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Key value for chemical safety assessment

Effects on fertility

Description of key information

no data

Link to relevant study records

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Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
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 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.)
Remarks:
Doses / Concentrations:
0, 80, 200 ppm in diet
Basis:
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
Gross pathological findings:
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:
mg/kg bw/day (nominal)
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: Generation: over all generations (migrated information)
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)
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, and 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 (abotu 15 mg/kg/day).

Endpoint:
toxicity to reproduction
Remarks:
other: repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2007
Reliability:
2 (reliable with restrictions)
Reason / purpose:
reference to same study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
64, 192, 576, 1728 and 5184 mg/m³ (10, 30, 90, 270 and 810 ppm)
Basis:
analytical conc.
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, sham-exposed
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified Generation not specified (migrated information)
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 (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. 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no recent guideline studies with carbon tetrachloride addressing the endpoint of fertility in experimental animals. Available studies are generally old and were performed to then-current scientific standards. Although somewhat more limited than studies done according to current guidelines, published results allow the conclusion that carbon tetrachloride, when administered at doses or concentrations which cause pronounced hepatotoxicity, may also affect (male) fertility (see further below).

 

In a three-generation study, groups of 24 albino rats were exposed by inhalation to carbon tetrachloride concentrations of 50, 100, 200 and 400 ppm for 5 days/week up to 10.5 months. The authors reported that fertility was impaired in the animals exposed to 200 and 400 ppm, but not at the lower concentrations. A decrease in fertility was observed at 200 and 400 ppm; however, since both sexes were exposed, it not clear whether this was due to effects on the males or females. Fertility was unaffected at 100 ppm, and increased at 50 ppm. Embryotoxic, foetotoxic, and teratogenic effects were not mentioned; liver cirrhosis was observed at 50 ppm (315 mg/m3) or higher, which was the LOAEC for rats in this study (Smyth et al.,1936; Smyth and Smyth, 1935).

 

In several publications (Chatterjee, 1966; Chatterjee, 1968; Kalla and Bansal, 1975), testicular atrophy, abnormality in the process of spermatogenesis, inhibition of oestrous cycle, and weight and vascularization decreases of ovary and uterus, were observed after carbon tetrachloride administration. The relevance of these studies for hazard assessment is questionable, as these studies were conducted by intraperitoneal administration of very high doses (> 2000 mg/kg bw).

 

No sperm head anomalies were seen in CBA*BALB/c mice after intraperitoneal administration of 0.1 to 1.5 mg carbon tetrachloride per kg body weight for 5 days (IPCS, 1999).

 

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.

 

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.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. 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.

 

In well-conducted chronic toxicity/carcinogenicity studies on carbon tetrachloride in rats and mice after oral or inhalation exposures, reported by the Japan Bioassay Research Center, effects were observed in reproductive organs at high dose levels, as described below.

 

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). 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). 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.

 

A set of three recent studies 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 (see below) used produced significant liver (and probably kidney) toxicity.

 

Sönmez et al. (2013) treated male Wistar rats with a single weekly oral dose of 0.25 mL carbon tetrachloride (in olive oil) per kg body weight for 10 weeks. This represents a weekly bolus of 375 mg/kg bw for 10 consecutive weeks. Ignoring potential bolus effects, this dose corresponds to a daily dose of 75 mg/kg bw/day (5 days/week). This dose level is far above the NOAEL for hepatotoxic effects of carbon tetrachloride observed in a number of studies. In this study, carbon tetrachloride administration caused significant decreases in absolute and relative weights of all reproductive organs studied; it can be assumed that body weights were also reduced by at least 10%.

 

In the study by Türk et al.(2013), carbon tetrachloride was again used as a model inducer of sperm damage, lipid peroxidation, and testicular apoptosis in male rats treated weekly with 0.25 mL/kg bw of carbon tetrachloride in olive oil by gavage for 10 weeks (corresponding to 75 mg/kg bw/day; 5 days/week). The aim of the study was to investigate whether pomegranate juice (PJ) consumption had an ameliorating effect on carbon tetrachloride-induced sperm damage and testicular apoptosis associated with the oxidative stress in male rats. Carbon tetrachloride administration at this high dose caused significant decreases in body and reproductive organ weights, sperm motility and concentration, and testicular catalase activity, significant increases in malondialdehyde (MDA) level, and abnormal sperm rate and apoptotic index, along with some histopathological damage, when compared with the control group. Again, the dose level used in this study was well above the dose level reported to be hepatotoxic in rats.

 

In a similar study by Yüce et al. (2013), the potential protective effects of cinnamon bark oil on effects of carbon tetrachloride on the male reproductive system were investigated. Again, carbon tetrachloride administration at this high dose (equivalent to 75 mg/kg bw/day) caused significant decreases in body and reproductive organ weights, testicular catalase activity, sperm motility and concentration, and significant increases in lipid peroxidation level, abnormal sperm rate, and apoptotic index, along with some histopathological damage compared with the control group.

