Reaction mass of carbon tetrachloride and chloroform and 1,2-dichloroethane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility were observed in repeated dose toxicity studies (oral, inhalation exposure).

The following NOAELs/NOAECs have been established for carbon tetrachloride in fertility studies:

•  Fertility (oral route): NOAEL is 10 mg/kg bw/day

•  Fertility (inhalation route): NOAEC is 630 mg/m3 (100 ppm)

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 2e -Study based on scientific principles but problems on analytical issues (exact concentrations are unknown). - very limited test item information and information on animal husbandry

Qualifier:

no guideline followed

Principles of method if other than guideline:

- 18 male and 18 female rats per dose group; at 2 months intervals 9 of the males of each dose group were mated with 2 females each, the other 9 males mating with 18 sterile (hysterostomized but normally cycling) females;
- the two male groups were switched between the fertile and the sterile female groups after each interval
- to evaluate the basic reproductive capacity of treated females they were mated with untreated males at the age of 6 wk and 2 months before the first mating with treated males

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: in-house breeding
- Age at study initiation: not reported
- Weight at study initiation: (P) Males: average = 130 g; Females: average = 110 g; (F1) not reported
- Fasting period before study: not reported
- Housing: normally: six animals per cages (stainless-steel), mating: 1 male + 2 females per cage, females with weight gain > 60 g after fertilization housed individually until post natal day 10
- Use of restrainers for preventing ingestion (if dermal): no, feeding study
- Diet (e.g. ad libitum): commercial mash, fumigated with the test item, weighted portions twice daily (20 % at 11 a.m., 80% at 7 p.m.)
- Water (e.g. ad libitum): not reported
- Acclimation period: in-house breeding


ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 7 - 10 d
- Mixing appropriate amounts with (Type of food):
3 Kg batches of commercial mash fumigated in hermetically sealed 3 L fumigation containers for 48 h to reach concentrations of 80 ± 5 ppm and 200 ± 20 ppm
mash was stored either in polyvinyl bags with polyamide inlet or glass jars with plastic screwcover with polyamide inlet. Loss of test item during storage (7-10 days) was tested to be < 5 %
test method: cold extraction method (Determination of fumigant residues in cereals and other foodstuffs: A multi-detection scheme for gas chromatography of solvent extracts. Journal of the Science of Food and Agriculture, Volume 20, Issue 9, Date: September 1969, Pages: 566-572, S. G. Heuser, K. A. Scudamore)
- Storage temperature of food: not reported

VEHICLE
- no vehicle used

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 10 d
- Proof of pregnancy: not reported
- replacement of unsuccessfull males not reported
- Further matings after two unsuccessful attempts: the altenating mating of half of the males with fertile and sterilized females allowed after 4 alternations the determination and elimination of fertile males and females
- After successful mating each pregnant female was caged in their original cages (6 animals per cage) until a dam reaches a weight gain of > 60 g post coitus hence she was housed individually until post natal day 10
- Any other deviations from standard protocol: see above

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

cold extraction method (Determination of fumigant residues in cereals and other foodstuffs: A multi-detection scheme for gas chromatography of solvent extracts. Journal of the Science of Food and Agriculture, Volume 20, Issue 9, Date: September 1969, Pages: 566-572, S. G. Heuser, K. A. Scudamore)

Duration of treatment / exposure:

2 years

Frequency of treatment:

twice daily via diet (20 % at 11 a.m., 80% at 7 p.m.)

Dose / conc.:

0 ppm (nominal)

Remarks:

nominal in diet

Dose / conc.:

80 ppm (nominal)

Remarks:

nominal in diet

Dose / conc.:

200 ppm (nominal)

Remarks:

nominal in diet

No. of animals per sex per dose:

18

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: doses were chosen based on a 6 wk range finding study
- Rationale for animal assignment (if not random): not reported

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until wk 13 and once every 2 wk thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumption was recorded weekly up to wk 13 and every second week thereafter
- as the housing was 6 per cage only an average determination was possible


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

Oestrous cyclicity (parental animals):

no analysis reported

Sperm parameters (parental animals):

no analysis reported

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not reported

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of pups, postnatal mortality, weight gain of total litters until post natal day 10,


GROSS EXAMINATION OF DEAD PUPS:
not reported

Postmortem examinations (parental animals):

- serum analysis: total protein, albumin, glucose, urea, cholesterol, uric acid, chloride, sodium, potassium and transaminases (AST ALT)

Statistics:

- Anova
- multiple range test

Reproductive indices:

not calculated

Offspring viability indices:

not calculated

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- around the age of 14 months the animals started to suffer from chronic respiratory disease
- see table 2 for details

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- No significant differences were found between the different groups in growth
- see table 1

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- No significant differences were found between the different groups in feed consumption and feed efficiency

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- see table 3 for details
- the difference between the high dose group (200 ppm) and the other groups in the overall number of parturitions was only due to a low number in the fourths pregnancy test round. In the fifths round the pregnancy rates in this group recovered to normal. So the findings in the fourth round were nterpreted as being incidential.

