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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test, OECD 471: positive (reference 7.6.1 -1).

Micronucleus test in vitro, OECD 487, screening: negative (reference 7.6.1 -2)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-09 to 2015-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
TA 98 (his D 3052, uvrB, rfa + R-factor)
TA 100 (his G 46, uvrB, rfa + R-factor)
TA 1535 (his G 46, uvrB, rfa)
TA 1537 (his C 3076, uvrB, rfa)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Male Wistar, HSdCpb:Wu rats, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (Merck KGaA, Darmstadt, Germany). The animals received drinking water and a standard diet ad libitum. The body weight of the animals used was 179 g ± 6.76 g.
- method of preparation of S9 mix : according to Ames
- quality controls of S9: Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch.
Test concentrations with justification for top dose:
with and without S9: 5.00, 15.8, 50.0, 158.0, 500.0, 1580.0, 5000.0 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: well solubility of the test item; recommended by the guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: Daunomycin; 2-Aminoanthracene
Rationale for test conditions:
According to the guidelines
Evaluation criteria:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The criteria provided in table 1 in "any other information on materials and methods", based upon the historical controls of the laboratory and statistical considerations, were established.

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

For valid data, the test material was considered to be positive or mutagenic if:
• the assay was to be considered valid, and
• a dose dependent (over at least two test material concentrations) increase in the number of re-vertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same lest system or
• "clear increases" occurred al least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed

Please refer to background material attached.

Conclusions:
Under the conditions of this study the test item is considered mutagenic in procaryotic cells.
Executive summary:

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). Treatments of all tester strains were performed in the absence and in the presence of S9 mix, using final concentrations of the test item between 5 and 5000 µg/plate, plus vehicle and positive controls. After test material exposure, no precipitation of the test material on the agar plates occurred and no toxicity to the bacteria was observed.

Daunomycin, sodium azide, 9-aminoacridine, and 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used as positive control in the presence of the metabolic activation and thus for testing the activity of the S9 mix.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions of this assay, there were relevant increases in revertant numbers after test item exposure observed in TA 100 in the absence of S9 mix in both series.

Therefore, the test material is considered mutagenic under the described experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-09-14
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Remarks:
Only short report avilable, only screening test was conducted
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
only screening test was conducted
Principles of method if other than guideline:
The in vitro micronucleus assay is a mutagenicity test system used for the detection of chemicals that induce the formation of small membrane-bound DNA fragments, such as micronuclei, in the cytoplasm of interphase cells. The assay has the potential to detect the activity of both clastogenic and aneugenic chemicals. Micronuclei were detected by using the In Vitro MicroFlow® method (Litron Laboratories, Rochester, NY, USA). The relevant limit of cytotoxicity is determined by the most sensitive parameter (RPD, RICC or RS).
GLP compliance:
no
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Metabolic activation system:
rodent liver S9 mix
Test concentrations with justification for top dose:
5, 10, 20, 25, 27.5, 30, 32, 35, 35.5, 40, 63.1, 112 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Grisoefulvin
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the conditions of this test the test item was not mutagenic.
Executive summary:

The objective of this screening assay was to assess the mutagenic potential of the test item. The in vitro micronucleus assay is a mutagenicity test system used for the detection of chemicals that induce the formation of small membrane-bound DNA fragments, such as micronuclei, in the cytoplasm of interphase cells. The assay has the potential to detect the activity of both clastogenic and aneugenic chemicals. Micronuclei were detected by using the In Vitro MicroFlow® method (Litron Laboratories, Rochester, NY, USA). The relevant limit of cytotoxicity is determined by the most sensitive parameter (RPD, RICC or RS).

The test item showed a very steep cytotoxicity response. The test item showed a micronucleus induction at one concentration near the limit of cytotoxicity only, which could not be reproduced in a second experiment. Therefore, this effect is considered to be of no biological relevance. The test item was thus concluded to be not mutagenic in this screening test system in the absence or presence of the metabolic activation (S9 mix).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test (reference 7.6.1 -1)

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). Treatments of all tester strains were performed in the absence and in the presence of S9 mix, using final concentrations of the test item between 5 and 5000 µg/plate, plus vehicle and positive controls. After test material exposure, no precipitation of the test material on the agar plates occurred and no toxicity to the bacteria was observed.

Daunomycin, sodium azide, 9-aminoacridine, and 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used as positive control in the presence of the metabolic activation and thus for testing the activity of the S9 mix. Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions of this assay, there were relevant increases in revertant numbers after test item exposure observed in TA 100 in the absence of S9 mix in both series.

Therefore, the test material is considered mutagenic under the described experimental conditions.

Micronucleus Test in vitro, screening test (reference 7.6.1 -2)

The objective of this screening assay was to assess the mutagenic potential of the test item. The in vitro micronucleus assay is a mutagenicity test system used for the detection of chemicals that induce the formation of small membrane-bound DNA fragments, such as micronuclei, in the cytoplasm of interphase cells. The assay has the potential to detect the activity of both clastogenic and aneugenic chemicals. Micronuclei were detected by using the In Vitro MicroFlow® method (Litron Laboratories, Rochester, NY, USA). The relevant limit of cytotoxicity is determined by the most sensitive parameter (RPD, RICC or RS).

The test item showed a very steep cytotoxicity response. The test item showed a micronucleus induction at one concentration near the limit of cytotoxicity only, which could not be reproduced in a second experiment. Therefore, this effect is considered to be of no biological relevance. The test item was thus concluded to be not mutagenic in this screening test system in the absence or presence of the metabolic activation (S9 mix).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are not sufficient for final conclusion on classification under Regulation (EC) No 1272/2008. An in vitro mutation assay in bacteria revealed a positive result, whereas an in vitro screening test on cytogenicity in mammalian cells was negative. Based on the available data no conclusion can be made regarding classification and labelling as mutagen according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.

The test item is registered as transported isolated intermediate according to REACH Art. 17/18 .Consequently, there is no concern in regards to human or environmental exposure. Therefore, further hazard assessment is legally and scientifically not required.