 

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.

 

Short description of key information:

No effects on fertility were observed in repeated dose toxicity studies (oral, inhalation exposure). The NOAEL in an oral study with carbon tetrachloride is 10 mg/kg. The NOAEC in an inhalation fertility study is 100 ppm (630 mg/m3). There are no studies on fertility by dermal 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)

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

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only as short abstract available
Qualifier:
no guideline followed
Principles of method if other than guideline:
- treatment of pregnant female F344 rats with 150 mg/kg bw CTC in corn oil (gavage) on gestagation day 8
- monitoring of luteinizing hormone levels 0 - 20 h post treatment
- additional groups of animals treated with and without CTC on gestagation day 8 and with and without human choriongonadotropin (4 groups in total were analyzed for incidence of full liter resorption)
GLP compliance:
no
Limit test:
no
Remarks:
Doses / Concentrations:

Basis:

Abnormalities:
not specified
Developmental effects observed:
not specified
Executive summary:

The present study (Narotsky, 1995 a) analyzes the potential mode of action of CTC on full litter resorptions in rats:

"Full litter resorption (FLR), i.e., pregnancy loss, has been shown to occur in gravid F-344 rats following exposure to a of halogenated chemicals. Previous work showed that FLR is associated with reduced progesterone levels and is rescued by exogenous progesterone. To gain insight into the mechanism of this response, we administered 0 or 150 mg CTC/kg in corn oil by gavage on gestation day (GD) 8 and measured serum LH at 0, 5, 14, and 20 hr by repeated sampling from the lateral tail vein. Additional animals were treated with CTC (150 mg/kg on GD 8 ) + hCG (0.5 IU/rat/day in saline subcutaneously on GD 8-11), Or both CTC and human choriongonadotropin (hCG); controls received both vehicles. All animals were allowed to deliver and their litters were examined on postnatal days 1 and 6. Non gravid uteri were stained with 10% ammonium sulfide to detect sites of early resorption. All control dams successfully maintained their litters; however, 11 (65%) of 17 rats receiving only CTC had FLR. Dams whose litters were later found resorbed had dramatically reduced serum LH levels at 5 hr with significant reductions evident through 20 hr. Control dams had values of 0.22, 0.20, 0.21, and 0.16 ng LH/ml at 0, 5, 14, and 20 hr, respectively, whereas the corresponding values for dams with FLR were reduced by 17, 67, 69, and 65%. All 15 females receiving CTC, and hCG successfully maintained their litters; no adverse developmental effects were evident. These data indicate that CTC induced FLR is associated with reduced LH levels, and that hCG, apparently acting as a surrogate for LH, effectively rescues the pregnancy. Further work is planned to investigate the possible causes of reduced LH levels in halocarbon-induced pregnancy loss."

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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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


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

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
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.
Dose descriptor:
LOAEC
Effect level:
2.11 mg/L air (analytical)
Basis for effect level:
other: maternal toxicity
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.
Dose descriptor:
NOAEC
Effect level:
2.11 mg/L air (analytical)
Basis for effect level:
other: teratogenicity
Dose descriptor:
LOAEC
Effect level:
2.11 mg/L air (analytical)
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

- 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 /litterb

11±3

13±2

10±4

Live fetuses/litterb

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±0.34e

4.96±0.68e

Fetal crown-rump length (mm)c

43.7±1.0

42.2±1.0c

41.8±2.2e

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±3c

13±3c

13±3c

16±4c

18±4c

23±3

NDb

1000 ppm

23

19±3

9±3c

13±3c

14±3c

12±4c

12±5c

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±18b

281±25b

253±22b

21

397±24b

368±33b

336±57b

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

2nd

10

63±3

159±20b

NDc

4th

10

75±2

154±21b

81±5b

7th

10

57±

218±51b

ND

10th

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 ±3b

49 ±4b

Six days after last exposure, nonpregnant, (n=6)

Rel. liver weight, mg liver/g bw, mean±SD

34 ±3

39 ±5

48 ±3b

Six days after last exposure,
(n=
  )

Rel. liver weight, mg liver/g bw, mean±SD

37 ±3
(43)

40 ±3b
(22)

45 ±6b
(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

--

--

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEC
2 112 mg/m³
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no recent guideline studies addressing the endpoints of reproductive and developmental toxicity in experimental animals exposed by the oral or inhalation routes. Available studies are generally old and were performed to then-current scientific standards. Although somewhat more limited than studies done according to current guidelines, available data suggest that the foetus is not preferentially sensitive to carbon tetrachloride, and effects of carbon tetrachloride on foetal development and post-natal survival are secondary to maternal toxicity.

Inhalation studies

There is one developmental toxicity study using the inhalation route of exposure.In the study by Schwetz et al. (1974), 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.

