Carbon tetrachloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study along OECD GL 414, 2 doses only, exposure gestation days 6 - 15, no evaluation details given.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1974

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Carbon tetrachloride

EC Number:

200-262-8

EC Name:

Carbon tetrachloride

Cas Number:

56-23-5

Molecular formula:

CCl4

IUPAC Name:

tetrachloromethane

Details on test material:

- Name of test material (as cited in study report): carbon tetra chloride
- Lot/batch No.: Lot No. 9256, Burdick & Jackson Lab. Inc., Muskegon, Michigan

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Spartan
- Age at study initiation:
- Weight at study initiation: average 250 g
- Fasting period before study: not reported
- Housing: wire bottom cages
- Diet (e.g. ad libitum): Purina Rat Chow, Ralston Purina Co. St. Louis, Missouri
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, temperature not reported
- Humidity (%): controlled, exact humidity not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): controlled, exact light cycle not reported

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: 3.7 m³ stainless steel, cubical dynamic exposure chamber
- Method of holding animals in test chamber: not reported
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating vapours: by metering the liquid at known rates into a temperature controlled evaporating flask and diluting this vapours with filtered room air at a rate calculated to give the desired concentration. The nominal concentration of each compound in the chamber atmosphere was calculated from the ratio of the material delivery rate to the rate of total air flow through the chamber.
- Temperature, humidity, pressure in air chamber: not reported
- Air flow rate: not reportes
- Air change rate: not reported
- Method of particle size determination: not applicable
- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used: 3 times daily using a Beckman IR10 infrared spectrophotometer and in addition by using combustion conductivity analysis
- Samples taken from breathing zone: yes


VEHICLE (if applicable)
- no vehicle used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical concentration was determined by analyzing the chamber atmosphere 3 times daily using a Beckman IR10 infrared spectrophotometer with a multipath gas cell having a variable path length of 1-10 m and a volume of 4.5 liters. In addition, the concentration in the chambers was continuously monitored using combustion conductivity analysis to assure the absence of significant deviations from the desired level.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not reported
- Length of cohabitation: not reported.
- Further matings after two unsuccessful attempts: not reported
- Verification of same strain and source of both sexes: not reported
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none reported

Duration of treatment / exposure:

7 hours

Frequency of treatment:

daily from gestation day 6 to 15

Duration of test:

in total 20 days (pregnancy duration)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
300 and 1000 ppm (2137 and 6425 mg/m3).
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
334 and 1004 ppm (2137 and 6425 mg/m3).
Basis:
analytical conc.

No. of animals per sex per dose:

litter size 22 -23

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): not reported

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
- Time schedule: not reported


DETAILED CLINICAL OBSERVATIONS: No data



BODY WEIGHT: Yes
- Time schedule for examinations: not reported


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption of each animal was measured at 2-day intervals throughout the experimental period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: in addition to the reproductive organs: livers, possibly others as well, but not reported

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

Fetal examinations:

- External examinations: Yes, but not detailed
- Soft tissue examinations: Yes, but not detailed
- Skeletal examinations: Yes, but not detailed
- Head examinations: Yes, but not detailed
see table 2 for details

Statistics:

Fisher Exact Probability test and ANOVA + Dunnett's test

Indices:

not calculated

Historical control data:

not reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- Feed consumption of rats at both dose levels was reduced throughout the 10 day exposure period (see Tables 3 and 4 of attached document).
Body weight gain was reduced in a dose dependent manner in both treated groups.
- Hepatotoxicity is reflected by significant increases in SGPT activities during all exposure days, but not thereafter (see Table 5 of attached document). Gross changes in the appearance of the liver characteristic of CTC intoxication (pale, mottled livers) were evident immediately following the last exposure but were not evident 6 days later. The relative liver weight of exposed rats was significantly increased at both doses while absolute liver weight was not always (see Table 6 of attached document).
- There were no gross observations during exposure on demeanor or or signs of toxicity.

