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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
publication
Title:
NTP Technical Report on the Toxicity Studies of trans-1,2-Dichloroethylene (CAS No. 156-60-5); Administered in Microcapsules in Feed to F344/N Rats and B6C3F1 Mice
Author:
National Toxicology Program
Year:
2002
Bibliographic source:
National Toxicology Program Toxicity Report Series Number 55
Report date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
mice dosed in 14-week feeding study
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-dichloroethylene
EC Number:
205-860-2
EC Name:
trans-dichloroethylene
Cas Number:
156-60-5
Molecular formula:
C2H2Cl2
IUPAC Name:
arsenic
Details on test material:
- Purity: ≥ 99%

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks
- Housing: 1 (male) or 5 (female) per cage (Polycarbonate)
- Diet: NIH-07 open formula pelleted diet, ad libitum, changed once or twice (female mice) per week
- Water: Tap water via automatic watering system, ad libitum
- Acclimation period: 12 (females) or 13 days (males)

ENVIRONMENTAL CONDITIONS
- Temperature: 22.2°C ± 1.7°C
- Humidity: 50% ± 15%
- Air changes: at least 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: food-grade, modified corn starch (CAPSUL®) and reagent-grade sucrose (80:20)
Details on exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION: Microcapsules loaded with the test item and placebos (empty microcapsules) were prepared by the analytical chemistry laboratory with a proprietary process using food-grade, modified corn starch (CAPSUL®) and reagent-grade sucrose (80:20) to produce dry microspheres; the outer surfaces of the microcapsules were dusted with food-grade, hydrophobic, modified corn starch. Following microencapsulation of the test item, the analytical chemistry laboratory tested the microencapsulated chemical for conformance to specifications. The microcapsules were examined microscopically for appearance. Conformance to particle size specifications (with no more than 1% of particles having diameters greater than 420 μm) was determined by passing placebo and loaded microcapsules through U.S. standard sieves (numbers 30, 40, 60, 80, 100, 120, and PAN). The chemical loads (amount of test item in the starch/sugar matrix) of freshly prepared microcapsules and of microcapsules stored under a variety of conditions were determined with GC. Major peak comparisons of the neat and microencapsulated chemical and 9-month stability studies were also performed with GC.
Duration of treatment / exposure:
14 consecutive weeks
Frequency of treatment:
continuous in feed
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
3125, 6250, 12500, 25000, or 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10/sex/concentration
Control animals:
yes, concurrent vehicle
Positive control(s):
None

Examinations

Tissues and cell types examined:
PCEs and NCEs taken from peripheral blood samples obtained from male and female mice
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The test item was selected for subchronic toxicity studies based on the results of preliminary acute toxicity studies reported in other publications and its prevalence in industrial applications. In these studies, the most significant effect was a 20% reduction in final mean body weight of male rats exposed to 50000 ppm of the test item. Based on this information, the highest dose for the 14-week study was 50000 ppm.


DETAILS OF SLIDE PREPARATION:
At the end of the 14-week exposure, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS:
Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group. In addition, 1000 polychromatic erythrocytes (PCEs) were scored per animal to determine the percentage of PCEs in the total erythrocyte population.

Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean.
The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the untreated control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single exposed group was less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials (as noted above). Results of the 14-week study were accepted without repeat tests, because additional test data could not be obtained. Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined

Any other information on results incl. tables

There were no treatment-related deaths of mice exposed to the test item. The final mean body weights and body weight gains of males and females in the 50000 ppm groups and the body weight gains of females in the 12500 and 25000 ppm groups were significantly less than those of the vehicle controls. Feed consumption by the exposed groups was similar to that by the vehicle controls. Exposure concentrations of 3125, 6250, 12500, 25000, and 50000 ppm resulted in average daily doses of 480, 920, 1900, 3850, and 8065 mg/kg for males and 450, 915, 1830, 3760, and 7925 mg/kg for females. There were no clinical findings of toxicity.

In addition, no effect on the percentage of micronucleated polychromatic erythrocytes among the total erythrocyte population was observed, indicating no inhibition or stimulation of erythropoiesis in the bone marrow of exposed mice.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Negative in a peripheral blood micronucleus test in male and female mice when administered in feed for 14 weeks
Executive summary:

A peripheral blood micronucleus study was conducted in mice to determine whether the test material induces an increase in the frequency of normochromatic erythrocytes (NCEs) and polychromatic erythrocytes (PCEs) in blood.  In this study, groups of 10 male and 10 female mice per dose group were fed up to 50000 ppm of test item in the diet for 14 consecutive weeks.  Blood smears were prepared at the end of the 14 week exposure then 2000 NCEs and 1000 PCEs per animal were evaluated for the presence of micronuclei. 

The test item administered in microcapsules in feed for 14 weeks did not increase the frequency of NCEs in the peripheral blood of male or female mice. In addition, no effect on the percentage of micronucleated polychromatic erythrocytes among the total erythrocyte population was observed, indicating no inhibition or stimulation of erythropoiesis in the bone marrow of exposed mice.