6-(2-chloro-6-cyano-4-nitrophenylazo)-4-methoxy-3-[N-(methoxycarbonylmethyl)-N-(1-methoxycarbonylethyl)amino]acetanilide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 1999 to 22 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP methodology followed and the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted July 21, 1997, "Mammalian Erythrocyte Micronucleus Test". used to performed the experiment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

relative humidity was lower than 30 % (lowest value 21 %) and test article of two deliveries (S 160511 pre-experiments and S 160521 micronucleus test) were used.(These deviations had no influence on the integrity and validity of the study)

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-500-8

EC Name:

-

Cas Number:

204277-61-2

Molecular formula:

Hill formula: C23 H23 Cl N6 O8 CAS formula: C23 H23 Cl N6 O8

IUPAC Name:

methyl 2-({4-[2-(2-chloro-6-cyano-4-nitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)propanoate

Test material form:

other: Solid

Details on test material:

None

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation: Males mean value 32.6 g (SD +/- 2.7g); Females mean value 25.4 g (SD +/- 2.0 g)
- Assigned to test groups randomly: yes, the animals were distributed into the test groups at random and identified by cage number
- Fasting period before study: Approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: Single
- Diet : pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3°C
- Humidity (%): relative humidity 21-46 %
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Name: polyethylene glycol 400 (PEG 400)
- Supplier catalogue no. : MERCK KGaA, D-64293 Darmstadt 9726 (gas chromatography grade)
- Route and Frequency of Administration: orally, once
- Volume Administered: 10 ml/kg b.w.

Details on exposure:

Test material. a single standard dose of 10ml/kg body weight oralls (200, 670 and 2000 mg/kg b.w.)
Vehicle control: Orally, once, 10ml/kg body weight-
Positive control: Orally, once, 40 mg/kg b.w.

Duration of treatment / exposure:

24 hours and 48 hours

Frequency of treatment:

Once

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 670 and 2000 mg/kg b.w.
Basis:
nominal conc.

No. of animals per sex per dose:

Ten animals (5 males and 5 females) per test roup were evaluated.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: CPA; Cyclophosphamide
- Supplier: SIGMA-Aldrich Vertriebs-GmbH, D-82041 Deisenhofen
- Catalogue no.: 21.870-7 (purity: at least 98 %)
- Dissolved in: deionised water
- Dosing: 40 mg/kg b.w.
- Route and Frequency of Administration: orally, once
- Volume Administered: 10 ml/kg b.w.

Solution prepared on day of administration. The stability of CPA at room temperature is good. At 25°C only 3.5 % of its potency is lost after 24 hours

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes

Details of tissue and slide preparation:

Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made
from each bone marrow sample.

Evaluation criteria:

- A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
- A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
-

Statistics:

Evaluation criteria can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

(did not induce micronuclei as determined by micronucleus test in the bone marrow cells of the mouse)

Toxicity:

yes

Remarks:

Slight toxicity at 500 and 2000 mg/kg b.w. (reduction of spontaneous activity)

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control (PEG 400) serves as negative control

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
FAT 41024/B, did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of FAT 41024/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This experiment was performed according to the OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and follows GLP methodology.

The test article, FAT 41024/B, was formulated in polyethylene glycol 400 (PEG 400). PEG 400 was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs.

The following dose levels of the test article were investigated:

- 24 h preparation interval: 200, 670, and 2000 mg/kg b.w..

- 48 h preparation interval: 2000 mg/kg b.w..

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test article the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 41'024/B had no cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biological relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a substantial increase of induced micronucleus frequency.

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. Therefore, FAT 41024/B is considered to be non-mutagenic in this micronucleus assay.