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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Exposure 7 h/day rather than 6 h/day.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroform
EC Number:
200-663-8
EC Name:
Chloroform
Cas Number:
67-66-3
Molecular formula:
CHCl3
IUPAC Name:
trichloromethane
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 or test system 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:
air
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.
Target concentrations were 0, 3, 10 and 30 ppm; the actual delivered concentrations of chloroform were 0, 3.1, 10.7, or 30.2 ppm (0, 15, 52.2, or 147 mg/m3).
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. Day of mating (identified based on the presence of a sperm plug) was defined as gestation day 1.
Duration of treatment / exposure:
From day 7 to 16 after mating (day of mating is GD 1).
Frequency of treatment:
7 hours per day
Duration of test:
Killing of animals, Caesarian section and autopsy on Day 21 of pregnancy
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/m³ air (analytical)
Dose / conc.:
52.2 mg/m³ air (analytical)
Dose / conc.:
147 mg/m³ air (analytical)
No. of animals per sex per dose:
20 pregnant females per concentration group
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. Pregnancy status of dams without fetuses was confirmed via 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 skeletal anomalies. The remaining foetuses were fixed in Bouin's solution, cut into cross sections and examined under a stereomicroscope for organ (visceral) 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

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a slight reduction in weight gain during the exposure period in dams of the 10 and 30 ppm groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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.

Maternal developmental toxicity

Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
All dams in the treated groups carried live fetuses to term with the exception of one dam in the 30 ppm group, which presented with a total litter resorption having 13 empty implantation sites.
Description (incidence and severity):
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 no fetuses; 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.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
15 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: reduction in BW gain at the next two higher levels tested

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
It was noted 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 mean fetal weight, although statistically significantly lower in the 30 ppm group, was not really different from the fetal weight in the control group and was still within the normal range.
Description (incidence and severity):
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.
Details on embryotoxic / teratogenic effects:
One dam exposed to 30 ppm chloroform via inhalation exhibited only empty implantation sites (i.e., no fetuses were present). A statistically significant increase in the incidence of fetuses with body weights <3 grams and in the incidence of fetuses with slight or no ossification of individual skull bones was observed in the 30-ppm exposed group when compared with controls. The incidence of fetuses with body weights <3 grams was increased in a concentration-related fashion (3.2%, 14.2%, 24%, and 26.9% at 0, 3, 10, and 30-ppm, respectively); this trend did not appear to be due to variations in litter size. However, when the litter was used as the statistical unit of comparison, only litters from the high-concentration group had a significant number of fetuses weighing 3 grams or less. The mean crown-rump length, although statistically significantly lower in the 30 ppm group, was not really different from the crown-rump length in the control group, and was still within the normal range.
A significant increase in the incidence of fetuses with ossification of less than two caudal vertebral centers was observed at all concentrations. A dose response was observed for the incidence of litters with this effect; however, the effect was not statistically significant. Finally, all exposure groups exhibited a significant increase in the incidence of both litters and fetuses with nonossified or weakly ossified sternebrae; however, there was no statistically significant concentration-related trend for this effect. Even though there were increases in low-weight fetuses at the two lowest concentrations, this effect was not considered adverse. No indication of a teratogenic effect of chloroform was found in the foetuses examined.
The skeletal ossification of the foetuses of the dams exposed to chloroform corresponded to the stage of pregnancy. The statistically greater number of foetuses in the 30 ppm group, as compared to the control group, with poorly or not yet ossified cranial bones, and the not yet or only poorly ossified sternebrae and less ossified caudal vertebrae in all three compound groups is not relevant for the evaluation of chloroform, as the number of affected foetuses lies within the range of the control values. As these show, the number of control foetuses in which sternebrae or caudal vertebrae were poorly or not yet ossified was extremely low in comparision with the previous control values. The lack of a relationship between dose and
effect and the fact that, in the previous study (1989), concentrations of 30 to 300 ppm had not affected skeletal ossification in foetuses also tend to contradict a causal relationship with exposure to chloroform.
Therefore, the NOAEC for developmental effects in this study was 10.7 ppm (52.2 mg/m3) based on the weight of evidence of the data, including comparison to historical controls and the higher concentration study (1989).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
52.2 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased fetal weight and ossification at the next higher level

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
147 mg/m³ air (analytical)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

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:
Based on decreased body weight gain in dams a NOAEC of 3 ppm could be identified. However, even though there were increases in low-weight fetuses at the two lowest concentrations, this effect was not considered adverse. No indication of a teratogenic effect of chloroform was found in the foetuses examined. Therefore, the NOAEC for developmental effects in this study was 10.7 ppm (52.2 mg/m3) based on the weight of evidence of the data, including comparison to historical controls and the higher concentration study (1989).
Executive summary:

In this supplementary study, groups consisting of 20 female Wistar rats were exposed to chloroform in concentrations of 3, 10 or 30 ppm for 7 hours daily between day 7 and day 16 of pregnancy. A simultaneous control group of the same size inhaled air without the addition of the test compound. On day 21 of pregnancy, the dams were killed and delivered by caesarian section. The foetuses were then examined morphologically for developmental anomalies.

The examinations revealed that repeated inhalation of chloroform in a concentration of 3 ppm during the sensitive phase of embryofoetal organogenesis did not lead to an impairment of the general state of health, nor were there adverse effects upon the feed consumption and body-weight development of the dams or the intrauterine development of the conceptuses. After concentrations of 10 ppm and 30 ppm the dams displayed a slight reduction in feed consumption and body weight development. The foetuses were slightly stunted. On exposure to 30 ppm all embryonic primordia of one dam died in utero. The findings observed in the dams and conceptuses after exposure to 30 ppm correspond with those determined in the previous study (1989) at this concentration.

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. Concentrations of 10 ppm

and above led to slight adverse effects on feed consumption and bodyweight development in the dams. Intrauterine embryonic death seemed to occur at concentrations of

30 ppm and above. The intensity of the maternal and embryonal findings was dose-dependent. No indication of a teratogenic effect of chloroform was found in the foetuses examined. The toxic effect of chloroform on dams and conceptuses which was described in the literature was confirmed, although some findings were different, but no teratogenic potential was observed.

On the basis of the results of the embryotoxicity studies, it can therefore be concluded that the NOAEC for chloroform in the rat, in the case of inhalative exposure, lies at 3 ppm (15 mg/m3) for maternal toxicity and 10 ppm (52.2 mg/m3) for fetotoxicity.

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