 

As described in an abstract of an unpublished doctoral dissertation, pregnant Sprague-Dawley rats were exposed to ambient air or 250 ppm (1575 mg/m3) carbon tetrachloride vapour for 8 hours/day on GDs 10–15. There were no adverse effects on maternal body weight, litter size, the ratio of live to still births, or the incidence of skeletal abnormalities (Gilman, 1971, as referenced in EPA, 2010 and IPCS, 1999).

 Oral studies

No teratogenic effects (morphological anomalies) were reported in rats following maternal oral exposure to carbon tetrachloride on 1 or 2 successive days of gestation beginning between gestational days 7 and 11. Total resorption of fetuses was reported at maternally toxic doses. Doses of 1,400 mg/kg bw/day during gestation caused marked maternal toxicity in rats, and total resorption of fetuses in some animals, but no adverse effects in surviving litters (Wilson 1954, as referenced by EPA, 2010 and ATSDR, 2005).

 

Narotsky and Kavlock (1995) reported the results of a developmental toxicity screening study in rats with carbon tetrachloride. Groups of 16-23 timed-pregnant F344 rats were treated with 112.5 or 150 mg/kg bw/day of carbon tetrachloride by oral gavage in corn oil on gestational days 6–19. Maternal body weight was monitored periodically throughout gestation. The dams were allowed to litter. Pups were examined on postnatal days (PNDs) 1, 3, and 6 and weighed on PNDs 1 and 6. The number of females actually pregnant in each group was 13, 9, and 14 in the control-, low-, and high-dose groups, respectively. Both doses of carbon tetrachloride caused maternal weight loss (4–8%) early in the treatment period and reduced extra-uterine weight gain (35–45% lower than controls) over the treatment period as a whole. The incidence of full-litter resorption was markedly increased in both groups administered carbon tetrachloride: 4/9 (44%) and 10/14 (71%) in the 112.5 and 150 mg/kg-day groups, respectively (versus 0/13 in controls). As a result, prenatal loss (reported as percent loss per litter) was significantly increased in both treated dose groups. Implantation sites of the resorbed litters were not grossly visible in most cases, requiring ammonium sulfide stain to find them. This suggested to the researchers that the resorptions occurred early in pregnancy. Among dams that maintained their pregnancies, resorptions were not increased nor were postnatal losses. Pup body weight was not markedly affected by treatment. No malformations were associated with carbon tetrachloride exposure.

 

In follow-up investigations, these researchers suggested that the all-or-none nature of the observed resorptions points to a maternally-mediated response, and reported evidence that the response is associated with reduced levels of progesterone and luteinizing hormone (LH) in the dams. When a single dose of carbon tetrachloride (150 mg/kg bw) was administered to pregnant Fischer 344 rats on gestation day 6, 7, 8, 10, or 12, full litter resorption was observed in 36–72% of dams treated on gestation days 6–10; none was seen in dams treated on gestation day 12. In animals receiving a single oral dose of 150 mg/kg bw carbon tetrachloride on GD 8, serum LH levels were significantly reduced (by 17–69% at intervals up to 20 hours post-dosing) in animals with full-litter resorption; no adverse developmental outcomes were observed in animals that received carbon tetrachloride and human chorionic gonadotropin, which acted as an LH surrogate. No developmental toxicity was observed in the surviving litters (Narotsky et al., 1997a; EPA, 2010).

 

In a further 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.

 

Mice appear to be less vulnerable than rats to carbon tetrachloride-induced maternal and foetal toxicity. Oral administration of carbon tetrachloride to pregnant B6D2F1mice at 82.6 or 826 mg/kg bw/day for 5 consecutive days beginning on gestation day 1, 6, or 11 had no effect on maternal body weight, liver or kidney weight, or pregnancy. No malformations or effects on pup weight or crown–rump length were observed, and development of the pups was normal (Health Canada, 2011; EPA, 2010; ATSDR, 2005).

 

In a collaborative study by Takayama et al. (1995), sixteen substances with a broad variety of modes of action (mainly drugs), were investigated. The study aimed to determine the optimal period and parameters for detection of male fertility disorders in rats. Although carbon tetrachloride was not included in this study, it gives interesting information on various fertility endpoints. The results indicate that the presence of histopathological changes in the testis precedes changes in sperm counts and other effects on fertility indexes and, thus, could be the most sensitive parameter for detecting male infertility induced by drugs and other substances. Furthermore, the authors suggest that the male premating treatment period can be shortened from 9 to 4 weeks without any loss in assay predictiveness.

 

Toxicity to reproduction: other studies

Additional information

not applicable

Justification for classification or non-classification

Based on the results of the above mentioned studies on the effects on fertility or the developmental toxicity/teratogenicity of CTC, no classification is warranted, neither for fertility neither for developmental toxicity.