ORGAN WEIGHTS (PARENTAL ANIMALS)
non data

GROSS PATHOLOGY (PARENTAL ANIMALS)
no data

HISTOPATHOLOGY (PARENTAL ANIMALS)
no data

OTHER FINDINGS (PARENTAL ANIMALS): serum biochemistry
- no significant differences between the control and the treatment groups except for the serum-protein values, which were significantly (P < 0.05) higher in the male rats of the control and low-dose groups compared to the other groups.
The most pronounced difference was found in the globulin fraction, indicating a more advanced stage of chronic disease than in the other animals as confirmed by post mortem analysis of the respiratorytract of these animals.
- The biochemical tests did not reveal any effect either on liver function, as indicated by transaminases and cholesterol values, or on kidney function as shown by the urea and uric acid levels.
- see table 4 for details

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant adverse effects found at any dose level; 15 mg/kg corresponds to the dose of 200 ppm in diet.

Remarks on result:

other: over all generations

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- reported to be unaffected
- as no standard deviation is given to the average values this cannot be checked

CLINICAL SIGNS (OFFSPRING)
no data

BODY WEIGHT (OFFSPRING) (until post natal day 10)
- reported to be unaffected
- as no standard deviation is given to the average values this cannot be checked

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
no data

HISTOPATHOLOGY (OFFSPRING)


OTHER FINDINGS (OFFSPRING)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Reproductive effects observed:

not specified

- Table 1: Mean body weights of male and female rats fed mash fumigated with CTC

		Body weight (g) of rats fed diet containing CTC (ppm)
Duration of treatment (wk)	No fumigant (control)	80	200
Males
0	130	131	130
1	160	160	158
2	192	190	184
3	218	218	210
4	244	250	239
5	271	275	263
6	297	301	290
7	313	314	304
8	328	334	322
9	340	346	337
10	354	362	348
11	362	366	356
12	370	373	362
13	376	380	367
Females
0	110	110	110
1	133	133	130
2	153	151	152
3	170	169	169
4	189	186	186
5	202	197	197

- Table 2: Survival of male and female rats fed mesh fumigated with CTC in a 2-yr feeding study

Duration of study (months)	No. of survivors in groups fed diet containing
	No fumigant (control)		CCl4(ppm)
			80		200
	M	F	M	F	M	F
0	18	18	18	18	18	18
9	17	17	18	18	18	18
12	16	16	17	18	17	18
15	16	16	16	17	16	18
18	15	15	15	16	11	16
21	7	12	8	10	6	10
24	4	9	3	5	4	7

- Table 3: Reproductive performance of female rats, fed mash fumigated with CTC

	Values for groups fed diet containing
	No fumigant (control)	CTC (ppm)
Parameter		80	200
Pregnancy 1
No. of females mated	18	18	18
No. pregnant (% of total)	83	89	72
No. with litters (% of total)	72	89	67
Mean litter size	10.3	12	11.8
Mortality of young (%)
At birth	16.4	0	8.5
At weaning	6.0	11.4	9.2
Mean body weight of young (g)
At birth	5.4	5.1	5.0
At weaning	14.5	13.0	13.3
Pregnancy 2-5
No. of females mated	66	72	72
No. pregnant (% of total)	58	70	56
No. with litters (% of total)*	53	64	44
Mean litter size	9.4	9.2	9.8
Mortality of young (%) at 10 days	9.8	23	11.3
Mean body weight of young (g)
At birth	5.7	5.5	5.4
At weaning	14.2	15.9	14.3

* No. of females producing litters declined sharply in pregnancies 4 and 5 (i.e. in females more than 1 yr. old)

