Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-Jan-1997 to 27-Jan-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 471)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 26-May-1983
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)
Remarks:
CCR Cytotest Cell Research GmbH & Co. KG, 64380 Roftdorf, In den Leppsteinswiesen 19
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
422-600-5
EC Name:
-
Cas Number:
73936-91-1
Molecular formula:
C29H35N3O
IUPAC Name:
2-(2H-1,2,3-benzotriazol-2-yl)-6-(2-phenylpropan-2-yl)-4-(2,4,4-trimethylpentan-2-yl)phenol
Details on test material:
- Physical state: whitish, solid
- Analytical purity: 99.35-99.45 %
- Storage: at room temperature, away from light

Method

Target gene:
trp, his
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from phenobarbital (i.p.) and beta-naphthoflavone (p.o.) induced male Wistar rats
Test concentrations with justification for top dose:
33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate
Vehicle / solvent:
On the day of the experiment, the test item was dissolved in acetone. The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Remarks:
all controls were concurrent
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (experiment I), preincubation (experiment II and IIb)

DURATION
- Preincubation period: 1 hour
- Exposure duration: 48 hours
- Expression time: at least 48 hours

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was assessed as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

POSITIVE CONTROLS
without S9 mix:
sodium azide for strains TA 1535, TA 100 (10 µg/plate)
4-nitro-o-phenylene-diamine for strains TA 98 (10 µg/plate) and TA 1537 (50 µg/plate)
methyl methane sulfonate for strain WP2uvrA (5 µl/plate)
with S9 mix:
2-aminoanthracene for strains TA 1535, TA 1537, TA 98, TA 100 (2.5 µg/plate) and WP2 uvrA (10 µg/plate)
Evaluation criteria:
The generally accepted conditions for the evaluation of the results were:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
A test item was considered as positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration was induced. A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any one of the test points was considered non-mutagenic in this system.
A test item was considered as mutagenic if in the strains TA 98, TA 100 and WP2 uvrA the number of reversions were at least twice as high and in the strains TA 1535 and TA 1537 were at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
The data were not evaluated statistically.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar from 333.3 up to 5000.0 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. According to the results of this pre-experiment the concentrations applied in the main experiments were chosen.

Any other information on results incl. tables

Experimental Result

Without S9 Mix

Revertants/plate (mean from three plates)

TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Concentration
µg/plate
I II I II I II I II I II
Negative control 17 18 11 11 20 17 141 104 44 28
Solvent control 16 15 9 9 18 18 139 102 39 32
Positive control 1167 1109 134 128 430 568 1213 1102 1086 993
33.3 16 13 11 7 21 14 147 103 34 37
100 16 16 8 9 20 16 142 108 42 39
333.3 17 14 12 7 24 13 146 89 39 33
1000 13 14 11 7 26 16 145 106 46 35
2500 17 10 8 8 27 13 134 94 39 35
5000 16 9 9 8 24 25 129 110 45 34

With S9 -Mix

Revertants/plate (mean from three plates)
TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Concentration
µg/plate
I II I II I II I II I II IIa
Negative control 18 19 17 15 33 33 158 147 44 32 38
Solvent control 16 13 15 12 30 25 154 119 52 28 33
Positive control 275 118 92 70 1119 482 1244 604 236 146 174
33.3 17 16 13 9 24 26 151 131 47 30 32
100 17 18 14 14 30 24 138 119 53 30 31
333.3 17 14 16 12 27 28 149 137 58 41 39
1000 19 14 16 12 31 26 152 132 50 52 35
2500 17 16 16 14 27 32 139 156 57 67 38
5000 17 14 14 16 24 40 156 164 52 50 31

I = Experiment I (standard plate test)

II and IIa = Experiment II and IIa (preincubation method)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test article is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A GLP-compliant Ames study following OECD guideline 471 was performed to investigate the test article's potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and an additional experiment (experiment IIa) following the preincubation procedure with the Escherichia coli strain WP2 uvrA in the presence of metabolic activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in both experiments. No toxic effects occurred in the test groups with and without metabolic activation. No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed after treatment with test material at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix). A slight increase in the number of revertants occurred in the second experiment in strain WP2 uvrA in the presence of metabolic activation exceeding the threshold of twice the number of the corresponding solvent control at 2500 µg/plate. This effect could not be verified under identical conditions in an additional experiment (experiment IIa) in strain WP2 uvrA in the presence of metabolic activation. Therefore, the slight increase observed in the second experiment was judged to be caused by statistical fluctuations of the rather low numbers of revertant colonies and does not represent a mutagenic effect. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.