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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out under the conditions of GLP and in accordance with the OECD Guideline for Testing of Chemicals No. 414 "Teratogenicity", 1981 with no restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chloroform, reagent grade of 99.0-99.4 % purity, Batch-No. 012 K13819245

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: company's own breeding (Hoe:WISKf(SPF71))
- Age at study initiation: 65-70 days
- Weight at study initiation: 195 +/- 9 g
- Fasting period before study: no data
- Housing: one per cage in plastic cages on wood shavings; in pairs in a metal-grid cage, separated by a metal partition dividing (during 10 days of exposure)
- Diet (e.g. ad libitum): standard diet Altromin 1310 pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 to 23 °C; 21 to 22.5 °C (during 10 days of exposure)
- Humidity (%): 56 to 70 %; 42 to 74 % (during 10 days of exposure)
- Air changes (per hr): 16 to 20 changes per hour; 10 changes per hour (during 10 days of exposure)
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Chloroform was continuously applied by means of a slow infusion injector onto an evaporation head and was vaporised at 80 °C. A flow of 800 L/h fed the air-gas mixture into the inhalation chamber via a connecting tube.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of chloroform in the inhalation chamber was measured using a Miran 80 single-beam photometer. Concentrations were measured every 30 minutes.
Details on mating procedure:
Female rats in oestrus were mated with fertile males overnight (15.30 to 7.30 hours) before the start of the study.
Duration of treatment / exposure:
from Day 7 to 16 after mating
Frequency of treatment:
7 hours per day
Duration of test:
Killing of animals, Caesarian section and autopsy on Day 21 of pregnancy
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10, 30 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
20 pregnant females
Control animals:
yes, concurrent no treatment

Examinations

Maternal examinations:
Daily examination of behaviour and general physical conditions; body weight and feed consumption monitored once a week. The dams were dissected and their organs examined macroscopically. The heart, liver, kidneys and spleen were weighed.
Ovaries and uterine content:
The uterus was opened on Day 21 after mating and the live and dead conceptuses, embryonic primordia under resorption, placentae and corpora lutea in the ovaries were counted and examined macroscopically. The diameter of the embryonic resorption sites and the placental weights were determined. The presence of iron was demonstrated by staining the uterus with ammonium sulphide.
Fetal examinations:
Foetuses were examined for signs of life, outward appearance and detectable anomalies. The body weight of foetuses was determined and the crown-rump length was measured. The sex was determined at autopsy.
Half of the foetuses from each litter and those found dead in utero were fixed in alcohol, dissected under a magnifying glass, eviscerated and cleared in potassium hydroxide solution. Skeletons were stained with alazarin red S and examined under a stereomicroscope with regard to developmental stage and anomalies.
The remaining foetuses were fixed in Bouin's solution, cut into cross sections and examined under a stereomicroscope for organ anomalies.
Statistics:
In the comparison with the simultaneous control group, a standard MANOVA with sequentially rejective multiple comparisons was used to evaluate the body and organ weights, while a non-parametric linear model of Puri and Sen (1985) with sequentially rejective multiple comparisons was used to evaluated the relative feed consumption.
Implants and corpora lutea were analysed using the Mantel-Haenszel chi-squared test, as were the quotas of live and dead foetuses and of embryonic primordia under resorption. Foetal weights, crown-rump lengths and placental weights were evaluated by analysis of variance which had been corrected by the random litter effect. In the procedures mentioned, a 5 % probability of error per parameter group was adhered to. The findings obtained at autopsy and at body cross-section and skeletal examination of the foetuses were evaluated separately for the foetuses and for the litters by the exact Fisher test.
Historical control data:
To compare the study data with previous control data, normal ranges were calculated for groups. They are fixed in such a way that, at probability of 95 %, they contain at least 75 % of the values of a group control of animals. The parallelogram method of Abt (1982) was used to evaluate body and organ weights. The parallelopiped analysis of Wald (1943) was used to evaluate body weight development and feed consumption of the dams and the litter means of the foetal bodyweight, crown-rump length and placental weight. Corpora lutea, implants and survival rates were compared with univariate normal ranges after Wilks (1942). For the quotas of live and dead foetuses and conceptuses under resorption, the ranges were determined by triangular analysis after Rosenkranz (1988).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The dams in the 30 ppm group displayed a slight reduction in feed consumption during the entire exposure period, this being rather more marked in the first week than in the second. There was a slight reduction in weight gain during the exposure period in dams of the 10 and 30 ppm groups. 20 dams in the 3 and 10 ppm groups and 19 dams in the 30 ppm group carried live foetuses to term. One dam in the 30 ppm group had not foetuses; instead there were merely 13 empty implantation sites in utero. Three dams in the 3 ppm group had a fairly high number of necrotic biastocysts (35.7 to 80 %). Two dams in the 10 ppm group had an extremely large number of corpora lutea (20 and 23) in the ovaries. In the dam of the 30 ppm group which had been found to have only empty implantation sites in the uterus, the corpora lutea were small and could not be determined exactly.
The final autopsy revealed that one dam in the 3 ppm group, three dams in the 10 ppm group and four dams in the 30 ppm group had moderate to severe unilateral or bilateral renal pelvic dilatation. In the 30 ppm group, there was a very slight gain in kidney weights as compared to the control dams, but this observation remained within the range of the historical control data.

