2-[[4-[(2-cyanoethyl)ethylamino]phenyl]azo]-5-nitrobenzonitrile

Genetic toxicity: in vivo

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:

October 26 to November 18, 1998

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:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 21st, July 1997

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: approximately 7 weeks.
- Weight at study initiation: males mean 33.5 g (32.0 - 36.0 g); females mean 27.1 g (24.0 - 29.0 g).
- Assigned to test groups randomly: randomization schemes 98.0890 and 98.089.
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate.
- Diet: rat/mice diet ssniff® R/M-H (V 1534), ad libitum.
- Water: tap water in plastic bottles, ad libitum.
- Acclimation period: 5 days under study conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 50 ± 20 %
- Photoperiod: 12 hours daily light.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

SOLVENT
- Solubility: suspension in Tylose HEC 4000 (0.5% w/v).
- Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v)', signed October 26th, 1998

Details on exposure:

- Formulation of test compound: on the days of administration the test substance was suspended in Tylose HEC 4000 (0.5 % w/v) at the appropriate concentration. A magnetic stirrer was used to keep the prepaation homogeneous until dosing had been completed.

DOSE SELECTION
In a preliminary dose range finding study, oral administration of 2000 mg test item per kg body weight did not cause any toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

Frequency of treatment:

Twice at an interval of 24 hours

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Endoxan containing cyclophosphamide, dissolved in distilled water.
- Dose: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows: 5 minutes in methanol; 5 minutes in May-Grünwald's solution; brief rinsing twice in distilled water; 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise); rinsing in distilled water; drying; and coating with Entellan®.

METHOD OF ANALYSIS
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group.
A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronudeated polychromatic erythrocytes in the dose group of test item was within the normal range of the negative control groups. No statistically significant increase of micronudeated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20 % of the control values.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Any other information on results incl. tables

Summary tables and statistics

Sex	Dose mg/kg bw	Killing time	No animals	No poly counted	Poly/Ery		Poly with MN
					mean	mean SD	mean	(%) mean	mean SD
male	0 - Control	24 hrs	5	2000	0.48	0.07	1.6	0.08	0.03
male	2000 test item	24 hrs	5	2000	0.48	0.04	2.2	0.11	0.11
male	50 - Endoxan	24 hrs	5	2000	0.42	0.04	60.6	3.03	0.69
female	0 - Control	24 hrs	5	2000	0.54	0.05	1.2	0.06	0.07
female	2000 test item	24 hrs	5	2000	0.53	0.06	2.4	0.12	0.06
female	50 - Endoxan	24 hrs	5	2000	0.44	0.05	53.8	2.69	0.59

Sex	Dose mg/kg bw	Killing time	No animals	No poly counted	Poly/Ery		Poly with MN			Mu I.
					mean	mean SD	mean	(%) mean	mean SD
pooled	0 - Control	24 hrs	10	2000	0.51	0.07	1.4	0.1	0.05	1
pooled	2000 test item	24 hrs	10	2000	0.51	0.06	2.3	0.1	0.09	1.6
pooled	50 - Endoxan	24 hrs	10	2000	0.43	0.04	57.20*	2.9	0.63	40.9

Mut. I. = Mutagenic index

Control = Vehicle Tylose HEC 4000 (0.5 % w/v))

* significantly different from control (p < 0.05)

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the results indicate that test item is not mutagenic in the micronucleus test.

Executive summary:

The micronucleus test was carried out with test substance, which was suspended in Tylose HEC 4000 (0.5 % w/v); it was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study). According to the test procedure, the animals were killed 24 hours after administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with test item and was not less than 20 % of the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Conclusion

Under the conditions of the present study, the results indicate that test item is not mutagenic in the micronucleus test.