2,6-di-tert-butyl-4-nonylphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro of 2,6-Di-tert-butyl-4-nonylphenol was assessed by 3 tests.

In a bacterial reverse mutation assay according to OECD guideline 471 with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 tested at 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation, 2,6-Di-tert-butyl-4-nonylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.

In an in vitro mammalian cell micronucleus assay according to OECD guideline 487 in Chinese hamster V79 cells, micronuclei frequencies were evaluated in a short-term exposure experiment of 4h exposure at concentrations of 5.0, 10, 20 and 40 µg/mL without metabolic activation and 20, 25 and 50 µg/mL with metabolic activation: and in a long-term exposure experiment of 24 h exposure at concentrations of 2.5, 5.0 and 10 µg/mL without metabolic activation. 2,6-Di-tert-butyl-4-nonylphenol did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

In an in vitro mammalian cell gene mutation test according to OECD guideline 476 in Chinese Hamster V79 cells 2,6-Di-tert-butyl-4-nonylphenol was tested in a 4 h-short-term exposure assay at concentrations of 5, 10, 25, 50, 100 and 150 µg/mL without metabolic activation and at concentrations of 0.5, 1.0, 2.5, 5, 10, 25 and 50 µg/mL with metabolic activation. In the described mutagenicity test under the experimental conditions reported, the test item 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

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:

2016

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:

adopted 21st July 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch: 222375101
- Expiration date of the lot/batch: 02 May 2018
- Purity test date: 02 September 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions:

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Cytokinesis block (if used):

histidine

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and (3-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Pre-experiment for toxicity: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment I and II: 31.6,100, 316, 1000, 2500 and 5000 µg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

A. dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I in agar (plate incorporation)
Experiment II preincubation

DURATION Experiment I (plate incorporation)
- Preincubation period: No
- Exposure duration: at 37 °C for at least 48 h in the dark
- Selection time (if incubation with a selection agent): at least 48 h

DURATION Experiment II (preincubation)
- Preincubation period: for 60 min at 37 °C
- Exposure duration: 60 min plus at least 48 h
- Selection time (if incubation with a selection agent): 60 min plus at least 48 h


SELECTION AGENT (mutation assays): 10.5 mg L-histidine x HCI x H2O

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Any supplementary information relevant to cytotoxicity:

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent controI (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
-a clear and dose-related increase in the number of revertants occurs and/or
-a biologically relevant positive response for at least one of the dose groups occurs
-in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
-if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
-if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation observed at 2500 µg/plate and higher in all tester strains used in Experiment I and II

RANGE-FINDING/SCREENING STUDIES:The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment.
Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
No toxicity was observed.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (2013-2015):
Without S9:
TA 98: 4-NOPD 10µg: min 141 max 2213 mean 444.5 SD 180.0
TA 100: NaN3 10 µg: min 132 max 1498 mean 627.6 SD 2255.3
TA 1535: NaN3 10 µg: min 34 max 1854 mean 795.5 SD 323.7
TA 1537: 4-NOPD 40µg: min 32 max 273 mean 92.0 SD 24.8
TA 102: MMS 1µL: min 162 max 3321 mean 1801.0 SD 483.5

With S9:
TA 98: 2-AA 2.5 µg: min 113 max 3606 mean 2126.7 SD 679.0
TA 100: 2-AA 2.5 µg: min 169 max 3132 mean 1786.6 SD 494.6
TA 1535: 2-AA 2.5 µg: min 20 max 1384 mean 103.4 SD 64.9
TA 1537: 2-AA 2.5 µg: min 26 max 489 mean 221.8 SD 92.2
TA 102: 2-AA 10 µg: min 137 max 1520 mean 748.8 SD 176.3

- Negative (solvent/vehicle) historical control data (2013-2015):
Without S9:
TA 98: min 13 max 54 mean 23.1 SD 6.1
TA 100: min 49 max 139 mean 89.2 SD 13.2
TA 1535: min 4 max 39 mean 12.0 SD 5.8
TA 1537: min 2 max 35 mean 7.4 SD 2.6
TA 102: min 141 max 472 mean 251.4 SD 55.6

