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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
GLP compliance:
yes
Remarks:
CCR, Cytotest Cell ResearchGmbH & CO. KG, D-6101 Rossdorf, Germany
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 2,2'-([1,1'-biphenyl]-4,4'-diyldivinylene)bis(benzenesulphonate)
EC Number:
248-421-0
EC Name:
Disodium 2,2'-([1,1'-biphenyl]-4,4'-diyldivinylene)bis(benzenesulphonate)
Cas Number:
27344-41-8
Molecular formula:
C28H22O6S2.2Na
IUPAC Name:
disodium 2,2'-([1,1'-biphenyl]-4,4'-diyldivinylene)bis(benzenesulphonate)
Details on test material:
- Name of test material (as cited in study report): FAT 65029/G
- Physical state: solid, yellow
- Analytical purity: about 90%
- Impurities (identity and concentrations): 7 % NaCl, 3 % H2O
- Lot/batch No.: EN 372980, Op. 247/248
- Storage condition of test material: room temperature, light protected

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Fullinsdorf, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: yes 18 hours before treatment
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: The vehicle was chosen to its nontoxicity for the animals
- Amount of vehicle (if gavage or dermal): 10ml/kg
Duration of treatment / exposure:
single administration
Frequency of treatment:
once
Post exposure period:
24, 48, and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral-gavage
- Doses / concentrations: 40mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. 2 male and 2 female mice received 5000 mg/kg bw test substance. All treated animals expressed slight toxic reactions; reduction of spontaneous activity followed by apathy. The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test substance had cytotoxic properties.

DETAILS OF SLIDE PREPARATION: The smear was air-dried and then stained with May-Gruenwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic
erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
All treated animals expressed slight toxic reactions; reduction of spontaneous activity followed by apathy.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: All treated animals expressed slight toxic reactions; reduction of spontaneous activity followed by apathy.
- Evidence of cytotoxicity in tissue analyzed: The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test substance had cytotoxic properties.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at preparation intervals 24 hours and 72 hours after application of the test article. Biometric analysis of the results demonstrated a significant increase of the micronucleus frequency after administration of the test article at preparation interval 48 hours (p< 0.05). This statistical significance is considered to be of minor importance:
1. The corresponding actual negative control rate in this study was very low (0.02%).
The mean historical negative control value obtained within the last 12 months was 0.73 %. The range was 0.04% - 0.12%.
2. The frequency of 0.09 % PCEs with micronuclei after treatment with 5000 mg/kg b.w. test article is within the range of the historical control data presented.
3. The value of 0.09% deviates not substantially from the data obtained at preparation intervals 24 h (0.07 %) and 72 h (0.07 %) .
Therefore, the statistical significance is not considered to be an indication for an induced mutagenic effect due to the test article.
- Ratio of PCE/NCE (for Micronucleus assay): see table

Any other information on results incl. tables

Table 1: Summary of results:

test group

dose

(mg/kg bw)

sampling time (h)

% PCEs with micronuclei

range

PCE/NCE

solvent

0

24

0.11

0-5

1000/987

test article

5000

24

0.07

0-2

1000/1143

CPA

40

24

1.24

6-21

1000/1143

solvent

0

48

0.02

0-1

10000/939

test article

5000

48

0.09*

0-2

1000/1161

solvent

0

72

0.08

0-2

1000/877

test article

5000

72

0.07

0-3

1000/1010

* p < 0.05

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

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