- Table 4: Terminal serum analyses in rats fed mash fumigated with CTC for 2yrs

Serum component	Mean values
	No fumigant (control)	CCl4
		80	200
		Males
Glucose (mg/100ml)	128.0+15.1	139.0+23.0	102.8+6.5
Protein (g/100ml)	7.6+0.09	7.5+0.5	7.1+0.13
Albumin (g/100ml)	3.5+0.06	3.5+0	3.3+0.06
Globulin (g/100ml)	4.1+0.15	4.0+0.5	3.7+0.11
Urea (mg/100ml)	46.5+4.0	48.5+7.5	45.8+3.6
Uric acid (mg/100ml)	1.37+0.06	1.34+0.05	1.48+0.06
Cholesterol (mg/100ml)	103.3+3.3	166.5+20.5	115.7+12.1
GOT(IU)	103.8+12.8	100.5+10.5	109.3+13.4
GPT(IU)	20.0+2.9	22.5+2.5	19.0+2.9
		Males
Glucose (mg/100ml)	97.5+5.0	89.7+2.3	94.6+3.6
Protein (g/100ml)	6.9+0.30	7.6+0.34	7.0+0.18
Albumin (g/100ml)	3.8+0.35	3.5+0.35	3.6+0.09
Globulin (g/100ml)	3.1+0.33	4.0+0.18	3.4+0.17
Urea (mg/100ml)	43.8+5.3	50.6+5.5	43.4+1.5
Uric acid (mg/100ml)	1.60+0.11	1.87+0.29	1.47+0.22
Cholesterol (mg/100ml)	87.3+9.6	65.6+4.7	86.7+9.4
GOT(IU)	119.3+26.0	145.0+25.4	158.6+25.4
GPT(IU)	115.9+3.6	20.5+0.5	25.8+3.3

Conclusions:

The present study (Alumot 1976) reports for CTC a NOAEL of 15 mg/Kg bw after chronic oral dietary administration (2 yrs) in the rat concerning fertility. No guideline was followed and the determined concentrations are only rough estimates.

Executive summary:

The potential of CTC to adversely affect the health and the fertility of rats (unspecified for the strain) was analysed in a chronic 2 year feeding study with food concentrations of 80 and 200 ppm. Body weight development and food consumption were monitored during the course of the study. At the end of treatment, clinical chemistry parameters were investigated. Females (age 3 months) were mated in with untreated males 6 weeks after the start of treatment to test their basic reproductive capacity. At a 2 months intervals 9 of the males of each dose group were mated with 2 treated females each, the other 9 males mating with 18 sterile untreated females. Over the 2 year period the fertility in all females decreased constantly so in total treated males and females were mated 4 times with each other (plus females once with untreated males). The offspring was examined for litter size, viability, bodyweight and body weight gain up to post natal day 10. The parental animals were analyzed for biochemical parameters (total protein, albumin, glucose, urea, cholesterol, uric acid, chloride, sodium, potassium, AST and ALT) after study termination.

The treatment groups did not differ in any of the above stated parameters from the control group except for the number of parturitions in the high dose group in the fourth mating. As the parturition rate in this group recovered to normal in the 5th mating, this result was judged to be incidential. Based on these findings the highest dose was decided to be a NOAEL (about 15 mg/kg/day).

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 413

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan, Inc. (Kanagawa)
- Housing: individually in stainless steel wire hanging cages (150 mm x 216 mm x 176 mm)
- Diet (e.g. ad libitum): sterilized commercial pellet diet (CRF-1, Orienal Yeast Co., Tokyo) ad libidum
- Water (e.g. ad libitum): sterilized drinking water, ad libidum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.5 - 25.1°C
- Humidity (%): 52.3 - 57.1%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 / 12

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: exposure chamber (4.3 cbm, glass and stainless steal) maximal load: 50 stainless steal wire mesh cages
- Method of holding animals in test chamber: stainless steal wire mesh cages, animals individualIy housed
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating vapor: vapor-air mixture was generated by bubbling clean air through liquid of CTC in a temperature-regulated glass flask, and by cooling it through a thermostatted condensor.The airflow containing the saturated vapor was diluted with clean air and then warmed in a thermostatted circulator which served to stabilize the vapor concentration by complete gasification of CTC and by buffering the pressure difference between the glass flask and the inhalation chamber. The vapor-air mixture flow rate was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to the inhalation exposure chamber.
- Temperature, humidity, pressure in air chamber: 22 ± 2 °C, 55 ± 10 %, - 50 to - 150 Pa
- Air flow rate: 860 l/min
- Air change rate: 12 changes/h
- Method of particle size determination: not applicable (vapor of volatile solvent)
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of CTC vapor were monitored by gas chromatography once every 15 min.
- Samples taken from breathing zone: yes

VEHICLE
- No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography, sampling every 15 min during exposure, no further details given

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hrs/day, 5 days/week

Details on study schedule:

- Dose selection rationale: the lowest inhalation concentration of 64 mg/m³ (10 ppm) was selected in consideration of the validity of the present occupational exposure limit (OEL) value for CCld
- Rationale for animal assignment (if not random): not reported
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported

