Chloroform

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/m³ air (analytical)

Doses / concentrations 2

Dose / conc.:

52.2 mg/m³ air (analytical)

Doses / concentrations 3

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 14^th 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 13^th – 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 7^th cervical vertebra – short – unilateral	Var Number	0	1	3	0
	%	0	0.8	2.2	0
Extra rib: at 1^st 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 1^st to 5^th toes non-ossified	Ret Number	0	0	1	0
	%	0	0	0.7	0
Hindpaw  - phalanx: phalanx III of 1^st to 5^th 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.