With S9:
TA 98: min 13 max 61 mean 29.9 SD 7.0
TA 100: min 67 max 162 mean 98.3 SD 14.2
TA 1535: min 4 max 32 mean 9.4 SD 3.7
TA 1537: min 3 max 36 mean 7.4 SD 2.8
TA 102: min 91 max 586 mean 317.7 SD 76.6


- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: Precipitation observed at 2500 µg/plate and higher

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not cause gene mutations by
base pair changes or frameshifts in the genome of the tester strains used.
Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic in th is bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 2,6-Di-tert-butyl-4-nonylphenol for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Precipitation was observed in all tester strains used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2,6-Di-tert-butyl-4-nonylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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:

adopted July 29, 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation in Chinese Hamster V79 Cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch: 222375101
- Expiration date of the lot/batch: 02 May 2018
- Purity test date: 02 September 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions:

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate and their high cloning efficiency of untreated cells, usually more than 50%.
These facts are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index:
12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Methods for maintenance in cell culture if applicable: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37°C incubation temperature.
For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 µM
hypoxanthin and 5 µM thymidine.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37°C incubation temperature.
Complete Culture Medium:
MEM medium supplemented with
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
Treatment Medium:
MEM medium supplemented with
o % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
Selective Medium:
MEM medium supplemented with
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
11 µg/mL thioguanine (TG)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. Each cell batch was routinely checked for mycoplasma infections (PCR)
- Periodically checked for karyotype stability: Not specified
- Periodically 'cleansed' against high spontaneous background: yes. For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthin and 5 µM thymidine.

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Pre-test for toxicity: 25, 50, 100, 200, 300, 500,1000, 2000 µg/mL
Main test:
without metabolic activation: 5, 10, 25, 50, 100 and 150 µg/mL
with metabolic activation: 0.5, 1.0, 2.5, 5, 10, 25 and 50 µg/mL
The selection of the concentrations used in the main experiment was based on data from the pre-experiments according to OECD guideline 476. Precipitation of the test item was noted at a concentration of 150 µg/mL (without metabolic concentration) and at a concentration of 50 µg/mL (with metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility lest DMSO was be used as solvent. The solvent was compatible with the survival of the cells and the activity of the S9 mix.

Untreated negative controls:

yes

Remarks:

treatment medium, duplicate cultures

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, duplicate cultures

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): Approximately 5.0 x 10E6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% FBS) in a culture flask, respectively.

DURATION (main experiments)
- Exposure duration: 4h (short-term exposure)
- Expression time (cells in growth medium): incubation for an appropriate time (6-7 days)
- Selection time (if incubation with a selection agent): incubation for an appropriate time (7-12 days)
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine (TG)

NUMBER OF REPLICATIONS:
2 (duplicate) for determination of toxicity (cloning efficiency in non-selective medium)
5 for determination of mutagenicity (cloning efficiency in selective medium)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the expression period (after 7 to 9 days) for selection the mutants, about 4 x 10E5 cells for each treatment group were seeded in cell culture petri dishes (diameter 90 mm) with selection medium containing 11 µg/mL thioguanine (TG) for further incubation.
The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium to determine the cloning efficiencies.
After incubation for an appropriate time (7-12 days) colonies were fixed with methanol, stained with Giemsa and counted. The mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.

NUMBER OF CELLS EVALUATED: Not applicable


DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is observed as soon as the relative survival decreased below 70%.
- Any supplementary information relevant to cytotoxicity: A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment without metabolic activation.
Without metabolic activation the relative survival was 32% for the highest concentration (150 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 50 µg/mL with a relative survival of 138%.

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
-none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
-there is no concentration-related increase when evaluated with an appropriate trend-test
-all results are inside the distribution of the historical negative contral data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined -at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
-the increase is concentration-related when evaluated with an appropriate trend test, and
-any of the results are outside the distribution of the historical negative control data.
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation the relative survival was 32% for the highest concentration (150 µg/mL) evaluated

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

The highest biologically relevant concentration evaluated with metabolic activation was 50 µg/mL with a relative survival of 138% .