Dose / conc.:

10 ppm (analytical)

Remarks:

64 mg/m³

Dose / conc.:

30 ppm (analytical)

Remarks:

192 mg/m³

Dose / conc.:

90 ppm (analytical)

Remarks:

576 mg/m³

Dose / conc.:

270 ppm (analytical)

Remarks:

1728 mg/m³

Dose / conc.:

810 ppm (analytical)

Remarks:

5184 mg/m³

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, sham-exposed

Basis for effect level:

other: See 'Remarks'

Remarks on result:

other: See 'Remarks'

Remarks:

F344/DuCrj rats (10/sex/group) were exposed (whole body) to 10, 30, 90, 270, or 810 ppm carbon tetrachloride vapor for 6 hours/day, 5 days/week for 13 weeks (Naganoet al., 2007b). Males at 270 or 810 ppm had significantly reduced absolute testes weights, but relative weights were similar to those in controls. Females at 810 ppm had significant reductions in absolute and relative ovary weights. Gross pathology revealed an increase in seminal vesicle atrophy in animals treated with 810 ppm (Japan Bioassay Research Center, undated). The low concentration of 10 ppm (63 mg/m3) was, however, a LOAEC for hepatic effects based on increased liver weight and histopathology. From additional information received about the Nagano studies, it might be prudently concluded that, if any changes in the estrous cycle occurred, it would have resulted in histopathology in reproductive organs in females. It can be concluded that such effects only are seen at very high doses inducing pronounced hepatotoxicity and mortality.   

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Executive summary:

F344/DuCrj rats (10/sex/group) were exposed (whole body) to 10, 30, 90, 270, or 810 ppm carbon tetrachloride vapor for 6 hours/day, 5 days/week for 13 weeks (Nagano et al., 2007b). Males at 270 or 810 ppm had significantly reduced absolute testes weights, but relative weights were similar to those in controls. Females at 810 ppm had significant reductions in absolute and relative ovary weights. Gross pathology revealed an increase in seminal vesicle atrophy in animals treated with 810 ppm (Japan Bioassay Research Center, undated). The low concentration of 10 ppm (63 mg/m³) was, however, a LOAEC for hepatic effects based on increased liver weight and histopathology. From additional information received about the Nagano studies, it might be prudently concluded that, if any changes in the estrous cycle occurred, it would have resulted in histopathology in reproductive organs in females. It can be concluded that such effects only are seen at very high doses inducing pronounced hepatotoxicity and mortality. 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

630 mg/m³

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Available data suggest that carbon tetrachloride, when administered at doses or concentrations which cause pronounced hepatotoxicity, may also affect (male) fertility (see further below) by oral route and by inhalation. 

Oral exposure:

In a 2 -year Alumot et al.(1976) conducted a 2-year feeding study in which rats were given doses of 0, 5-8, or 15-25 mg/kg bw/day (0, 80, 200 ppm nominal in diet)of carbon tetrachloride. Rats were fed food, fumigated with carbon tetrachloride, during short periods per day. Based on calculated food intake and due to measured losses, dose levels were reduced to 10-18 mg/kg bw.In this study, 18 males and 18 females were used per dose group. Females were first mated with untreated males, 6 weeks after the start of the treatment, to test their basic reproductive capacity. Then the treated rats were mated 5 times at 2-month intervals:at each interval 9 of the males of each dose group were mated with 2 females each, the other 9 males mating with 18 sterile untreated females. The two male groups were switched between the fertile and the sterile female groups after each interval. Although this study is a pre-guideline study, and therefore not fulfilling all requirements of the currently accepted OECD guidelines, this study presented information on effects on fertility. The authors did not observe any adverse effects on fertility in either males or females. No adverse effects attributable to either treated males or treated females were observed in terms of pregnancy rate, delivery rate, live litter size, offspring weight or mortality. The highest dose (200 ppm in the diet), estimated to be equivalent to an intake of 10-18 mg carbon tetrachloride/kg bw/day, also did not cause liver damage or alterations to the body weight of the rats. Although the study certainly has limitations, the results indicate that the tested doses of carbon tetrachloride had no effects on fertility. A NOAEL of 10 mg/kg bw/day (the lower of the corrected values) for parental toxicity and reproductive toxicity can be derived using default assumptions.

