10 ppm.
Group 4 (high dose, Batch A and B): 20 ppm.

- Rationale for animal assignment: Random.

-Other:
The rat is a universally accepted species in reproductive studies; in addition, Huntingdon Research Centre Ltd has background control data on reproductive performance in pre-natal toxicity studies for rats of the strain employed.

The route of administration (inhalation exposure) was selected by the Sponsor as this was a potential route of exposure to man.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (during exposure in 30-minute intervals).
- Cage side observations checked in table [No.1] were included.


DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
at arrival and on Days 3, 6, 8, 10, 12, 14, 16, 18 and 20 of gestation.

FOOD CONSUMPTION: Yes
Time schedule for examinations:
- group mean values (g/rat/day) on Days: 3-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-19 of gestation.

WATER CONSUMPTION: Yes
- Time schedule for examinations:
- group mean values (g/rat/day): daily (Day 3 to Day 19 of gestation).


POST-MORTEM EXAMINATIONS: Yes
from all adult animals:
- Sacrifice on gestation day 20
- Organs examined: No data.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Number and distribution of live young
Number and distribution of embryofoetal deaths
Individual foetal weight from which the litter weight was calculated
Foetal abnormalities
Fetal examinations:
- External examinations: Yes: all per litter.
- Soft tissue examinations: Yes: half per litter.
- Skeletal examinations: Yes: half per litter.
- Head examinations: No.

Statistics:
Group mean values for maternal bodyweights and litter parameters have been calculated using all animals with live young at terminal sacrifice. All values expressed as a percentage were first calculated within the litter and the group values derived as a mean of individual litter percentages.

Group values for food and water were calculated from cage values and include all animals.

Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters and results are presented in relevant tables of this report:
- body weight change
- food consumption
- water consumption
- litter data
- foetal changes

Dependent on the heterogeneity of variance between treatment groups,
- parametric tests, analysis of variance (Snedecor and Cochran, 1967) followed by Williams' test (Williams, 1971/72) or
- non-parametric tests, Kruskal-Wallis (Hollander and Wolfe, 1968) followed by Shirley's test (Shirley, 1977)
were used to analyse these data, as most appropriate.

For litter data and skeletal variants the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used.

Malformations and anomalies were analysed in terms of litters with affected foetuses, using a one-tailed permutation test (Edgington 1980).

Where 75 % or more of the value for a given variable are the same, a Fisher's exact test (Fisher, 1950) was used, when considered necessary.
Historical control data:
See Appendix 3.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs observed at 10 and 20 ppm during the 6-hour exposure periods (as noted from outside die exposure chambers) were principally related to irritancy i.e. half-closed/closed eyelids, noted generally between 0.5 and 1.5 hours after exposure began from the first day of treatment (Day 6 post coitum) at 20 ppm and from the third day at 10 ppm. At 5 ppm, no treatment-related clinical signs were observed during exposure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 20 ppm, bodyweight gain was reduced following the start of treatment (Day 6) through to termination at Day 20, attaining statistical significance (P<-0.01) throughout.
Bodyweight gain at 5 and 10 ppm was essentially similar to controls (P >0.05).

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Half-closed/closed eyes were seen at the 20 and 10 ppm treatment levels during the exposure periods, although this sign was considered to be due to an irritant effect of the vapour and was not of major toxicological importance.

There was clear evidence of a significant treatment-related toxic response in adult females at 20 ppm, principally manifested as reduced bodyweight gain, impaired food intake and increased water consumption. The only possible treatment-related maternal effect at 5 or 10 ppm was a marginal reduction in food intake after initiation of treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
0.014 mg/L air (analytical)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Amongst litter parameters, litter and mean foetal weight were slightly lower at 20 ppm. It is therefore considered that the no adverse effect level for all litter parameters is 10 ppm. However, in utero survival and morphological development appeared unaffected at exposure levels up to and including 20 ppm and it can be concluded that methacrolein does not induce any specific or permanent alteration in embryofoetal development in the rat.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
0.028 mg/L air (analytical)
Basis for effect level:
other: slight reduction of the mean fetal weight in the 20 ppm group

Fetal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
no teratogenicity

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In this assessment of the effect of methacrolein on pregnancy and in utero development of the rat, daily target doses of 0 (Control), 5, 10 and 20 ppm were administered to groups of 25 time-mated females by whole-body inhalation exposure for 6 hours each day from Day 6 to Day 15 post coitum inclusive. On Day 20, all animals were killed and subjected to post mortem examination; litter values were determnined and foetuses were subsequently examined for visceral and skeletal changes.

Clear evidence of maternal toxicity was observed at 20 ppm, principally manifested as reduced weight gain and food intake. In addition, microscopic changes to the nasal turbinates, larynx, trachea, lungs and liver were observed at this exposure level in the preliminary study (HRC Report No. BGH 42/921663). At 10 ppm, food intake was slightly reduced with microscopic changes to the nasal turbinates observed in the preliminary study. With the possible exeption of marginally lower food intake at 5 ppm, no clear maternal effects were seen at this exposure level and 5 ppm was considered to be the no adverse effect level for adult females.

Amongst litter parameters, litter and mean foetal weight were slightly lower at 20 ppm. It is therefore considered that the no adverse effect level for all litter parameters is 10 ppm. However, in utero survival and morphological development appeared unaffected at exposure levels up to and including 20 ppm and it can be concluded that methacrolein does not induce any specific or permanent alteration in embryofoetal development in rat.