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test item was dissolved in DMSO, processed by ultrasound for 15 min and diluted prior 10 treatment. The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).
- Precipitation: Precipitation of the test item was noted at a concentration of 150 µg/mL (without metabolic concentration) and at a concentration of 50 µg/mL (with metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined in a pre-experiment. Eight concentrations [25, 50, 100, 200, 300, 500,1000, 2000 µg/mL] were tested with and without metabolic activation.
Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is observed as soon as the relative survival decreased below 70%.
Approximately 10.0 x 10E6 cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h (short term exposure) the treatment medium (MEM without serum) containing the test item was removed and the cells were washed twice with PBS, trypsinised and counted with a cell counter. For each treatment group two 25 cm2 flasks have been seeded with approx. 200 cells to determine cloning efficiencies (viability). After incubation for an appropriate time (7-9 days) colonies were fixed with methanol, stained with Giemsa and counted.
The relative survival was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
*EMS:
Mean of mutants/10E6 cells: 306
Minimum of mutants/10E6 cells: 193
Maximum of mutants/10E6 cells: 631
Standard deviation: 81.70
Relative standard deviation number of control values: 26.70
Number of control values: 40
*DMBA:
Mean of mutants/10E6 cells: 389
Minimum of mutants/10E6 cells: 132
Maximum of mutants/10E6 cells: 709
Standard deviation: 172.01
Relative standard deviation number of control values :44.26
Number of control values: 40
- Negative (solvent/vehicle) historical control data:
*without S9:
Mean of mutants/10E6 cells: 23
Minimum of mutants/10E6 cells: 9
Maximum of mutants/10E6 cells: 36
Standard deviation: 7.98
Relative standard deviation number of control values: 34.66
Number of control values: 32
*with S9:
Mean of mutants/10E6 cells: 24
Minimum of mutants/10E6 cells: 8
Maximum of mutants/10E6 cells: 37
Standard deviation: 7.37
Relative standard deviation number of control values: 30.88
Number of control values: 42

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:
Pre-Test for Toxicity: Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is observed as soon as the relative survival decreased below 70%. Approximately 10.0 x 10E6 cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h (short term exposure) the treatment medium (MEM without serum) containing the test item was removed and the cells were washed twice with PBS, trypsinised and counted with a cell counter. For each treatment group two 25 cm2 flasks have been seeded with approx. 200 cells to determine cloning efficiencies (viability). After incubation for an appropriate time (7-9 days) colonies were fixed with methanol, stained with Giemsa and counted.
The relative survival was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.
Main tests: In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6-7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.
Cytotoxicity will be evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment will be adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%).

CE[%] = (number of clones) / (number of cells plated) x 100

Adjusted CE [%] = CE x (number of cells at the end of treatment) / (number of cells at the beginning of treatment)

RS [%] = (adjusted CE in treated culture) / (adjusted CE in the negative / solvent control) x 100

Conclusions:

In the described mutagenicity test under the experimental conditions reported, the test item 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item 2,6-Di-tert-butyl-4-nonylphenol was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments. The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:

5, 10, 25, 50, 100 and 150 µg/mL

and with metabolic activation:

0.5, 1.0, 2.5, 5, 10, 25 and 50 µg/mL

Precipitation of the test item was noted at a concentration of 150 µg/mL (without metabolic activation) and at a concentration of 50 µg/mL (with metabolic activation).

Biologically relevant growth inhibition (relative survival < 70%) was observed in the experiment without metabolic activation. The relative survival was 32% for the highest concentration (150 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 50 µg/mL with a relative survival of 138%.

No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). All mutant values are within the historical data base of the test facility.

Without and with metabolic activation no biologically relevant increase of mutant frequencies was observed.