 

Inhalation exposure:

In an inhalation study by Adams et al.(1952), male Wistar-derived rats were exposed to 5, 10, 25, 50, 100, 200, or 400 ppm carbon tetrachloride for 7 h per day, 5 days per week, for 24–29 weeks. At 200 ppm, testes weight was decreased, and some tubules showed complete atrophy of the germinal elements. Moderate to marked degeneration of testicular germinal epithelium was observed at 400 ppm. The concentrations producing testicular degeneration were highly toxic as demonstrated by high mortality (9/15 and 13/15 animals died prematurely at 200 and 400 ppm, respectively). The NOAEC for rats in this study was 5 ppm (32 mg/m3), based on liver effects seen at 10 ppm or higher.

 In well-conducted chronic toxicity/carcinogenicity studies on carbon tetrachloride in rats and mice after inhalation exposures, reported by the Japan Bioassay Research Center (Nagano et al. 2007b), effects were observed in reproductive organs at high dose levels, as described in the CTC dossier.

 

F344/DuCrj rats (10/sex/group) were exposed (whole body) to 10, 30, 90, 270, or 810 ppm carbon tetrachloride vapor for 6 hours/day, 5 days/week for 13 weeks (Nagano et al., 2007a & 2007b). Only males at 270 or 810 ppm had significantly reduced absolute testes weights, but relative weights were similar to those in controls. Females at 810 ppm had significant reductions in absolute and relative ovary weights. Gross pathology revealed an increase in seminal vesicle atrophy in males treated with 810 ppm (Japan Bioassay Research Center, undated). The low concentration of 10 ppm (63 mg/m3) was, however, a LOAEC for hepatic effects based on increased liver weight and histopathology.

 

Groups of F344/DuCrj rats (50/sex/group) were exposed (whole-body) to 5, 25, or 125 ppm carbon tetrachloride vapor for 6 hours/day, 5 days/week for 104 weeks (Nagano et al., 2007a ; not reported in this dossier). In animals which died prematurely or were sacrificed in moribund condition, an increase in testes atrophy was observed at 25 ppm or higher. Relative testes weight was decreased in surviving animals at 125 ppm (Japan Bioassay Research Center, undated). Under the conditions of this study, however, the NOAEC for systemic toxicity was found to be 5 ppm (32 mg/m3) based on liver toxicity at 25 ppm, according to the study authors.

 

From additional information received about the Nagano studies, it might be prudently concluded that, if any changes in the estrous cycle occurred, it would have resulted in histopathology in reproductive organs in females. It can be concluded that such effects only are seen at very high doses inducing pronounced hepatotoxicity and mortality. 

 

The same group also conducted a 2-year study using Crj:BDF1 mice under the same exposure conditions. Histopathology revealed ceroid deposition in the ovaries of mice that were exposed to 125 ppm and dilatation of the uterus at 25 ppm and higher. In male mice, absolute and relative testicular weights were increased at 25 ppm (there was just one surviving animal at 125 ppm) (Japan Bioassay Research Center, undated). Under the conditions of this study, the NOAEC for systemic toxicity was found to be 5 ppm (32 mg/m3), again based on liver toxicity, according to the study authors, equivalent with the NOAEL for reproductive organs.

Based on all of the studies conducted by inhalation and available in the CTC dossier and only those described above are explaining that the NOAEC of 100 ppm (630 mg/m3) was determined by inhalation for the fertility.

 

A set of three recent studies (not reported in this dossier) performed by the same group of researchers (Sönmez et al., 2013; Türk et al., 2013; Yüce et al. 2013), indicated effects on the rat testis induced by high doses of carbon tetrachloride. It should be noted, however, that these studies were not designed to evaluate the toxic properties of carbon tetrachloride, but were aimed to study the effects of antioxidants on oxidative stress in the testes using high doses of carbon tetrachloride as a model compound. An oxidative stress mechanism for testicular damage was hypothesised by the formation of free oxygen radicals which might have a high affinity to cell membrane lipids leading to tissue damage of testis and effects on sperm during maturation. Thus, the investigators did not investigate any other organs mainly because they did not consider it of relevance for their research model. However, due to the well-developed database on hepatic effects of carbon tetrachloride it is considered as extremely likely that the very high oral doses used produced significant liver (and probably kidney) toxicity.

The above studies have very little relevance for the reproductive risk assessment of carbon tetrachloride because specific effects (oxidative stress) were studied at a very high dose of carbon tetrachloride which is known to produce significant hepatotoxicity.

Dermal exposure:

no data

For additional information on carbon tetrachloride, its dossier is available on the ECHA website.

Effects on developmental toxicity

Description of key information

The following NOAELs/NOAECs have been established for carbon tetrachloride in developmental toxicity studies:

•  Developmental toxicity (oral route): NOAEL is 25 mg/kg bw/day

•  Developmental toxicity (inhalation route): LOAEC is 2112 mg/m3 (334 ppm)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, well- documented publication/study report which meets basic scientific principles, nevertheless the maternal toxicity was not fully assessed: no full gross macroscopic examination was performed on any dams, only uteri of females that did not deliver were stained to detect full-litter resorptions "FLR".

