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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vtro Bacterial Reverse Mutation studies are available for the Substance. The conclusions are summarised as follows:

in vitro Bacterial Reverse Mutation studies (2016) = Not mutagenic (+ & - S9)

in vitro Bacterial Reverse Mutation studies (2001) = Not mutagenic (+ & - S9)

Based on the weight of evidence the registered Substance was considered to be non-mutagenic in bacteria with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Oct 2015 to 23 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Refer to the main study report
GLP compliance:
yes
Remarks:
Refer to the main study report
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.
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:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate for all test conditions
Mutagenicity assay: 33.3, 100, 333, 1000, 3333 and 5000 µg per plate (for TA98, TA1537 and WP2A uvrA) and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 µg per plate (for TA100 and TA1535)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test substance, 2 aminoanthracene, 2-nitrofluorene, 9-aminoacridine, methyl methanesulfonate); water (sodium azide)
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 35.8ºC for 10 minutes in the solubility test conducted at BioReliance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay

NUMBER OF CELLS EVALUATED: >/= 0.3 * 10^8 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.
Evaluation criteria:
The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported.

For the test article to be evaluated positive (mutagenic), it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity, either background or as a reduction in revertant count, was observed beginning at 100 or at 1000 µg per plate with tester strains TA100 and TA1535 in the presence and absence of S9 activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, The Substance did not induce reverse mutations at selected loci of several tester strains in either the presence or absence of Aroclor induced rat liver S9. A confirmatory (independent repeat) assay was not required as the study was concluded to be unequivocally negative
Executive summary:

The test Substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl Sulfoxide (DMSO) was used as the vehicle. All criteria for a valid study were met as described in the protocol.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate of the test material was observed from3333-5000 µg per plate. Toxicity, either background or as a reduction in revertant count, was observed beginning at 33.3, 66.7, 100 or 333 µg per plate with tester strains TA100 and TA1535 in the presence and absence of S9 activation. No toxicity was observed in other tester strains treated. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate (for TA98, TA1537 and WP2AuvrA) and 1000 µg per plate (for TA100 and TA1535).

In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 µg per plate (for TA98, TA1537 and WP2AuvrA) and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 µg per plate (for TA100 and TA1535). Precipitate was observed from 3333-5000 µg per plate. Toxicity, either background or as a reduction in revertant count, was observed beginning at 100 or at 1000 µg per plate with test strains TA100 and TA1535 in the presence and absence of S9 activation. No toxicity was observed in other tester strains treated. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

All criteria for a valid study were met as described in the protocol. Under the conditions of this study, test Substance was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimurium and at the tryptophan locus of Escherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April 2000 - 20 April 2000
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Expiration Date: September 2004
Description: Amber Solid
Storage Condition: Room Temperature
Target gene:
The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Storage: Frozen (< — 70° C) in broth with 8% DMSO, after an overnight incubation at 37 ± 2° C.
Metabolic activation:
with and without
Metabolic activation system:
Homogenate from the livers of Aroclor 1254 pretreated Sprague Dawley Rats (S9)
Test concentrations with justification for top dose:
Prior to initiation of the initial mutagenicity assay, a rangefinding test was performed to determine appropriate doses for the assay.
Pinpoint background (a sign of toxicity) was observed on plates at 50 µg/plate with metabolic activation and at 5 µg/plate without metabolic activation.
Revertants were unable to be distinguished from the background at 5000 µg/plate with metabolic activation and at 2000 µg/plate without metabolic activation.
Based on the results of this rangefinding test, test concentrations of 50, 15.6, 5.0, 1.6, and 0.5 12g/plate (+S9), and 10, 3.2, 1.0, 0.3, and 0.1 µg/plate (-S9) were selected for the assay.
Vehicle / solvent:
Tetrahydrofuran (THF) was determined to be the vehicle of choice for the doses required for the assay.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
mitomycin C
other: 2-Aminoanthracene, N-Methyl-N-Nitro-N-Nitrosoguanidin, Danthron
Details on test system and experimental conditions:
Frozen tester strains were thawed and inoculated into a nutrient broth culture one day prior to
dosing and incubated at 37±2°C for 8-16 hours. The number of cells per culture, following a 10 hour incubation, was determined to be 2.24 x 109 cells/mL.