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
It was noticeable that in the 10 ppm and 30 ppm groups, 24 and 26.9 % of the foetuses had body weights below 3.0 g, whereas in the control and 3 ppm groups, only 3.2 and 14.2 % of the foetuses had body weights below 3.0 g.
The morphological examination revealed no abnormalities in the control or exposure groups, with one exception of an internal hydrocephalus in a live foetus of the 3 ppm group.
Numerous foetuses in all exposure groups showed poorly or not yet ossified cranial bones and/or sternebrae, and several foetuses also exhibited fewer than two ossified caudal vertebral centres and or missing ossification of the 5th metacarpal bone. Poorly or not yet ossified vertebral arches and or centres, ribs and phalanges were observed only in a few foetuses in various exposure groups. In many foetuses there was no ossification of the 5th metacarpal bone. The number of control foetuses in which fewer than two caudal vertebral centres and sternebrae were only poorly or not yet ossified was small. On comparison of the findings observed in the compound and control groups, it can be seen that the number of foetuses in the 30 ppm group with poorly ossified cranial bones was statistically higher when compared to the number of affected control foetuses. The same was true for the foetuses in the three exposure groups which exhibited poor ossification of the caudal vertebrae and sternebrae. The number of foetuses in the 10 ppm group which displayed waved and or thickened ribs was greater when compared to the control foetuses. All findings, nevertheless, lay within the range of the historically found spontaneous rate.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
3 ppm
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Survey of results during gestation and at Caesarian section

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of pregnancies

Total

20

20

20

20

Number of females at term with live foetuses

Total

20

20

20

19

Number of corpora lutea

Total

290 N

291 N

301 N

267 N

Mean

14.5

15.6

15.1

14.1

S.D.

1.4

1.7

2.9

1.4

Number of implantations

Total

259 N

255 N

273 N

254 N

Mean

13.0

12.8

13.7

13.4

S.D.

2.2

3.5

2.9

1.5

Pre-implantation loss %

Mean

10.87

13.68

9.41

4.83

Post-implantation loss %

Mean

4.55

3.17

5.80

6.11

Number of total intrauterine deaths

Total

11 N

8 N

15 N

16 N

Mean

0.55

0.40

0.75

0.84

S.D.

0.89

0.60

1.02

1.42

Number of live foetuses

Total

248 N

247 N

258 N

238 N

Mean

12.4

12.4

12.9

12.5

S.D.

2.4

3.5

3.0

1.9

N: within the normal range

Table 2: Survey of results in live foetuses at Caesarian section

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of foetuses

Total

248 N

247 N

258 N

238 N

% of implantations

Mean

95.45

96.83

94.20

93.89

Males

%

47.58

44.13

50.39

50.84

Body weight (in g)

Mean

3.4 N

3.2 N

3.2 N

3.2 N *

S.D.

0.3

0.3

0.3

0.3

Crown/Rump length (in mm)

Mean

35.8 N

35.5 N

34.4 N

34.8 N *

S.D.

2.0

2.1

2.6

1.9

Placental weight (in g)

Mean

0.48 N

0.45 N

0.48 N

0.45 N

S.D.

0.07

0.07

0.09

0.07

N: within the normal range; * implants and corpora lutea were analysed using the Mantel-Haenszel chi-squared test, as were the quotas of live and dead foetuses and of embryonic primordia under resorption. Foetal weights, crown-rump lengths and placental weights were evaluated by analysis of variance which had been corrected by the random litter effect. In the procedures, a 5 % probability of error per parameter group was adhered to.

Table 3: Observations for individual foetuses - external or visceral defects found at autopsy

Observations

Type

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

Number of foetuses examined

Total

130

129

135

123

External: retarded foetus

Number

1

2

4

1

%

0.8

1.6

3.0

0.8

External/visceral: no abnormalities detected

Number

121

120

129

112

%

93.1

93.0

95.6

91.1

Blood in thoracic cavity

Min Number

0

2

0

0

%

0

1.6

0

0

Blood in abdominal cavity

Min Number

0

0

0

1

%

0

0

0

0.8

Kidney: pelvis distended – uni- or bilateral

Min Number

8

5

2

9

%

6.2

3.9

1.5

7.3

Skeleton: no abnormalities detected

Number

35

42

36

31

%

26.9

32.6

26.7

25.2

Skull: splitting of bone on parietal bone – left

Min Number

1

0

0

0

%

0.8

0

0

0

Skull: individual skull bones – slight or non-ossification

Ret Number

42

47

48

60 *

%

32.3

36.4

35.6

48.8

Thoracic vert. arch: weakly ossified – amidst normally ossified thoracic vertebrae – right

