Benzenamine, N-phenyl-, styrenated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2uvrA, negative ± metabolic activation (bacterial reverse mutation assay, OED 471, GLP)

Genetic toxicity in vitro: Gene mutation in mammalian cells: V79 cells, HPRT-assay, negative ± metabolic activation (according to OECD 476, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2016 - 02 June 2016 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes”. Adopted 28 July 2015.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

In principle Guideline OECD 476 will satisfy the requirements of the following guideline: Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

In principle Guideline OECD 476 will satisfy the requirements of the following guideline: US EPA OPPTS 870.5300

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by the Department of Health of the Government of the United Kingdom

Type of assay:

other: forward gene mutation assay in mammalian cells

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22.
- Methods for maintenance in cell culture if applicable: Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% foetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: see above
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes: Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Enzyme Fraction: Phenobarbital/beta-Naphthoflavone induced rat liver S9 (Sprague-Dawley, male)

Test concentrations with justification for top dose:

The test item was considered to be an UVCB and therefore the maximum recommended dose concentration was 5000 μg/mL. Hence, the dose levels of test item used in the preliminary cytotoxicity test were 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiment. The dose range of test item was 5 to 80 μg/mL in the absence of metabolic activation (S9) and 5 to 60 μg/mL in the presence of S9.
The maximum dose level selected for the main mutagenicity experiment was the lowest precipitating dose level in the absence of S9 (80 μg/mL) and in the presence of S9 the dose selection was based on the toxicity seen in the preliminary toxicity test and was 60 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethyl benzanthracene (DMBA), stereochemistry not specified

Remarks:

Solvent for positive controls: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Cells were seeded at 1 x 10exp7 cells/225 cm² flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 10exp6 available for dosing in each flask using a parallel flask.

DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 to 5 to maintain logarithmic growth
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): approx 14 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-Thioguanine (6-TG)

STAIN: 10% Giemsa solution

NUMBER OF REPLICATIONS: triplicates for cloning efficiency, 10 replicates per group for mutation frequency

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Flasks were fixed with methanol and stained with Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

standard requirements

Evaluation criteria:

Interpretation of Results

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

Statistical analysis
When there is no indication of any increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Evaporation from medium: none stated
- Water solubility:
- Precipitation: A precipitate of the test item was observed at the end of exposure at and above 78.13 μg/mL in the 4-hour exposure group in both the absence and presence of S9.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Cytotoxicity Test
A dose range of 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL were used in the preliminary cytotoxicity test. The maximum dose tested was the maximum recommended dose level for an UVCB substance.
A precipitate of the test item was observed at the end of exposure at and above 78.13 μg/mL in the 4-hour exposure group in both the absence and presence of S9. There was modest toxicity demonstrated in the absence of S9 and there was a 57% reduction in cloning efficiency at 5000 μg/mL and a 15% reduction in cloning efficiency at the lowest precipitating dose level (78.13 μg/mL) when compared to the vehicle control. The toxicity demonstrated in the presence of S9 was much more marked and there was 43% and 98% reduction in cloning efficiency at 39.06 μg/mL and 78.13 μg/mL, respectively.
The maximum dose level selected for the main mutagenicity experiment was the lowest precipitating dose level in the absence of S9 (80 μg/mL) and in the presence of S9 the dose selection was based on the toxicity seen in the preliminary toxicity test and was 60 μg/mL.

Conclusions:

The study was conducted under GLP according to OECD guideline 476 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 gene mutations in V79 cells.
The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this GLP OECD 476 guideline study is to assess the potential mutagenicity of Benzenamine, N-phenyl-, styrenated, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

 

Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (dimethyl sulphoxide) and positive controls in the presence and absence of an S9 metabolic activation system.

The dose levels used in the Main Mutagenicity Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate in the absence of S9 and toxicity in the presence of S9. The dose levels selected for the Main Test were 5, 10, 20, 40, 60, 80 μg/mL over 4-hours without S9, and 5, 10, 20, 40, 45, 50, 60 μg/mL over 4-hours with S9 (2%).