No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 487, "In Vitro Mammalian Cell Micronucleus Test", adopted 29 July, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch: 222375101
- Expiration date of the lot/batch: 02 May 2018
- Purity test date: 02 September 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions:

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: These cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions. These facts were necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index:
- Methods for maintenance in cell culture if applicable: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.
*Complete Culture Medium
MEM medium supplemented with:
10% fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
2mM L-glutamine
2.5 µg/mL amphotericin
25 mM HEPES
*Treatment Medium (short-term exposure)
Complete culture medium with 0% FBS.
*After Treatment Medium / Treatment Medium (long-term exposure)
Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.
- Properly maintained: [yes/no)
- Periodically checked for Mycoplasma contamination: yes. Routine checking of mycoplasma infections were carried out before freezing.
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Cytokinesis block (if used):

1.5 µg/mL cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Pre-experiment for toxicity: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 µg/mL
The concentration of 2000 µg/mL was considered to be the highest test concentration to be used in this test system following the recommendation of the corresponding OECD testing guideline 487.
The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment
Experiment I (toxicity):
without metabolic activation: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.5, 10, 20, 30, 40 and 50 µg/mL
with metabolic activation: 10, 12.5, 15, 17.5, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350 and 400 µg/mL
Experiment II (toxicity):
without metabolic activation: 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10, 25 and 50 µg/mL
Experiment I (micronuclei frequencies: short-term exposure 4 h):
without metabolic activation: 5.0, 10, 20 and 40 μg/mL
with metabolic activation: 20, 25 and 50 μg/mL
Experiment II (micronuclei frequencies: long-term exposure 24 h):
without metabolic activation: 2.5, 5.0 and 10 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium. Therefore the test item was dissolved in dimethylsulfoxide (DMSO) and diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

cell culture mledium

Negative solvent / vehicle controls:

yes

Remarks:

cell culture medium with 1% DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: cochicine dissolved in MEM at concentrations of 0.08 and 1.5 µg/mL without S9 (aneugenic control)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Approx. 50 000 cells were seeded per cell culture flask.

DURATION Experiment I
- Attachment period: approximately 48 h (=time after seeding, before exposure)
- Exposure duration: 4 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
DURATION Experiment II
- Attachment period: approximately 48 h (=time after seeding, before exposure)
- Exposure duration: 24 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 23 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): 1.5 µg/mL cytochalasin B

STAIN (for cytogenetic assays): acridine orange solution

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: according to the criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered

DETERMINATION OF CYTOTOXICITY
- Method: other: As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells
CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3)) / n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells

The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures:

% Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))

CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric χ² test. The p value was used as a limit in judging for significance levels in comparison with the concurrent solvent control.
Statistical significance at the 5% level (p < 0.05) was evaluated by the χ² test for trend. The p value was used as a limit in judging for significance levels.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Experiment I

Cytotoxicity / choice of top concentrations:

other: no increase of the cytostasis above 30%

Remarks:

Experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment I

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No increase in cytostasis above 30% up to 5.0 µg/mKL; cysostasis of 34% , 43% and 61% at 10 µg/mL, 20 µg/mL and 40 µg/mL respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment II

Cytotoxicity / choice of top concentrations:

other: No increase of the cytostasis above 30% was noted up to 5.0 µg/mL. At 10 µg/mL a cytostasis of 61% was noted.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:For the maximum concentration without metabolic activation the pH value was determined:
Exp I test item 50µg/mL: pH 7.4; Exp II test item 50µg/mL: pH 7.4
- Effects of osmolality: For the maximum concentration without metabolic activation the osmolality (in comparison to negative (solvent) control) was determined:
Exp. I Solvent control 473 mOsm/kg; test item 50µg/mL 474 mOsm/kg; Exp. II Solvent control 470 mOsm/kg; test item 50µg/mL 476 mOsm/kg
- Precipitation: The test item was dissolved in DMSO and rediluted in cell culture medium (MEM medium) at a ratio of 1:100 to achieve the final test item concentrations. Precipitation of the test item was noted at 20 μg/mL and higher without metabolic activation and at 50 μg/mL and higher with metabolic activation in experiment I. No precipitation of the test item was observed in the concentrations groups evaluated in experiment II.

