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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Tolerance and acceptable daily intake of chlorinated fumigants in the rat diet.

Author:

Alumot E, Nachtomi E, Mandel E, Holstein P.

Year:

1976

Bibliographic source:

Food Cosmet Toxicol., vol. 14, no. 2, p. 105-10.

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Carbon tetrachloride

EC Number:

200-262-8

EC Name:

Carbon tetrachloride

Cas Number:

56-23-5

Molecular formula:

CCl4

IUPAC Name:

tetrachloromethane

Details on test material:

- Name of test material (as cited in study report): carbon tetrachloride
- no further details given

Test animals

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

Administration / exposure

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.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

80 ppm (nominal)

Remarks:

nominal in diet

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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)

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

- 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

Applicant's summary and conclusion

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).