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

: see below

Principles of method if other than guideline:

- deviations from OECD TG 414: 12-14 dams per dose, treatment on gestagation days 6 - 15, narrow dose range, delivery allowed and pups and dams sacrificed and examined on post natal day 6
- comparison of 2 vehicles (cornoil and amulphor)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Indianapolis, IN, U.S.A.)
- Age at study initiation: not reported
- Weight at study initiation: average = 161.35 g
- Fasting period before study: not reported
- Housing: not reported
- Diet (e.g. ad libitum): Purina Lab Chow 5001, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: animals were delivered timed-pregnant by the supplier


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 1.1
- Humidity (%): 50 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: corn oil or 10% aqueous emulphor EL-620

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
dosing formulations prepared at appropriate concentrations to provide the desired dose when administered at 1 ml formulation/kg body weight
vehicles: either corn oil or 10% aqueous emulphor EL-620 in distilled deionized water
individual doses were calculated based on the individual body weight on gestation day 6


VEHICLE
- Justification for use and choice of vehicle (if other than water): cornoil is a standard vehicle for volatile organic solvents, emulphor was tested in comparison to detect vehicle based effects
- Concentration in vehicle: 0 - 75 mg/mL
- Amount of vehicle (if gavage): 1 ml formulation/kg body weight
- Lot/batch no. (if required): not reported
- Purity: not reported
- Supplier: corn oil: Sigma Chemical Co., St. Louis, MO, U.S.A.; 10% aqueous emulphor EL-620: castor oil ethoxylated; Rhone Poulenc, Cranbury,
NJ, U.S.A.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- no detailed information
- animals were delivered timed pregnant

Duration of treatment / exposure:

gestation day 6 - 15 (copulatory plug or vaginal sperm = gestation day 0)

Frequency of treatment:

once daily

Duration of test:

gestation day 0 to post natal day 6 (= 28 days)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- 2 sets of experiments (replicates), but cumulated for statistic analysis
- 12 - 14 dams per dose group (cumulated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of previous work where corn oil was used as the vehicle. In those studies CTC caused Full Litter Resorption "FLR", reduced pup weights, and maternal toxicity at 112.5 and 150 mg/kg/day.
- Rationale for animal assignment (if not random): equal distribution of body weights among groups

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: "troughout the experimental period"

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 5, 6, 8, 10, 13, 16 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post natal day 6 (theoretically gestation day 27)
- Organs examined: uteri of females that did not deliver stained with 10 % ammonium sulfide

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No, dams were allowed tom deliver
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes, but details not reported
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Examination for full litter resorptions (FLRs): Yes

Fetal examinations:

Pups were analysed as dams were allowed to deliver:
pups were analyzed for their weight and individually examined on post natal days 1 and 6

Statistics:

- dams that died or had only one implant were excluded from statistical analyses
- continuous variables
were evaluated bv. analv.s is of variance (ANOVA) or covariance (Kleinbaum, D. G., Kupper, L. L., and Muller, K. E. (1988). AppliedRegression Analysis and Other Multivariable Methods, 2nd ed. PWS-Kent, Boston, MA.) using the general linear models (GLM) procedure on SAS (SAS Institute Inc. (1988). SAS/STAT User's Guide, Release 6.03 Edition. SAS Institute, Cary, NC.).
- Gestation lengths in each replicate were ranked according to the time and stage that parturition was observed; GLM analyses were applied to the ranked data
- Pup weights were analyzed as litter means with the number of live PD 1 pups as a covariate.
- the number of implants was used as a covariate in the analysis of litter size.
- When a significant treatment effect was detected, Student's t test on leastsquares means was used for painvise comparisons between individual CTC treated groups and their same-vehicle control and between groups receiving different vehicles at the same dosage
- The incidences of FLR in groups receiving the same dosage but different vehicles were compared using Fisher's Exact Test. In addition, ANOVA was used to compare replicates and to evaluate possible vehicle-dose interactions on maternal weight gain during GD 6-8 as well as on the incidence of FLR
- The BMD was defined as the lower 95% confidence limit of the administered dose predicted to cause a 5% increase in response (Allen et al., 1994). For endpoints with a zero background rate, this predicted dose is the estimated 5% effect dose (ED05). Teralog software (Howe, 1994) was used to calculate the ED05 and BMD for FLR using a generalization of a log-logistic model (Kupper et al., 1986). Intralitter correlations and litter-size variables were set to zero and, due to the all-or-none nature of
the FLR endpoint, litter sizes were set to one.

