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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not genotoxic (Ames test)

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:
From May 04 to 26, 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:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
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:
liver microsomal preparation (S9-mix)
Test concentrations with justification for top dose:
Dose selection
Experiment 1 (plate incorporation method)
The test item was tested using the following method: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were initially assayed in triplicate against each tester strain, using the direct plate incorporation method. However, several of the bacterial tester strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) or poor revertant colony frequency and, therefore a number of strains had to be repeated employing an amended test item dose range as follows:
Salmonella strain TA100 (presence and absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strains TA98, TA1537, TA1535 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Experiment 2 – Pre-Incubation Method
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows: All Salmonella strains (absence of S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate. Salmonella strains TA1535 and TA100 (presence of S9) and E.coli strain WP2uvrA (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate. Salmonella strains TA1537 and TA98 (presence of S9) and E.coli strain WP2uvrA (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
in the absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation method in Experiment 1 and pre-incubation method in Experiment 2

DURATION
- Preincubation period: 20 min in Experiment 2
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

PROCEDURE
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the solvent vehicle or test item formulation or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical Analysis
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
other: TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp.1: 50 µg/plate without S9 mix, 150 µg/plate with S9 mix. Exp.2: starting from 5 µg/plate without S9 mix and from 50 µg with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The vehicle (acetone) control plates gave 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Cytotoxicity

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method) the test item induced toxicity evident as visible reductions in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of metabolic activation (S9-mix) and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit of the test item was employed as the maximum dose level in the second mutation test. The test item induced a stronger toxic response after employing the pre-incubation method in the second mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 5 μg/plate (TA100, TA1535, TA98 and TA1537) and 15 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial lawns were noted from 50 μg/plate (TA1535), 150 μg/plate (TA100, TA98 and TA1537) and 500 μg/plate (WP2uvrA). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Conclusions:
Not genotoxic.
Executive summary:

Method

The substance was tested for mutagenic effects in vitro in Salmonella typhimurium and Escherichia coli strains, according to the OECD guidelines 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to ten dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was initially 1.5 to 5000 μg/plate. However, several tester strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and a number of strains had to be repeated employing amended doses ranging between 0.15 and 5000 μg/plate. The second experiment was performed at a later date (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.015 and 1500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Conclusion

Not genotoxic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance was tested for mutagenic effects in vitro in Salmonella typhimurium and Escherichia coli strains, according to the OECD guidelines 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to ten dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was initially 1.5 to 5000 μg/plate. However, several tester strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and a number of strains had to be repeated employing amended doses ranging between 0.15 and 5000 μg/plate. The second experiment was performed at a later date (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.015 and 1500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated bacterial reverse mutation tests.

The substance did not create gene mutations in the strains of Salmonella typhimurium and E. Coli under the performed test, therefore according to the 3.5. of the CLP Regulation EC n.1272/2008, it cannot be classified as mutagenic for germ cells.

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