Ret Number

0

0

0

1

%

0

0

0

0.8

Fragmented thoracic vert. centra

Min Number

1

0

0

0

%

0.8

0

0

0

Lumbar vert. arch: weakly ossified – uni- or bilateral

Ret Number

1

0

3

2

%

0.8

0

2.2

1.6

Sacral vert. arch centra: non-ossified

Ret Number

0

1

1

0

%

0

0.8

0.7

0

Caudal vert. centra: ossification of less than 2 vertebral centres

Ret Number

4

14 *

16 *

14 *

%

3.1

10.9

11.9

11.4

Extra vertebra/extra rib: anlage of a 14 thoracic vertebra with an analogous 14th rib – short and/or normally long – uni- or bilateral

Var Number

1

0

1

2

%

0.8

0

0.7

1.6

Sternebrae: fragmented, longitudinally displaced or dysplasia

Min Number

5

1

4

2

%

3.8

0.8

3.0

1.6

Sternebrae: non-ossified or weakly ossified

Ret Number

7

32 *

35 *

18 *

%

5.4

24.8

25.9

14.6

Rib: weakly ossified – distal part – left or bilateral

Ret Number

0

0

1

0

%

0

0

0.7

0

Rib: shortened 13th – left

Min Number

1

0

0

0

%

0.8

0

0

0

Rib: wavy and/or thickened

Min Number

10

11

22 *

15

%

7.7

8.5

16.3

12.2

Extra rib: at 7th cervical vertebra – short – unilateral

Var Number

0

1

3

0

%

0

0.8

2.2

0

Extra rib: at 1st lumbar vertebra – short – uni- or bilateral

Var Number

52

41

39

30

%

40.0

31.8

28.9

24.4

Pectoral girdle: scapula bent costad and/or shortened – right or bilateral

Min Number

0

1

3

1

%

0

0.8

2.2

0.8

Forepaw: non-ossified metacarpal 5

Ret Number

13

13

21

11

%

10.0

10.1

15.6

8.9

Forepaw – phalanx: phalanx III of 1st to 5th toes non-ossified

Ret Number

0

0

1

0

%

0

0

0.7

0

Hindpaw  - phalanx: phalanx III of 1st to 5th toes non-ossified

Ret Number

0

1

2

0

%

0

0.8

1.5

0

* p 0.05 one sided obtained with Fisher's exact test: group 1 compared with groups 2, 3, 4

Table 4: Observations for individual foetuses - external or visceral defects obtained at body cross-section examination

Observations

Group 1: 0 ppm

Group 2: 3 ppm

Group 3: 10 ppm

Group 4: 30 ppm

# of foetuses examined

Total

118

118

123

115

External/visceral: no abnormalities detected

Number

109

108

111

110

%

92.4

91.5

90.2

95.7

Brain: Hydrocephalus internus

Maj Number

0

1

0

0

%

0

0.8

0

0

Blood in abdominal cavity

Min Number

2

3

1

1

%

1.7

2.5

0.8

0.9

Liver: Lobus dexter or lobus sinister - haematoma

Min Number

1

2

1

0

%

0.8

1.7

0.8

0

Kidney: blood in kidney and vicinity – left – or haematoma in kidney – right

Min Number

1

0

0

1

%

0.8

0

0

0.8

Kidney: pelvis distended – uni- or bilateral

Min Number

5

5

8

3

%

4.2

4.2

6.5

2.6

Kidney/ureter: pelvis and ureter distended – left of bilateral

Min Number

0

1

2

0

%

0

0.8

1.6

0

Applicant's summary and conclusion

Conclusions:
It can be concluded that, under the study conditions selected, the inhalation of chloroform in a concentration of 3 ppm is tolerated by both pregnant rats and conceptuses.
Executive summary:

A study of the embryotoxicity of chloroform to Wistar rats was carried out under the conditions of GLP and in accordance with OECD guideline No. 414 with no restrictions.

Pregnant Wistar rats were exposed to chloroform vapours at concentrations of 3, 10 and 30 ppm for 7 hours per day during 10 days (Day 7 to 16 after mating). All animals were killed on Day 21 of pregnancy and dams and foetuses were examined. Concentrations of 10 ppm and above led to slight adverse effects on feed consumption and body weight development in the dams and slight retardation in the conceptuses. Intrauterine embryonic death seemed to occur at concentrations of 30 ppm and above. The intensity of maternal and embryonic findings was dose-dependent. No indication of a teratogenic effect of chloroform was found in the foetuses examined.

It can be concluded that, under the study conditions selected, the inhalation of chloroform in a concentration of 3 ppm (14.7 mg/m3) is tolerated by both pregnant rats and conceptuses.