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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria (Ames)

Key study:

In a study performed the standardised guideline OECD 471, under GLP conditions, the test material was considered to be non-mutagenic.

 

In-vitro micronucleus

Key study:

In a study performed the standardised guideline OECD 487, under GLP conditions, the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

 

In-vitro Micronucleus

Key study:

In a study performed the standardised guideline OECD 490, under GLP conditions, the test item was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

 

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:
10 December 1996 to 16 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Description: Dark amber viscous liquid
Purity: 100%
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:
S9 Rat liver, induced with Aroclor
Test concentrations with justification for top dose:
Test 1 and Test 2: 0, 50, 150, 500, 1500 and 500 µg/plate.
5000 µg is the standard top dose recommended in the guidelines.
Vehicle / solvent:
Acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Key result
Species / strain:
S. typhimurium, other: TA 100, A 1535, TA 1537 and TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test material was considered to be non-mutagenic under the test conditions of this test.
Executive summary:

Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA 100, and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, 50, 150, 500, 1500, and 5000 ug/plate, in triplicate, both with and without the addition of a rat liver S9 metabolising system. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains, and fresh test material formulations.

The dose range was determined in a preliminary toxicity assay in which the test material at dose levels from 50 to 5000 µg/plate caused no visible reduction in the growth of the bacterial lawn at any dose level, either with or without metabolic activation.  A precipitate was observed at and above 1500 µg/plate, but did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system. The test material was considered to be non-mutagenic under the conditions of this test.  

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Purity: > 93%
Description: Yellow translucent viscous liquid
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 10 to 150 µg/mL for all three exposure groups.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine
Details on test system and experimental conditions:
The in vitro mammalian cell micronucleus assay was conducted by exposing HPBL to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.
Evaluation criteria:
The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
lymphocytes: human
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:
not applicable
Positive controls validity:
valid
Conclusions:
No significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9.
Executive summary:

The test substance was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle.

 

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 10 to 150 µg/mL for all three exposure groups.

 

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was not observed at any dose in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 75 µg/mL in all three exposure groups. The doses selected for evaluation of micronuclei were 10, 20, and 75 µg/mL for all three exposure groups. 

No significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

 

These results indicate the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Description: Yellow translucent viscous liquid
Purity: >93%
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 13.7, 27.3, 54.7, 109, 219, 438, 875, 1750 and 3500 µg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle.

Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 6.84, 13.7, 27.3, 54.7 and 109 µg/mL (for all treatment conditions).
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
The mammalian mutation assay was conducted by exposing L5178Y/TK+/- cells to appropriate concentrations of test substance, as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.
Evaluation criteria:
The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10 4), and was expressed as TFT-resistant mutants/106 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥90 mutants/106 clonable cells at the Aberdeen meeting in 2003 (Moore et al., 2006).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
≥109 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
These results indicate the test item was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.
Executive summary:

The test substance was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Ethanol was used as the vehicle.

 

In the preliminary toxicity assay, the concentrations tested were 13.7, 27.3, 54.7, 109, 219, 438, 875, 1750 and 3500 µg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥109 µg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 28, 74 and 56% at concentrations of 3500 µg/mL (4-hour treatment with S9), 3500 µg/mL (4-hour treatment without S9) and 3500 µg/mL (24-hour treatment without S9), respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 6.84, 13.7, 27.3, 54.7 and 109 µg/mL for all treatment conditions.

 

In the definitive mutagenicity assay, visible precipitate was observed at concentration ≥109 µg/mL at the beginning of treatment and end of treatment. Cultures treated at all concentrations of 4-hour treatment with S9, 4-hour treatment without S9 and 24-hour treatment without S9 exhibited 87 to 105%, 97 to 109% and 81 to 106% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 87 to 113% (4 hour treatment with S9), 83 to 111% (4-hour treatment without S9) and 62 to 88% (24 hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under any treatment condition. 

 

These results indicate the test item was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification