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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-di-tert-butyl-4-nonylphenol
EC Number:
224-320-7
EC Name:
2,6-di-tert-butyl-4-nonylphenol
Cas Number:
4306-88-1
Molecular formula:
C23H40O
IUPAC Name:
2,6-di-tert-butyl-4-nonylphenol
Test material form:
liquid
Details on test material:
- Density: 0.895 g/cm3
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:


Method

Target gene:
histidine
Species / strain
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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

Applicant's summary and conclusion

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.