Effect levels (maternal animals)

Dose descriptor:

LOAEC

Effect level:

2.11 mg/L air (analytical)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Reproductive parameters (see Table 1 of attached document):
Exposure to Carbon terachloride had no effect on reproductive parameters except for reduced foetal body weight and crown-rump length.
- Foetal examinations (see Table 2 of attached document):
A significant incidence of subcutanous edema was observed at 300 ppm but not at 1000 ppm. The incidence of sternebral anomalies was significantly increased in the foetuses exposed to 1000 ppm.

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

2.11 mg/L air (analytical)

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

LOAEC

Effect level:

2.11 mg/L air (analytical)

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

- Analytical concentration: in inhalation chambers: mean of 334 ± 48 and 1004 ± 90 ppm for the 300 and 1000 ppm groups, respectively.

- Table 1: Effect of inhaled carbon tetrachloride on observations made at Cesarean Section

Statistic	Air control	Carbon	tetrachloride
CTC concentration	--	334 ppm	1004 ppm
No. of litters	43	22	23
% pregnancy	91(43/47)	92(22/24)	82(22/28
Corp. lutea/dam	14±2	14±2	14±3
Implantation sites /litter^b	11±3	13±2	10±4
Live fetuses/litter^b	11±3	12±2	9±4
% resorptions/impl. sites	7(34/492)	6(16/284)	10(25/239)
% litters with resorption	44(19/43)	41(9/22)	48(11/23)
Litters totally resorbed	0/43	0/22	1/23
Resorptions/litter with resorptions	1.8(34/19)	1.8(16/9)	2.3(25/11)
Sex ratio, M/F	56:44	55:45	55:45
Fetal body weight (g)^c	5.64±0.34	5.29±0.34^e	4.96±0.68^e
Fetal crown-rump length (mm)^c	43.7±1.0	42.2±1.0^c	41.8±2.2^e

b Mean ± SD

c Mean of Litter means ±SD

e Significantly different from control by an analysis of variance and Dunnett’s test, p<0.05

- Table 2: Effect of inhaled carbon tetrachloride on the incidence of fetal anomalies among rat litters

	Air control	Carbon 334 ppm	Tetrachloride 1004 ppm
Number of litters examined	22	22	22
	% of Litters affected (No. of litters)
Gross	(0)	(0)	(0)
Skeletal
Skull anomalies (delayed ossific.)	12(5)	9(2)	9(2)
Lumbar ribs or spurs	24(10)	41(9)	27(6)
Vertebral anomalies (bipartite centra)	21(9)	27(6)	14(3)
Sternebral anomalies (unpooled controls)	61(14)	68(15)
(bipartite; delayed ossific.) (unp. cont.)	11(2)		59(13)^c
Total skeletal	58(25)	91(20)^c	68(15)
Soft tissue
Subcuatneous oedema	33(14)	59(13)^c	50(11)
Dilated Ureters	12(5)	14(3)	5(1)
Total soft tissue	51(22)	68(15	59(13)

c Incidence significantly different from control by the Fisher Exact Probability test, p<0.05

- Table 3: Effect of Carbon tetrachloride on rat maternal feed consumption (g/rat/day ± SD)

		Feed consumption on day
	n	4-5	6-7	8-9	10-11	12-13	14-15	16-17	18-19
Control	43	20±3	20±3	20±3	23±2	23±2	23±3	25±5	27±3
300 ppm	22	18±2	12±3^c	13±3^c	13±3^c	16±4^c	18±4^c	23±3	ND^b
1000 ppm	23	19±3	9±3^c	13±3^c	14±3^c	12±4^c	12±5^c	23±7	28±7

ND not determined

c Significantly different from control by Dunnett’s test p<0.05

- Table 4: Effect of Carbon tetrachloride on maternal rat body weight (g, mean ± SD)

	Control	334 ppm	1004 ppm
No. of dams	43	22	23
Gestation day
6	283±17	290±21	275±19
13	317±18^b	281±25^b	253±22^b
21	397±24^b	368±33^b	336±57^b

b Significantly different from control by Dunnett’s test, p<0.05

- Table 5: Effect of inhaled Carbon tetrachloride on SGPT activiy in rats (SE units ±SD)

