Benzenamine, N-phenyl-, styrenated

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-03-08 - 1993-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26. May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L383A, Annex V, B12 dated December 29, 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Environmental 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

GLP compliance:

yes (incl. QA statement)

Remarks:

HESSISCHES MINISTERIUM FÜR UMWELT, ENERGIE UND BUNDESANGELEGENHEITEN

Type of assay:

other: mammalian bone marrow cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, styrenated

EC Number:

270-485-3

EC Name:

Benzenamine, N-phenyl-, styrenated

Cas Number:

68442-68-2

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

4-(1-phenylethyl)-N-[4-(1-phenylethyl)phenyl]aniline

Test material form:

liquid: viscous

Details on test material:

- Storage: 4°C, moisture protected

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, D-97633 Sulzfeld 1
- Age at study initiation: minimum 10 weeks (at start of acclimatization)
- Weight at study initiation: 29.8 - 43.3 g (start of treatment)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: single, in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen), with granulated soft wood bedding (ALTROMIN, D-32770 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, D-32770 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 + 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: 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
- Concentration of test material in vehicle: 40, 120, and 400 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg b.w.

Details on exposure:

Intraperitoneal application

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

16, 24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

400 other: mg/kg bw

Remarks:

Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 16 h and 48 h after treatment.

Doses / concentrations 2

Dose / conc.:

1 200 other: mg/kg bw

Remarks:

Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 16 h and 48 h after treatment.

Doses / concentrations 3

Dose / conc.:

4 000 other: mg/kg bw

Remarks:

Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 16 h and 48 h after treatment.

No. of animals per sex per dose:

6 / sex / test group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intreperitoneally, once, dissolved in physiological saline
- Doses / concentrations: 30 mg/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20°C only 1 % of CPA is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:

bone marrow; polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose of the test article or the highest dose that can be formulated and administered reproducibly should be administered.
The volume should be compatible with physiological space available .
The maximum tolerated dose level was determined as the dose that caused toxic reactions without having grave effects on survival within 48 hours (pre-experiment).
Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 16 h and 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
During the study period the animals received feed and water ad libitum.
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 from animals treated with the highest dose was done 16, 24 and 48 hours after treatment. Bone marrow samples from animals treated with the low and medium dose were taken only at preparation interval 24 hours.

DETAILS OF SLIDE PREPARATION:
Preparation of the Animals:
The survived 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 1,500 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-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.

METHOD OF ANALYSIS:
Analysis of Cells:
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.
Five animals per sex and group were evaluated as described. The microscopic slides of the remaining animals were scored if an animal died in a test group (same time and dose group, same sex).

OTHER:
The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, negative and positive control. The micronucleated cells per thousand and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal.

Evaluation criteria:

EVALUATION OF RESULTS
A test article is considered positive if, at any of the intervals, there is a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test article is considered negative if there is no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time interval. A test is also considered negative if there is a significant increase in that rate which, according to the laboratory's experience is within the range of negative controls.

Statistics:

The biometric evaluation can be performed by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The animals treated with 4000 mg/kg b.w. The test item expressed toxic reactions. Reduction of spontaneous activity and eyelid closure followed by apathy were observed. Four out of six treated females died unexpectedly in the test article group at 48 h.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: In a first pre-experiment 4 animals (2 males, 2 females) received intraperitoneally a single dose of 5000 mg/kg b.w. Styrolised diphenylamine formulated in corn oil. The volume administered was 10 ml/kg b.w.. Observation period was 48 hours. In a second pre-experiment 4 animals (2 males, 2 females) per group received intraperitoneally a single dose of 3000 or 4000 mg/kg b.w. Styrolised diphenylamine formulated in corn oil. The volume administered was 10 ml/kg b.w.. Observation period was 48 hours. For results see "Any other information on results incl. tables"
- Solubility:
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, eyelid closure, apathy, 1 death at 5000 mg/kg

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding negative controls there was no enhancement in the frequency of micronuclei at any preparation interval and dose level after application of the test article. The mean values of micronuclei observed after treatment with Styrolised diphenylamine were in the same range as compared to the negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): At preparation interval 24 h the mean number of normochromatic erythrocytes was slightly increased after treatment with 4000 mg/kg of the test article as compared to the mean value of NCEs of the negative control, indicating that Styrolised diphenylamine had weak cytotoxic properties.
- Statistical evaluation: A biometric evaluation of the results was not necessary to be performed because the mean micronucleus frequencies after administration of the test article were not above the mean negative control value.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 474 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Benzenamine, N-phenyl-, styrenated to induce micronuclei in the bone marrow of NMRI mice.
In comparison to the corresponding negative controls there was no enhancement in the frequency of micronuclei at any preparation interval and dose level after application of the test article. The mean values of micronuclei observed after treatment with Styrolised diphenylamine were in the same range as compared to the negative control group.
The present in vivo study may be used to fully cover the in vitro chromosome mutation endpoint in IUCLID chapter 7.6.1, as according to REACH Annex VIII column 2, the study does not usually need to be conducted if adequate data from an in vivo cytogenicity test are available, which is the case here.
Hence, the available study is fully sufficient to serve as a cytogenicity study, and the test item does not need to be considered as a clastogenic or aneugenic substance.

Executive summary:

This GLP OECD 474 guideline study was performed to investigate the potential of Styrolised diphenylamine 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 intraperitoneally was 10 ml/kg b.w. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

The occurrence of micronuclei in ten animals (5 males, 5 females) per test group (exception was the test article group at preparation interval 48 h in which only 2 females could be evaluated) was evaluated. 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 levels of the test article were investigated:

16 h preparation interval: 4000 mg/kg b.w.

24 h preparation interval: 400, 1200, and 4000 mg/kg b.w..

48 h preparation interval: 4000 mg/kg b.w..

In pre-experiments the highest dose administered was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. In the main experiment in the group prepared at 48 h four of the six females died, unexpectedly.

After treatment with the highest test article dose at preparation interval 24 hours the number of NCEs was slightly increased as compared to the corresponding negative control thus indicating that Styrolised diphenylamine had a weak cytotoxic effect.

In comparison to the negative control there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used.

30 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as positive control which induced a distinct increase of the 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 in bone marrow cells of the mouse. Therefore, Styrolised diphenylamine is considered to be non-mutagenic in this micronucleus assay.