RANGE-FINDING/SCREENING STUDIES: According to the used guideline the highest recommended dose is 2000 μg/mL. The test item was dissolved in DMSO and rediluted in cell culture medium at a ratio of 1:100 to achieve the final test item concentrations. Precipitation of the test item was noted at concentrations of 62.5 μg/mL and higher with and without metabolic activation. The highest dose group evaluated in the pre-experiment was 125 μg/mL without metabolic activation and 250 μg/mL with metabolic activation. The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: The CPBI (Cytokinesis Proliferation Block Index)was determined in 500 cells per culture for each test group.
CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3)) / n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Duplicate cultures were set up and 1000 binucleated cells per culture were scored for micronuclei.
- Indication whether binucleate or mononucleate where appropriate: binucleated cells

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (2011-2016):
*EMS (ethylmethanesulfonate):
Mean: 4.54
Standard deviation: 1.47
Relative standard deviation: 32.37
Minimum number of micronucleated cells (%): 2.10
Maximum number of micronucleated cells (%): 7.40
Lower control limit (95%, mean -2SD): 1.60
Upper control limit (95%, mean +2SD): 7.48
Number of assays: 62
*CPA(Cyclophophamide):
Mean: 3.86
Standard deviation: 1.07
Relative standard deviation: 27.71
Minimum number of micronucleated cells (%): 2.25
Maximum number of micronucleated cells (%): 6.25
Lower control limit (95%, mean -2SD): 1.72
Upper control limit (95%, mean +2SD): 6.00
Number of assays: 35
*Colchicine:
Mean: 3.93
Standard deviation: 2.20
Relative standard deviation: 55.91
Minimum number of micronucleated cells (%): 1.85
Maximum number of micronucleated cells (%): 12.30
Lower control limit (95%, mean -2SD): 0.00
Upper control limit (95%, mean +2SD): 8.32
Number of assays: 62
- Negative (solvent/vehicle) historical control data (2011-2016) :
*Negative control without metabolic activation:
Mean: 0.89
Standard deviation: 0.25
Relative standard deviation: 28.42
Minimum number of micronucleated cells (%): 0.45
Maximum number of micronucleated cells (%): 1.50
Lower control limit (95%, mean -2SD): 0.39
Upper control limit (95%, mean +2SD): 1.40
Number of assays: 62
*Negative control with metabolic activation:
Mean: 1.02
Standard deviation: 0.33
Relative standard deviation: 31.84
Minimum number of micronucleated cells (%): 0.50
Maximum number of micronucleated cells (%): 1.75
Lower control limit (95%, mean -2SD): 0.37
Upper control limit (95%, mean +2SD): 1.68
Number of assays: 35
*Solvent control without metabolic activation:
Mean: 1.00
Standard deviation: 0.27
Relative standard deviation: 27.30
Minimum number of micronucleated cells (%): 0.55
Maximum number of micronucleated cells (%): 1.40
Lower control limit (95%, mean -2SD): 0.45
Upper control limit (95%, mean +2SD): 1.55
Number of assays: 29
*Solvent control with metabolic activation:
Mean: 1.05
Standard deviation: 0.41
Relative standard deviation: 39.20
Minimum number of micronucleated cells (%): 0.55
Maximum number of micronucleated cells (%): 1.83
Lower control limit (95%, mean -2SD): 0.23
Upper control limit (95%, mean +2SD): 1.88
Number of assays: 15

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: Precipitation of the test item at 50 µg/mL and higher

Table 1: Summary: Experiment I and II, without metabolic activation

 

	Dose Group	Concen- tration [μg/mL]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro-nucleated Cells Frequency [%]	Historical Control Limits Negative Control	Precipitation	Statistical Significant Increase^a
Exp. I 4 h treatment, 24 h fixation interval	C	0	2000	12	88	1.10	0.39% - 1.40%	/	/
	S	0	4000	0	100	1.40		/	/
	6	5.0	2000	12	88	1.50		-	-
	9	10	2000	34	66	1.55		-	-
	10	20	2000	43	57	1.30		+	-
	12	40	2000	61	39	1.35		+	-
	EMS	900	2000	23	77	6.05		-	+
	Colc	1.5	2000	11	89	3.35		-	+
Exp. II 24 h treatment, 24 h fixation interval	C	0	2000	0*	113	1.45	0.39% - 1.40%	/	/
	S	0	2000	0	100	1.35		/	/
	6	2.5	2000	0*	107	1.35		-	-
	7	5.0	2000	15	85	1.30		-	-
	8	10	2000	61	39	0.60		-	(+)
	6	2.5	2000	0*	107	1.35		-	-
	7	5.0	2000	15	85	1.30		-	-
	EMS	900	2000	61	39	5.65		-	+
	Colc	0.08	2000	29	71	10.45		-	+