Day of exp.	N	Control	334 ppm	1004 ppm
2^nd	10	63±3	159±20b	NDc
4^th	10	75±2	154±21b	81±5b
7^th	10	57±	218±51b	ND
10^th	10	56±2	241±68b	154±11b
6 days after last exp.
Non-pregnant	6	57±3	52±5	46±4
Pregnant	10	72±3	62±3	ND

b Significantly different from control by Tukey’s test, p0.05

ND not determined

- Table 6: Effect of inhaled Carbon tetrachloride on absolute and relative liver weights

	Parameter	Control	334 ppm	1004 ppm
Following last exp., nonpregnant (n04)	Abs. liver weight, g, mean±SD	9.0 ±0.6	10.0 ±1.4	10.4 ±1.5
Six days after last Exposure, nonpregnant, (n=6)	Abs. liver weight, g, mean±SD	9.3 ±0.8	9.9 ±1.9	11.4 ±1.4
Six days after last Exposure, pregnant, (n=)	Abs. liver weight, g, mean±SD	14.7 ±1.2 (43)	14.9 ±2.1 (22)	14.8 ±2.4 (23)
Following last exposure, non pregnant (n=4)	Rel. liver weight, mg liver/g bw, mean±SD	34 ±1	43 ±3^b	49 ±4^b
Six days after last exposure, nonpregnant, (n=6)	Rel. liver weight, mg liver/g bw, mean±SD	34 ±3	39 ±5	48 ±3^b
Six days after last exposure, (n=   )	Rel. liver weight, mg liver/g bw, mean±SD	37 ±3 (43)	40 ±3^b (22)	45 ±6^b (23)

b Significantly different from control by Dunnett’s test, p< 0.05

- Table 7: Summary of the toxicity of inhaled Carbon tetrachloride to pregnant rats

	334 ppm	1004 ppm
Embryo- and fetotoxicity
Resorptions	--	--
Fetal body measurements	dec	dec
Incidence of:
Gross anomalies	--	--
Skeletal anomalies	inc	inc
Soft tissue anomalies	inc	--
Maternal toxicity
Weight gain during gestation	dec	dec
Feed consumption	dec	dec
Conception rate	--	--
No. of implantations	--	--
Litter size	--	--
SGPT activity	inc	inc
Absolute liver weight	--	--
Relative liver weight	inc	inc
Gross liver pathology	inc	inc
Demeanor	--	--

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of pregnant rats to carbon tetra chloride at levels of 300 and 1000 ppm resulted in maternal toxicity and retarded foetal development. No teratogenic effects were reported.

Executive summary:

Sprague Dawley rats were exposed by inhalation to 300 or 1000 ppm carbon tetrachloride for 7h/day on days 6-15 of pregnancy. During the dosing period, food consumption by the dams was markedly reduced, resulting in a mean decrease in body weight of 7% (300 ppm) and 15% (1000 ppm) by day 21 in comparison with controls. Evidence of maternal hepatotoxicity was seen in both groups; serum glutamic-pyruvic transaminase (SGPT) was significantly elevated during exposure but had returned to normal by day 21 when relative liver weights were significantly increased but absolute weights unchanged. There was no statistically significant effect on resorptions though 1/23 litters was fully resorbed in the 1000 ppm group. No gross external abnormalities were seen in any group. The data on internal and skeletal anomalies is estimated difficult to evaluate: only information on the number and percentage of litters affected is given, with no data on the numbers of fetuses affected. However, no significant increases in anomalies are reported, except for subcutaneous oedema in the 300 ppm group and sternebral anomalies in the 1000 ppm group. These increases are unlikely to be of any biological significance since oedema was not significantly elevated in the 1000 ppm group and the incidence of sternebral anomalies varied considerably in the two control groups. Fetal body weight and crown-rump length were significantly decreased in a dose related manner but this is not unexpected in view of the severe effect on food consumption in the dams.