The assay acceptance criteria were achieved, in both exposure groups, in terms of cloning efficiency, number of cells plated, etc. The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The test item demonstrated no significant increases in mutant frequency at any test item dose level, either with or without metabolic activation. The dose range used in the absence of S9 included the lowest precipitating dose level and in the presence of S9 included the appropriate range of toxicity including one dose level that induced 90% growth inhibition.

 

Benzenamine, N-phenyl-, styrenated was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-10-30 - 2001-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline 471, updated and adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature

Target gene:

his- or trp-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley

MEDIA USED
Culturing Broth. The broth used to grow overnight cultures of the tester strains was Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

0, 33.3, 100, 333, 1000, 3330, and 5000 µg/plate
Top dose as demanded in the guideline. No cytotoxicity was observed with either tester strain in either the presence or absence of S9 mix as evidenced by no dose-related decrease in the number of revertants per plate and a normal background lawn in the pre-test with 5000 µg/plate top dose.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article was immiscible in water at concentrations of 100, 50.0, and 25.0 mg per mL. The test article formed a solution in dimethylsulfoxide (DMSO) at a concentration of 100 mg per mL. For this reason, DMSO (CAS # 67-68-5, Acros Organics, Lot A012097501) was used as the vehicle.

Untreated negative controls:

yes

Remarks:

vehicle controls

Negative solvent / vehicle controls:

yes

Remarks:

50µl DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (2-AA); ICR-191 (CAS 17070-45-0)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 52 ± 4 hours

SELECTION AGENT (mutation assays): his or trp minimal agar

NUMBER OF REPLICATIONS: triplicate plates, 2 independent assays

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

Evaluation criteria:

Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated.
Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitate was noted at 1000 µg/plate and above

RANGE-FINDING/SCREENING STUDIES: Dose Rangefinding Assay
Doses tested in the mutagenicity assay were selected based on the results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 µg per plate, were tested. No cytotoxicity was observed with either tester strain in either the presence or absence of S9 mix as evidenced by no dose-related decrease in the number of revertants per plate and a normal background lawn.

HISTORICAL CONTROL DATA
See respective table. The results gained within the present test were within historical ranges.

Conclusions:

The study was conducted under GLP according to OECD guideline 471 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 gene mutations in bacteria.
The test item did not induce any significant or dose-related increases in mutant colonies in either the presence or absence of metabolic activation, which was confirmed in a second experiment. The test item was therefore considered to be non-mutagenic to bacteria under the conditions of this test.

Executive summary:

The objective of this study according to OECD 401 under GLP was to evaluate the test article, for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli tester strain WP2wvrA.

The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 µg per plate, one plate per dose, both in the presence and absence of S9 mix.

The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with six doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 33.3,100, 333, 1000,3330, and 5000 µg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment.

The results of the Salmonella-Escherichia coli / Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo: Chromosome aberrations (micronucleus test): negative (according to OECD 474, max. conc. 4000 mg/kg, intraperitoneal application to NMRI mice)

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26. May 1983

Deviations:

no

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

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

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.

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

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.

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.

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

There are several data available sufficiently covering all required endpoints, gene mutations in bacteria and mammalian cells in vitro, and micronuclei in vivo, and which are all assessed as Klimisch 1. All available data is consistently negative for genotoxic effects.

The test item was negative in an Ames Test and HPRT assay, and also negative in an in vivo Micronucleus Test in mice. So, all mode of actions for a genotoxic event were screened, and no alert for any genotoxic mode of action was found. No hazard for humans could be identified, especially when taking into account the negative results of a micronucleus study in mice, as there is no indication is given that the obtained results are not relevant for humans.

Hence, the data base is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from the test item, no data gaps could be identified and no additional testing is required.

Justification for classification or non-classification

All available test results for gene mutations in bacteria and mammalian cells in vitro, and micronuclei in vivo, are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified.