Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of the study.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data is summarized based on the available information from various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
2. Histidine
3. Histidine for Salmonella typhimurium and Tryptophan for E. coli
Species / strain / cell type:
S. typhimurium, other: S. typhimurium, other: TA102, TA100, TA98 and TA97a
Remarks:
2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
3
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2. Lyophilized rat liver S9 fraction induced by Aroclor 1254
3. S9 metabolic activation system
Test concentrations with justification for top dose:
2. 0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 μg/plate
3. +S9: 0, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate
-S9: 0, 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in Ethanol
3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test chemical was soluble in DMSO.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
mitomycin C
other: NPD (1 μg/plate). 2AF (10 μg/plate), 2AA (0.5 or 1 μg/plate)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
3
Details on test system and experimental conditions:
2. NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - each experiment was repeated at least once
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 1.2x10^9 bacteria per milliliter
- Test substance added - in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 72 h
- Harvest time after the end of treatment (sampling/recovery times): No data
3. METHOD OF TREATMENT/ EXPOSURE: standard plate incorporation method
Evaluation criteria:
2. A dose related increase in the number of revertants was noted whether it be twofold over background or not
3. Plates were observed for increase in the mean number of revertant colonies.
Statistics:
2. means ±SD
3. No data
Species / strain:
S. typhimurium, other: TA102, TA100, TA98 and TA97a
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
bacteria, other: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
2. RANGE-FINDING/SCREENING STUDIES (if applicable): A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either
the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay.
3. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Salmonella reverse mutation assay was performed to determine the mutagenic nature of the given test chemical on a tester strains of Salmonella typhimurium TA102, TA100, TA98 and TA97 with and without metabolic activation system extracted from Lyophilized rat liver induced by Aroclor 1254 at dose concentrations of 0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate. The Salmonella mutagenicity test was performed by the plate incorporation method. Briefly, 2 ml of top-agar was mixed with 100µl of an overnight grown culture of S. typhimurium, 100µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500µl of the phosphate buffer or the S9 mixture. Ethanol served as the negative (solvent) controls, while the positive control substances were: SA (0.5µg/plate), NPD (1µg/plate), MC (0.5µg/plate), 4-NQNO (1µg/plate), 2AF (10µg/plate), 2AA (0.5 or 1µg/plate), and B-(α)-P (50µg/plate). SA and MC were dissolved in distilled water and dimethyl sulfoxide (DMSO) was used as vehicle for the other positive controls. Plates were incubated at 37°C for 72 h in the dark and then scored for revertant his+ bacteria colonies. Every determination was made in triplicate and each experiment was repeated at least once in order to check the reproducibility of the results. A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. The given test chemical was non toxic at highest doses of 2000–2500µg/plate. No mutagenic effect was observed with the four bacterial tester strains, in the absence as well as in the presence of extrinsic metabolic activation. Hence, the given test chemical is not likely to classify as a gene mutant in vitro.

 

Another gene mutation toxicity study was performed for the given test chemical in accordance with OECD TG 471 using the standard plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA were treated with test chemical in DMSO (dimethyl sulfoxide) at dose levels of 0, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence of S9 and at dose levels of 0, 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the absence of S9. No increase in the mean number of revertant colonies was observed at any tested dose level in any tester strain in the presence or absence of S9 in the initial or the confirmatory assays. Under the conditions of the study, the given test chemical was considered to be not mutagenic, when tested on Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of the study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames assay:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Salmonella reverse mutation assay was performed to determine the mutagenic nature of the given test chemical on a tester strains of Salmonella typhimurium TA102, TA100, TA98 and TA97 with and without metabolic activation system extracted from Lyophilized rat liver induced by Aroclor 1254 at dose concentrations of 0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate. The Salmonella mutagenicity test was performed by the plate incorporation method. Briefly, 2 ml of top-agar was mixed with 100µl of an overnight grown culture of S. typhimurium, 100µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500µl of the phosphate buffer or the S9 mixture. Ethanol served as the negative (solvent) controls, while the positive control substances were: SA (0.5µg/plate), NPD (1µg/plate), MC (0.5µg/plate), 4-NQNO (1µg/plate), 2AF (10µg/plate), 2AA (0.5 or 1µg/plate), and B-(α)-P (50µg/plate). SA and MC were dissolved in distilled water and dimethyl sulfoxide (DMSO) was used as vehicle for the other positive controls. Plates were incubated at 37°C for 72 h in the dark and then scored for revertant his+ bacteria colonies. Every determination was made in triplicate and each experiment was repeated at least once in order to check the reproducibility of the results. A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. The given test chemical was non toxic at highest doses of 2000–2500µg/plate. No mutagenic effect was observed with the four bacterial tester strains, in the absence as well as in the presence of extrinsic metabolic activation. Hence, the given test chemical is not likely to classify as a gene mutant in vitro.

 

Another gene mutation toxicity study was performed for the given test chemical in accordance with OECD TG 471 using the standard plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA were treated with test chemical in DMSO (dimethyl sulfoxide) at dose levels of 0, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence of S9 and at dose levels of 0, 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the absence of S9. No increase in the mean number of revertant colonies was observed at any tested dose level in any tester strain in the presence or absence of S9 in the initial or the confirmatory assays. Under the conditions of the study, the given test chemical was considered to be not mutagenic, when tested on Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of the study.

 

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.