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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
Type of information:
experimental study
Remarks:
source of read across
Adequacy of study:
key study
Study period:
From March 10th to May 06, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
no
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: KIeintierfarm Madoerin AG, CH 4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: males 31 - 38 g, females 25 - 32 g.
- Housing: n Makrolon type-3 cages with wire mesh top and granulated softwood bedding.
- Diet: peIIeted standand Kliba no. 343 mouse diet, Batch 33/85 (Klingentalmuehle), ab Iibitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidit: 40 - 70 %
- Photoperiod: 12 hours artificial fluorescent light / 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
4 %, w/v carboxymethylcellulose sodium salt in distilled water
Details on exposure:
- Volume administrated: 20 ml/kg body weight

FORMULATION OF THE TEST ARTICLE
A suspension was prepared immediately before application by adding the test article to distilled water containing CMC (4 % w/v).
Homogeneity of the suspension was maintained during application using a magnelic stirrer.

DOSE SELECTION
The maximum tolerated dose based on acute oral toxicity data. A preliminany acute oral LD50 study (Iimit test; 1 dose, 3 males and 3 females per dose) performed with the same mouse strain as used in this study showed the following result after 15 days observation: 5000 mg/kg body weight, 0 mortality in 6 animals. The 5000 mg/kg hody weight was used in the current study as the maximurn tolerated dose.
Frequency of treatment:
Single application
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
three groups, each containing 18 males and 18 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Vehocle: dissolved in 0.9 %, w/v saline solution.
- Dose: 50 mg/kg body weight

Examinations

Tissues and cell types examined:
One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.
Details of tissue and slide preparation:
SACRIFICE
24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. AII mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue.

PREPARATION OF SUSPENSION
The proximal end of the femur was cut with scissors; the distal end was left intact. The needle of a plastic syringe containing 0.2 ml calf serum was inserted into the proximar part of the marrow canal.
The bone marrow cells were dispersed in 1.5 ml of calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 RPM (ca. 180-190 G) for 5 minutes.
The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. A small drop of the marrow, serum suspension was smeared on the slide, which was allowed to dry overnight. Two slides per animal were prepared. One to three days later, the smears were stained using the panoptic stain method developed by Pappenheim.

METHOD OF ANALYSIS AND EVALUATION
The slides were coded before microscopic analysis. If macroscopic evaluation revealed technical imperfections, the first slide was replaced by the second slide prepared. From each animal, one thousand (1000) polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000x), for the incidence of micronuclei. Micronuclei were evaluated according to the characteristics which have been described by Schmid. Some characteristics of micronuclei are round features of a sharp contour, sometimes slightly oval or ring-shaped, average size of 1/20 to 1/5 of the erythrocyte size and darkly blue-stained, comparable with the staining quality of nucleic material. Structures of irregular or fibrillar shape, of divergent staining properties of other characteristics not typical for a micronucleus (e.g. atypical focus properties) were not considered to be micronuclei.
Multiple micronuclei in one cell were registered and calculated as one micronucleated cell.
The calculated ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per slide, measured the toxic efficacy of the test article.
Polychromatic erythrocytes (PCE) can be differentiated from normochromatic erythrocytes (NCE) by their basophilic, blue- grey color compared to the acidophilic orange-pink colon of NCE. PCE also tend to be somewhat larger and have more diffuse boundaries.
Additional information chromatic erythrocytes could be obtained for micronuclei.
Evaluation criteria:
The frequencies of micronucleated polychromatic erythrocytes of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution was applied.
Estimation and testing were performed by maximum likelihood method . If a test article produced no statistically significant positive response at any one of the test points, it was considered to be non-mutagenic in this system.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in males
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
After a single application of the test article at 5000 mg/kg body weight by gavage no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male of female treated groups when compared with corresponding negative control groups. These results were found at the three examination times , 24, 48 and 72 hours post-application, respectively.
Reduced PCE/NCE ratios were observed in bone marrow preparations of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effect of the test article was observed.

POSITIVE CONTROL
The positive control groups receiving cyclophosphamide exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test. The dose used for this reference mutagen exhibited a toxic effect as should by the reduced PCE/NCE ratio.

CLINICAL EXAMINATION
No death occurred during the application period. Yellowish-coloured urine and faces as well as diarrhea was observed in all test article-treated animals up to 48 hours post-application. In male animals, slight sedation was observed 4 hours and ruffled fun 48 hours post-application.
The animals were free of clinical findings 56 hours post-application and later.

Applicant's summary and conclusion

Conclusions:
The test item is not considered to be mutagenic in the in vivo mouse micronucleus assay performed.
Executive summary:

The study with mice was performed to assess the potential mutagenic activity, induced by test item, through damage to the chromosomes on to the mitotic apparatus, according to OECD guideline 474.

The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The negative control group received the test article vehicle, i.e. 4 %, w/v carboxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article. The positive control groups received 50 mg/kg body weight cyclophosphamide dissolved in 0.9 %, w/v saline solution. All animals were given a single application of 20 ml/kg body weight by gavage.

24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.

The data were statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method.

Reduced PCE/NCE ratios were observed in bone marrow preparation of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effects of the test article was observed.

After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male or female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus. Therefore, test item is not considered to be mutagenic in the in vivo mouse micronucleus assay.