C: Negative Control (Culture medium)

S: Solvent Control (DMSO 1% v/v in culture medium)

^a: statistical significant increase compared to solvent control (χ² test , p<0.05).

+: significant increase; (+): significant decrease -: not significant

EMS: Ethylmethanesulfonate, Positive Control (without metabolic activation) [900μg/mL]

Colc: Colchicine, Positive Control (without metabolic activation) [1.5 and 0.08μg/mL]

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n

Relative Cell Growth: 100 x ((CBPI Test conc – 1) / (CBPI control -1))

c1: mononucleate cells

c2: binucleate cells

cx: multinucleate cells

n: total number of cells

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

 

Table 2: Summary: Experiment I, with metabolic activation

	Dose Group	Concen- tration [μg/mL]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro-nucleated Cells Frequency [%]	Historical Control Limits Negative Control	Precipitation	Statistical Significant Increase^a
Exp. I 4 h treatment, 24 h fixation interval	C	0	2000	9	91	1.65	0.37% - 1.68%	/	/
	S	0	2000	0	100	1.80		/	/
	5	20	2000	6	94	1.30		-	-
	6	25	2000	2	98	1.40		-	-
	7	50	2000	9	91	1.80		+	-
	CPA	2.5	2000	38	62	3.95		-	+

C: Negative Control (Culture medium)

S: Solvent Control (DMSO 1% v/v in culture medium)

^a: statistical significant increase compared to solvent control (χ² test , p<0.05).

+: significant; -: not significant

CPA: Cyclophosphamide, Positive Control (with metabolic activation) [2.5μg/mL]

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n

Relative Cell Growth: 100 x ((CBPI Test conc – 1) / (CBPI control -1))

c1: mononucleate cells

c2: binucleate cells

cx: multinucleate cells

n: total number of cells

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate 2,6-Di-tert-butyl-4-nonylphenol for a possible potential to induce micronuclei in Chinese hamster V79 cells an in vitro micronucleus assay was carried out. The following study design was performed:

	Without S9		With S9
	Exp. I	Exp. II	Exp. I
Exposure period	4 h	24 h	4 h
Cytochalasin B exposure	20 h	23 h	20 h
Preparation interval	24 h	24 h	24 h
Total culture period*	72 h	72 h	72 h

*Exposure started 48 h after culture initiation

 

The selection of the concentrations was based on data from the pre-experiment. In experiment I without metabolic activation 40 µg/mL and with metabolic activation 50 µg/mL were selected as the highest concentration for microscopic evaluation. In experiment II without metabolic activation 10 µg/mL were selected as the highest concentration for microscopic evaluation.

The following concentrations were evaluated for micronuclei frequencies:

Experiment I (short-term exposure 4 h):

without metabolic activation: 5.0, 10, 20 and 40 µg/mL

with metabolic activation: 20, 25 and 50 µg/mL

Experiment II (long-term exposure 24 h):

without metabolic activation: 2.5, 5.0 and 10 µg/mL

Precipitation of the test item was noted at 20 µg/mL and higher without metabolic activation and at 50 μg/mL and higher with metabolic activation in experiment I. No precipitation of the test item was observed in the concentrations groups evaluated in experiment II.

If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.

In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 5.0 µg/mL. At a concentration of 10 µg/mL a cytostasis of 34%, at a concentration of 20 µg/mL a cytostasis of 43% and at a concentration of 40 µg/mL a cytostasis of 61% was noted. In experiment I with metabolic activation no increase of the cytostasis above 30% was noted.