Indices:

no. of full litter resorptions (see table 1)

Historical control data:

not reported

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- no fatalities occured at any dose
- Piloerection was seen with dose-related frequency at 50 and 75 mg/Kg for both vehicles, but in more animals and for longer periods in the groups receiving corn oil.
- Kyphosis was observed only in females receiving 75 mg/Kg in corn oil
- Significant maternal weight loss on gestation days 6-8 evident at 75 mg/Kg with both vehicles and at 50 mg/Kg with emulphor only
- Between vehicle comparisons revealed a significantly greater effect on gestation days 6-8 body weight change in animals receiving 75 mg/Kg in corn oil compared to animals receiving emulphor

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- All litters exposed to 0 or 25 mg/Kg survived the experimental period
- at 50 and 75 mh/Kg FLRs both vehicle (corn oil: 42 and 67% at 50 and 75 mgkg, respectively; emulphor: 14 and 8% at 50 and 75 mgkg, respectively (see tables 1 and 2); at 75mg/kg, at 75 mg/Kg the incidence of FLR with the emulphor (8%) was significantly less than with corn oil (67%).
- For surviving litters, there were no effects on gestation length, pre- or postnatal survival, or pup morphology.
- Nondose-related reductions in pup weights on PD 1 were noted at 25 mgkg in corn oil compared to the vehicle control group (weight difference compensated at post natal day 6)
- Litters exposed to 25 or 50 mgkg in corn oil had significantly reduced pup weights on PD 1 compared to their emulphor counterparts. These nondose-related reductions in pup weight were not attributed to treatment.
- On PD 6, all of these pups had survived and their weights were comparable to those of controls.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

- Table 1: Data Summary for Rats treated by gavage on gestation days 6-15 with Bromodichloromethane (BDCM) or Carbon Tetrachloride (CTC)

Vehicle	Dose (mg/kg)	No. dams	Maternal DG-6 body weight (g)	No. litters deli­vered	Day-1 live pups	Pup weight (g)
						Day 1	Day 6
Corn oil	0	13	162.2 ± 3.2	13	8.5 ± 0.8	5.4 ± 0.1	9.5 ± 0.2
	25	13	160.2 ± 4.4	13	8.5 ± 0.6	5.2 ± 0.1	9.6 ± 0.2
	50	12	162.3 ± 4.4	7	9.6 ± 0.7	5.1 ± 0.1	9.5 ± 0.1
	75	12	160.3 ± 3.7	4	7.5 ± 1.2	5.4 ± 0.1	9.8 ± 0.4
Aqueous	0	12	163.9 ± 3.7	12	9.1 ± 0.7	5.3 ± 0.1	9.6 ± 0.3
	25	12	159.1 ± 3.3	12	8.3 ± 0.6	5.4 ± 0.1	9.8 ± 0.2
	50	14	159.7 ± 3.4	12	8.8 ± 0.9	5.4 ± 0.1	9.1 ± 0.3
	75	12	163.1 ± 4.0	11	8.4 ± 0.8	5.3 ± 0.1	9.4 ± 0.3

Values are group means ± SE. Pup weights are group means of the litter means

a: includes one litter that was delivered delayed and was not viable

- Table 2: Summary of Effect Levels for Full-Litter Resorption

Vehicle	NOAEL	LOAEL	Response at LOAEL	Estimated BMD05	Estimated BMDL05
	(mg/Kg bw)	(mg/Kg bw)	(%)	(mg/Kg bw)	(mg/Kg bw)
Corn oil	25	50	42	30	18.9
Emulphor	25	50	14	39.5	14.0

Conclusions:

The present study (Narotsky, 1997 b) reports for CTC a NOAEL of 25 mg/Kg bw in the rat concerning both the dams and the foetuses toxicities after oral application. The corresponding LOAEL is 50 mg/Kg bw.

