1,1'-Biphenyl, 4-methyl-4'-(3E)-3-penten-1-yl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Amest Test, OECD 471: negative (reference 7.6.1 -1)

Chromosome aberration test in vitro, OECD 473: negative (reference 7.6.1 -2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-08-13 to 2004-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRY operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
2. Series: 1.58, 5.00, 15.8, 50.0, and 158 µg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

other: daunomycine

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The criteria provided in table 1 in "any other information on materials and methods", based upon the historical controls of the laboratory and statistical considerations, were established.

Interpretations:
A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
• the assay was to be considered valid, and
• a dose dependent (over at least two test material concentrations) increase in the number of re-vertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same lest system or
• "clear increases" occurred al least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Please refer to background material attached.

Conclusions:

The test material was not mutagenic under the test conditions with and without metabolic activation from induced rat liver S9 mix.

Executive summary:

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coil WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in acetone and tested at concentrations ranging from 1.58 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations 158 µg/plate. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol, the test item was not mutagenic under the described experimental conditions. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-05-24 to 2006-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CHO cells, supplied by Dr S Galloway, West Point, PA, USA, are maintained at Covance Laboratories Limited in tissue culture flasks containing McCoy's 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 µg/mL gentamycin. They are subcultured regularly at low density, and before overgrowth occurs, to maintain low aberration frequencies. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability. The cells are screened for mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 of male rat liver induced with Aroclor 1254

Test concentrations with justification for top dose:

serie 1: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, and 160 µg / mL medium
serie 2: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, and 120 µg / mL medium

It may be noted that an initial trial of Experiment 1 was performed but this was aborted due to errors in dose selection, resulting in inappropriate toxicity profiles.
The data observed following 3+17 hour treatments in the presence of S-9 in Experiments 1 and 2 were considered equivocal. In order to clarify the data, a third, confirmatory experiment was performed in the presence of S-9.

serie 3: 10, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, and 140 µg / mL medium

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

- Treatment
Prior to the start of treatment, the cell sheets from 4 flasks were removed using trypsin/EDTA and a mean cell count obtained. This provided the starting (baseline) count for the calculation of toxicity (expressed as population doublings relative to controls) at the time of cell harvest. For the cytotoxicity range-finder experiment, 0.05 mL of culture medium was removed from all cultures prior to treatment, giving a pre-treatment volume of 9.4 mL per culture. S-9 mix or KCl (0.5 mL) was added appropriately as detailed previously. Duplicate cultures (A, B) were treated with the solvent and single cultures treated with the test article at appropriate concentrations (0.1 mL per culture). Positive control treatments were not included.
For the main experiments, S-9 mix or KCl (0.5 mL) was added appropriately as detailed previously. One set of quadruplicate cultures (A, B, C and D) for each of the treatment regimes was then treated with the solvent and one set of duplicate cultures with the test article (0.1 mL per culture). Additional duplicate cultures were treated with 0.1 mL of the positive control chemicals. All cultures were incubated at 37°C.
Treatment media remained on cultures receiving the continuous treatment until sampling, that is, 20 or 44 hours after the beginning of treatment. Cultures received pulse treatments (both in the absence and presence of S-9) for 3 hours only. They were then washed twice with sterile saline, and fresh medium containing foetal calf serum and gentamycin added. Cultures were incubated for a further 17 or 41 hours before harvesting.


- Harvesting
Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 æg/mL to arrest dividing cells in metaphase. The monolayers of these cultures were then removed using trypsin/EDTA. An aliquot of cell suspension from all cultures (with the exception of positive control treated cultures) was taken for determination of cell number by using a Coulter Counter. The remaining suspension from each flask was transferred to a plastic centrifuge tube and the cells pelleted by centrifuging at 200 x 'g' for 5 minutes. The supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37°C for 5 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 200 x 'g', 5 minutes) and resuspension. This procedure was repeated several times (centrifuging at approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean.


- Preparation of metaphase spreads
Cells were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative (if required) so as to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides. After the slides had dried on a warm plate the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in Gurr's pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

- Selection of doses for chromosome analysis
The doses selected for chromosome aberration analysis were selected on the basis of toxicity (expressed as population doublings relative to controls). Toxicity was assessed by two separate measures;

1) Population doublings relative to controls. Population doublings (PD) was calculated for each concentration as follows:
PD = [log (N / Xo)] / log 2

Where N = mean final cell count/culture at each concentration Xo = starting (baseline) count

2) Mitotic inhibition relative to controls; that is, percentage of cells in mitosis. Slides from enough dose levels from each treatment group were scored to determine whether chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls, (based on at least 1000 cells counted where possible), preferably dose-related.

- Rationale for dose selection:
The highest dose for chromosome analysis from cultures sampled at 20 hours should be one at which at least 50% (approximately) reduction in population doublings has occurred or should be the highest dose tested. Analysis of slides from highly cytotoxic concentrations is avoided, if possible. Slides from cultures treated with heavily precipitating doses are checked to confirm that the presence of precipitate does not preclude analysis.

