4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Genetic toxicity: in vivo

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

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 474, EEC Directive 84/449, L 251, B.12 and in accordance with the Principles of Good Laboratory Practices (GLP)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Enviromuental Protection Agency, Code of Federal Regulations, Title 40, Subpart F—Genetic Toxicity, Revision July 1, 1986 “In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.”

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

MORTRACE SB CONC.

IUPAC Name:

MORTRACE SB CONC.

Constituent 2

Reference substance name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

IUPAC Name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

Constituent 3

Reference substance name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

EC Number:

410-610-2

EC Name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Cas Number:

111850-24-9

Molecular formula:

C20 H25 N3 O3

IUPAC Name:

2,6-bis(butan-2-yl)-4-[2-(4-nitrophenyl)diazen-1-yl]phenol

Test material form:

other: dark red-brown liquid

Details on test material:

- Name of test material (as cited in study report): Mortrace SB Conc.
- Physical state: dark red-brown liquid
- Analytical purity: concentrate
- Lot/batch No.: MR 26592 SBC
- Expiry date: June 03, 1993
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature protected from light

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 grams
- Assigned to test groups randomly: yes
- Fasting period before study: Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum
- Housing: individually housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: recommeded by various regulatory agencies
- Amount of vehicle (if gavage or dermal): 20 ml/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in corn oil. The vehicle was chosen to its relative non—toxicity for the animals. All animals received a single standard volume of 20 ml/kg body weight orally.

Duration of treatment / exposure:

single

Frequency of treatment:

single

Post exposure period:

At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

Twelve animals, six males and six females, were treated per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): recommended by various regulatory agencies
- Route of administration: orally, once
- Doses / concentrations: 30 mg/kg body weight

Examinations

Tissues and cell types examined:

not applicable

Details of tissue and slide preparation:

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 5 ml syringe. The cell suspension was centrifuged at 1500 rpm 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-6100 Darmstadt)/Gieznsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

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 the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test article, is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

Statistics:

Standard statistical methods were employed

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY - In pre-experiments 4 animals (2 males, 2 females) received orally a single dose of 500, 1000, 2000 or 5000 mg/kg b.w., respectively, Mortrace SB Conc. formulated in corn oil and the volume administered was 20 ml/kg b.w.
Clinical signs of toxicity such as reduction of spontaneous activity, eyelid closure, apathy and abdominal position were noted in the 500 and 1000 mg/kg dose group. The above symptoms along with mortality (50%) was noted in the higher doses of 2000 and 5000 mg/kg bw.. On the basis of these results 1000 mg/kg b.w. Mortrace SB Conc. was estimated to be close to the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY -
In the micronucleus assay 7 out of 36 animals treated with Mortrace SB Conc. died. The mean number of normochromatic erythrocytes was not increased aftertreatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that Mortrace SB Conc. had no cytotoxic properties.
In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Mortrace SB conc. were in the same range as compared to the negative control groups.
30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus 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 in the bone marrow cells of the mouse.

Any other information on results incl. tables

None

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 in the bone marrow cells of the mouse.

Executive summary:

The test article Mortrace SB Conc. was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Twelve animals per test group were treated and if available (lethalities were observed after treatment with the test article), ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for inicronuclei.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) 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 NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 1000 mg/kg b.w..

In pre-experiments this dose level was estimated to be close to the maximum tolerated dose. The animals expressed toxic reactions. However, in the micronucleus assay 7 out of 36 animals treated with Mortrace SB Conc. died.

The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that Mortrace SB conc. had no cytotoxic properties.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Mortrace SB Conc. were in the same range as compared to the negative control groups.

30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus 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 in the bone marrow cells of the mouse.