In experiment II without metabolic activation no increase of the cytostasis above 30 % was noted up to a concentration of 5.0 µg/mL. At a concentration of 10 µg/mL a cytostasis of 61% was noted.

In the main experiment I with and without metabolic activation and in the main experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric χ² Test was performed to verify the results in both experiments. No statistically significant increase (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated.

The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.

Ethylmethanesulfonate (EMS, 900 µg/mL) and cyclophosphamide (CPA, 2.5 µg/mL) were used as clastogenic controls. Colchicine (0.08 and 1.5 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of micronucleus frequency. This demonstrates the validity of the assay.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A key Ames study was performed in order to investigate the potential of 2,6-Di-tert-butyl-4-nonylphenol for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with theSalmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 (Schreib, 2016). In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100,316, 1000, 2500 and 5000 µg/plate. Precipitation was observed in all tester strains used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2,6-Di-tert-butyl-4-nonylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met. In conclusion,it can be stated that during the described mutagenicity test and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.

A key in vitro Micronucleus assay was performed with 2,6-Di-tert-butyl-4-nonylphenol to study potential micronuclei in Chinese hamster V79 cells with (4h) and without (4h, 24 h)metabolic activation (Donath, 2017). The selection of the concentrations was based on data from the pre-experiment. In experiment I without metabolic activation 40 µg/mL and with metabolic activation 50 µg/mL were selected as the highest concentration for microscopic evaluation. In experiment II without metabolic activation 10 µg/mL were selected as the highest concentration for microscopic evaluation. The following concentrations were evaluated for micronuclei frequencies:

Experiment I (short-term exposure 4 h): without metabolic activation: 5.0, 10, 20 and 40 µg/mL; with metabolic activation: 20, 25 and 50 µg/mL

Experiment II (long-term exposure 24 h): without metabolic activation: 2.5, 5.0 and 10 µg/mL

Precipitation of the test item was noted at 20 µg/mL and higher without metabolic activation and at 50 μg/mL and higher with metabolic activation in experiment I. No precipitation of the test item was observed in the concentrations groups evaluated in experiment II.

The limit of cytoxicity is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.

In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 5.0 µg/mL. At a concentration of 10 µg/mL a cytostasis of 34%, at a concentration of 20 µg/mL a cytostasis of 43% and at a concentration of 40 µg/mL a cytostasis of 61% was noted. In experiment I with metabolic activation no increase of the cytostasis above 30% was noted. In experiment II without metabolic activation no increase of the cytostasis above 30 % was noted up to a concentration of 5.0 µg/mL. At a concentration of 10 µg/mL a cytostasis of 61% was noted. In the main experiment I with and without metabolic activation and in the main experiment II without metabolic activation no biologically or statistically significant relevant increase of the micronucleus frequency was noted after treatment with the test item. In conclusion, it can be stated that during the study described and under the experimental conditions reported, 2,6-Di-tert-butyl-4-nonylphenol did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.

Therefore, 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

A key study with 2,6-Di-tert-butyl-4-nonylphenol was performed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster with and without metabolic activation afert 4 h exposure (Wallner, 2016). The test item was investigated at the following concentrations without metabolic activation: 5, 10, 25, 50, 100 and 150 µg/mL and with metabolic activation: 0.5, 1.0, 2.5, 5, 10, 25 and 50 µg/mL. Precipitation of the test item was noted at a concentration of 150 µg/mL (without metabolic activation) and at a concentration of 50 µg/mL (with metabolic activation). Biologically relevant growth inhibition (relative survival < 70%) was observed in the experiment without metabolic activation. The relative survival was 32% for the highest concentration (150 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 50 µg/mL with a relative survival of 138%.

No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). All mutant values are within the historical data base of the test facility. Without and with metabolic activation no biologically relevant increase of mutant frequencies was observed. No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 2,6-Di-tert-butyl-4-nonylphenol is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Justification for classification or non-classification

No classification and labelling is needed for mutagenicity of 2,6-di-tert-butyl-4-nonylphenolaccording to Regulation No. 1272/2008 (CLP).