For treatments sampled at 20 hours, slides from the highest selected dose and at least two lower doses, such that a range of cytotoxicity from maximum to little or none is covered, were taken for microscope analysis. For treatments sampled at 44 hours, slides from the highest selected dose was taken for microscope analysis. This dose level was (toxicity or solubility permitting) usually the highest dose taken from the corresponding treatment at the 20-hour sampling time (but it may be noted that an additional, higher, dose was selected for the 3+41 hour treatment in the presence of S-9).

For each treatment regime, two solvent control cultures were analysed for chromosome aberrations. Slides from the remaining solvent control cultures were only to be analysed if considered necessary, for example, to help resolve an equivocal result. A single positive control dose level, which gives satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage, is analysed.
Cell count data and the results of dose selection are presented in the results section of this report.


- Scoring of aberrations
Slides from the selected treatments and from solvent and positive controls were coded using randomly generated letters by a person not connected with the scoring of the slides. Labels bearing only the study reference number, experiment number and the code were used to cover treatment details on the slides. Where possible, one hundred metaphases from each code were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) was noted on a slide, analysis may be terminated. Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN and is detailed in Appendix 2. Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid and no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment. Observations were recorded on raw data sheets with the microscope stage co-ordinates of any aberrant cell.
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures. Although physically located remote from the CLEH facility, all analysts participating in this study were subject to CLEH management and GLP control systems, and all slides and raw data were returned to CLEH for archiving on completion of their analysis.


-Treatment of data
After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorised as follows:

1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells.

The totals for category 2 in negative control cultures were compared with the current laboratory negative control (normal) ranges to determine whether the assay was acceptable or not. The proportion of cells in category 2 in test article treated cultures were also compared with normal ranges. The statistical significance of any data set was only to be taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentration exceeded the normal range. Under this condition, the statistical method used would be Fisher's exact test. Probability values of p £ 0.05 were to be accepted as significant. The proportions of cells in categories 1 and 3 were also examined in relation to historical negative control (normal) ranges and statistical analysis by Fisher’s exact test may be used.
The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p < 0.05 were to be accepted as significant.

Evaluation criteria:

Evaluation criteria
The CHO assay is considered valid if the following criteria are met:
1. the binomial dispersion test demonstrates acceptable heterogeneity between replicate cultures, and
2. the proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the normal range, and
3. at least 160 cells out of an intended 200 were analysable at each dose level, unless 10 or more cells showing structural aberrations other than gaps only were observed during analysis, and
4. the positive control chemicals induce statistically significant increases in the number of cells with structural aberrations.

A test article is considered as positive in this assay if:
1. the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.
3. a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
A test article is considered positive in this assay if all of the above criteria are met.
A test article is considered negative in this assay if none of the above criteria are met.

Data that do not fall into either of the above categories are judged on a case by case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance is taken into account, for example consistency of response within and between concentration levels and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test material was not clastogenic in this in vitro test system.

Executive summary:

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells, thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. In this study the clastogenic potential of the test material was evaluated by examining its effects on the chromosomes of CHO cells, cultured in vitro and treated in the absence and presence of a rat liver metabolising system (S-9). The test methodology in this study is in accordance with current literature and complies with the following regulatory guidelines: OECD Guideline 473 (1997) and the ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995) (6, 7, 8). The test material did not induce structural chromosome aberrations when tested at, or very close to, its limit of cytotoxicity following 20+0 and 44+0 hour treatments in the absence of S-9. In the same test system, the test material showed evidence of inducing structural aberrations when tested to the limit of cytotoxicity following 3+17 hour treatment in the presence of S-9 in one experiment out of four. However, the effect was not reproduced in three further independent experiments (two 3+17 hour treatments, one 3+41 hour treatment), therefore it was considered of highly questionable biological relevance. Sporadic increases in the numbers of numerical aberrations were observed following 3+17 hour treatment in the presence of S-9 and 44+0 hour treatment in the absence of S-9 (due to endoreduplicated cells) and 3+41 hour treatment in the presence of S-9 (due to polyploid cells). However, these increases did not appear to be dose-related. The detection of numerical aberrations is not the primary end-point of this assay, therefore these observations may be considered of questionable biological relevance.

In conclusion, the test material was not clastogenic in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Amest Test (reference 7.6.1 -1)

Investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Chromosome Aberration in vitro (reference 7.6.1 -2)

In a second test for clastogenicity, the test material was investigated in three experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in polyploidy. The test material did not induce structural chromosome aberrations when tested at, or very close to, its limit of cytotoxicity following 20+0 and 44+0 hour treatments in the absence of S-9. In the same test system, the test material showed evidence of inducing structural aberrations when tested to the limit of cytotoxicity following 3+17 hour treatment in the presence of S-9 in one experiment out of four. However, the effect was not reproduced in three further independent experiments (two 3+17 hour treatments, one 3+41 hour treatment), therefore it was considered of highly questionable biological relevance. Sporadic increases in the numbers of numerical aberrations were observed following 3+17 hour treatment in the presence of S-9 and 44+0 hour treatment in the absence of S-9 (due to endoreduplicated cells) and 3+41 hour treatment in the presence of S-9 (due to polyploid cells). However, these increases did not appear to be dose-related. The detection of numerical aberrations is not the primary end-point of this assay, therefore these observations may be considered of questionable biological relevance. The results obtained in this test indicate that the test material is not clastogenic in this in vitro test system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.