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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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".

Data source

Reference
Reference Type:
publication
Title:
Effect of dosing vehicle on the developmental toxicity of bromodichloromethane and carbon tetrachloride in rats.
Author:
Narotsky MG, Pegram RA, Kavlock RJ.
Year:
1997
Bibliographic source:
Fundam Appl Toxicol., vol. 40, no 1, p. 30-36.

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
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
- Physical state: liquid
- Analytical purity: 99.9+%
- Supplier: Fisher Scientific Co. (Fair Lawn, NJ, U.S.A.)
- Lot/batch No.: not reported
- Stability under test conditions: stable
- Storage condition of test material: not reported

Test animals

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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

- 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

Applicant's summary and conclusion

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).

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