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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG471): not mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2016 - 22 September 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:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254.
Test concentrations with justification for top dose:
Direct plate:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Pre-incubation:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 2:
TA 1535, TA 1537 and TA98 (without S9): 0.17, 0.55, 1.7, 5.4, 17 and 52 μg/plate
TA 1535, TA 1537 and TA98 (with S9): 1.7, 5.4, 17, 52, 164 and 512 μg/plate
TA 100 (without S9): 0.17, 0.55, 1.7, 5.4, 17 and 52 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
Not performed.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Direct plate: without S9 at concentrations of 164 μg/plate and upwards and with S9 no toxicity was observed. Pre-incubation: without S9-mix at concentrations of 52 μg/plate and upwards and with S9 at concentrations of 164 μg/plate and upwards.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Direct plate: without S9 at concentrations of 164 μg/plate and upwards and with S9 no toxicity was observed. Pre-incubation: without S9-mix at concentrations of 17 μg/plate and upwards and with S9 at the concentration of 512 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Direct plate: without S9 at conc. of 164 μg/plate and upwards and with S9 at conc. of 1600 μg/plate and upwards. Pre-incubation: without S9-mix at conc. of 17 μg/plate and upwards and with S9 at conc. of 164 μg/plate and upwards.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Direct plate: without S9 at conc. of 164 μg/plate and upwards and with S9 at the conc. of 5000 μg/plate and upwards. Pre-incubation: without S9-mix at conc. of 17 μg/plate and upwards and with S9 at the conc. of 512 μg/plate.
Vehicle controls validity:
valid
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:
cytotoxicity
Remarks:
Direct plate: no toxicity was observed. Pre-incubation: no toxicity was observed without S9-mix and with S9 toxicity was observed at conc. of 1600 μg/plate and upwards.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Direct plate: The test item precipitated at the concentrations of 1600 and/or 5000 μg/plate in the absence and presence of S9-mix in the strains TA1535, TA1537 and TA98. The test item did not precipitate in strain TA 100 and WP2uvrA.
Pre-incubation: The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix in strains TA 100 and WP2uvrA. In the tester strains TA1535, TA1537 and TA98 no precipitate was observed up to the highest dose level tested.

RANGE-FINDING/SCREENING STUDIES:
Direct plate: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in TA100 without S9 at concentrations of 164 μg/plate and upwards and with S9 no toxicity was observed. In addition, no toxicity was observed at any of the dose levels tested in strain WP2uvrA.
Pre-incubation: Toxicity was observed in TA100 without S9-mix at concentrations of 52 μg/plate and upwards and with S9 at concentrations of 164 μg/plate and upwards. In strain WP2uvrA no toxicity was observed without S9-mix and with S9 toxicity was observed at conc. of 1600 μg/plate and upwards.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 78 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907
Mean 785 228 653 387 1155 860
SD 167 105 290 143 370 323
n 1684 1662 1448 1536 1646 1686

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 892 1404 1263 342
SD 178 327 461 165
n 1650 1677 1370 1410
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Direct plate: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix, except in tester strain TA100 in the presence of S9-mix. In addition, no toxicity was observed at any of the dose levels tested in strain WP2uvrA.
- Pre-incubation: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence of S9-mix.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 - Ames (1997) and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in the Ames test in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in TA 98, TA 1535, TA 1537 and TA 100 in the absence of S9 -mix tester strains in the absence and presence of S9-mix. In tester strain TA100 in the presence of S9-mix and in tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In the second pre-incubation experiment, test item was initially tested in the dose range-finding study in TA 100 and WP2uvrA up to concentrations of 5000 μg/plate.

The test item was tested up to concentrations of 52 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix, and up to 512 μg/plate in the tester strains TA1535, TA1537 and TA98 in the presence of S9-mix in the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenic activity of the substance was evaluated in the Ames test in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in TA 98, TA 1535, TA 1537 and TA 100 in the absence of S9 -mix tester strains in the absence and presence of S9-mix. In tester strain TA100 in the presence of S9-mix and in tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In the second pre-incubation experiment, test item was initially tested in the dose range-finding study in TA 100 and WP2uvrA up to concentrations of 5000 μg/plate.

The test item was tested up to concentrations of 52 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix, and up to 512 μg/plate in the tester strains TA1535, TA1537 and TA98 in the presence of S9-mix in the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments (1272/2008).