Executive summary:

The potential of CTC to cause developmental toxicity was analyzed with a modified Prenatal Developmental Toxicity Test where dams were allowed to deliver and pups were analyzed on post natal day 6. Analysis were focused on full litter resorption as previous studies of the authors (Narotsky 1995) have discovered this endpoint to be the most sensitive developmental toxicity effect of CTC in Fischer 344 rats. In addition the effect of different vehicles on this endpoint should be evaluated. Pregnant Fischer 344 rats were treated with 0, 25, 50 and 75 mg/Kg bw CTC in either cornoil or 10 % aqueous emulphor (emulphor EL-620: castor oil ethoxylated) via gavage on gestation days 6 - 15. Dams were weighted on gestation days 5, 6, 8, 10, 13, 16 and 20, pups on post natal days 1 and 6. Dams were examined for clinical signs throughout the study period, pups were examined for clinical signs and obvious abberations on post natal day 1 and 6. Dams were sacrificed on post natal day 6 and uteri analyzed for inplantations sites. Uteri of dams that had not delivered were stained with 10% ammonium sulfide fordetection of full-litter resorptions. None of the dams died during the study. Maternal effects comprised piloerection at 50 and 75 mg/Kg for both vehicles (more severe with corn oil), kyphosis at 75 mg/Kg in corn oil and

maternal weight loss on gestation days 6 -8 evident at 75 mg/Kg with both vehicles and at 50 mg/Kg with emulphor only. Embryotoxic effects were obvious at 50 mg/Kg bw and higher as specified by the abundance of full litter resorptions (corn oil: 42 and 67% at 50 and 75 mg/kg, respectively; emulphor: 14 and 8% at 50 and 75 mg/kg, respectively) which were absent at 0 and 25 mg/Kg bw. Significant differences in pup weight effects on post natal day 1 in various dose groups are judged to be incidentially as they were only slight, not dose related and recovered until post natal day 6.

Based on these results a NOAEL of 25 mg/Kg bw and a LOAEL of 50 mg/Kg bw for developmental toxicity in the rat for CTC after oral administration were derived. The same NOAEL and LOAEL values were derived for maternal toxicity based on body weight effects which were nevertheless very slight, and for piloerection. In addition BMD05 and BMDL05 values were calculated for developmental toxicity from the data (corn oil: BMD05 = 30.0, BMDL05 = 18.9; emulphor: BMD05 = 39.5, BMDL05 = 14.0; all in mg/K bw).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEC

2 112 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

 Oral exposure

In a study by Narotsky et al.(1997b), carbon tetrachloride was administered to rats from GD 6 to GD 15 by oral gavage at 0, 25, 50 or 75 mg/kg bw. Full litter resorptions were observed early during the gestation. No developmental toxicity was evident in surviving litters. There was an association between the pregnancy loss and maternal body weight loss. This suggests that maternal toxicity plays a role in the observed effect on full litter resorptions. In particular, alterations in progesterone levels could be involved in these full litter resorptions. Altered progesterone levels may be due to hepatic toxicity induced by carbon tetrachloride since the liver plays a major role in the steroid biosynthesis and catabolism. Full litter resorptions only occurred at doses where maternal toxicity was already evident. The LOAEL for developmental effects was 50 mg/kg bw, based on an increase in litter loss by resorptions. The NOAEL in this study was 25 mg/kg bw. 

Inhalation exposure

In the study by Schwetz et al. (1974) (not reported in this dossier), groups of pregnant Sprague-Dawley rats (22-23 animals/group)were exposed by inhalation to targeted concentrations of 300 or 1000 ppmcarbon tetrachloride for 7h/day on days 6-15 of gestation. 

Maternal toxicity was observed in both exposure groups. Food consumption and body weights were significantly reduced compared with controls, and hepatotoxicity was indicated by significantly elevated serum ALT (four-fold increase over control), gross changes in liver appearance, and significantly increased relative liver weight (26% at 334 ppm and 44% at 1,004 ppm). There was no statistically significant effect on resorptions (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 are difficult to evaluate: only information on the number and percentage of litters affected is given, with no data on the numbers of foetuses affected. However, no significant increases in anomalies were reported, except for subcutaneous oedema in the 300 ppm group and sternebral anomalies in the 1000 ppm group. These increases were judged unlikely to be of biological significance since oedema was not significantly elevated in the 1000 ppm group and the incidence of sternebral anomalies varied considerably in the control groups. Foetal body weights and crown-rump length were significantly decreased in a concentration- related manner, but this is not unexpected in view of the severe effect on food consumption in the dams.No other increases in individual soft tissue or skeletal anomalies were reported, and there was no effect on conception rate, number of implantations, litter size, or number of resorptions. 

In this study, the LOAEC was 334 ppm (2112 mg/m³; measured concentration), both for maternal and developmental toxicity.

Dermal Exposure

No study available

For additional information on carbon tetrachloride, its dossier is available on the ECHA website

Justification for classification or non-classification

Harmonized classification

The registered substance and its constituent carbon tetrachloride have no harmonized classification regarding reproductive or developmental toxicity according to the Regulation (EC) No 1272/2008 (CLP).

Self-classification:

Based on the available data, no additional classification is proposed for reproductive or developmental toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

There were no data regarding dermal route for this constituent.