Samples of bacteria (0.1 mL), followed by vehicle (25 1AL for THE or 100 1AL for DMSO or water), appropriate test substance dilution (25 11,L), or appropriate positive control substance dilution (100 pL), and 0.5 mL of S9 mix (+S9) or saline (-S9), were added to sterile glass test tubes containing molten top agar. The mixture was vortexed and immediately poured on plates containing a layer of minimal agar medium. After the top agar solidified the plates were inverted and incubated at 37± 2°C for approximately 2 days.
Evaluation criteria:
For tester strains TA98, TA1535, and TA1537, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than three times the mean number of spontaneous revertants on the vehicle control plates. For tester strains TA100 and TA102, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than two times the mean number of spontaneous revertants on the vehicle control plates.
A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance, including at least one positive dose.
For each strain, a lack of a positive response in the positive control group will render that portion of the test invalid.
Toxicity was defined as a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control. When the mean revertant colony count for a test substance concentration is greater than or equal to two or three times the vehicle control, toxicity may also be defined as a greater than 50% reduction in the mean number of revertant colonies at concentrations higher than the concentration that induced the largest increase in revertant colonies.
A positive result in this assay indicates that, under the test conditions, the test substance induced point mutations by base changes or frameshifts in the genome of Salmonella typhimurium.
A negative result indicates that, under the test conditions, the test substance was not mutagenic in Salmonella typhimurium.
Statistics:
The mean revertant colony count and standard deviation were determined for each dose point (Snedecor and Cochran, 1989).
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Light background
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Light background
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
light background
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Decrease in number of revertants compared to vehicle control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
REduction in the number of revertants compared to the vehicle control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test Substance was not mutagenic in any strain of Salmonella typhimurium tested.

See attached background documents

Conclusions:
The test Substance was not mutagenic in any strain of Salmonella typhimurium tested.
Executive summary:

This study was conducted in order to evaluate the mutagenic potential of the test substance in five selected strains of Salmonella typhimurium. The test substance was diluted in tetrahydrofuran (THF) and tested in both an initial and repeat assay. Based on the results of a rangefinding assay, test concentrations of 50, 15.6, 5.0, 1.6, and 0.5 µg/plate with metabolic activation (+S9) and test concentrations of 10, 3.2, 1.0, 0.3, and 0.1 pig/plate without metabolic activation (-S9) were selected for the initial assay. The assay was repeated in order to confirm the response observed in the initial assay. A 25 µL, sample of each concentration was tested with and without metabolic activation (rat liver S9 mix) following the procedures outlined by Maron and Ames (1983). Two positive controls and two vehicle controls were tested concurrently for each strain. The positive controls and their vehicles were tested using a 100 µL sample.

 

The test substance did not induce positive points in mean number of revertant colonies (equal to or greater than two or three times the vehicle control). A >50% reduction in the mean number of revertant colonies compared to the vehicle control was observed in the initial assay at 1011g/plate (­S9) in TA1537, at 0.5 µg/plate (+S9) in TA1537, and at > 15.6 µg/plate (+S9) in TA1537 and in the repeat assay at 1µg/plate (-S9) in TA1535. Light background was observed in the initial assay at > 0.3 µg/plate (-S9) in TA98, > 3.2 µg/plate (-S9) in TA100, 10 µg/plate (-S9) in TA102, at > 3.2 µg/plate (-S9) in TA1537, 50 µg/plate (+S9) in TA100, and in one plate at 5 µg/plate (+S9) in TA102. Light background was also observed in the repeat assay at 10 pig/plate (-S9) in TA98 and TA100, > 5µg/plate (+S9) in TA100 and 5 µg/plate (+S9) in TA102. These reductions and background abnormalities were considered to be a biologically significant indication of toxicity.

 

Based on the results of this assay, the test substance was not mutagenic in any strain ofSalmonella typhimuriumtested.

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

Genetic toxicity in vivo

Description of key information

Justification for selection of genetic toxicity endpoint
Studies were conducted on the registered substance. In accordance with Annex VII of REACH, no further testing was performed since the in vitro gene mutation study in bacteria returned a negative result.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of genetic toxicity endpoint
Studies were conducted by a GLP accredited laboratory using OECD Testing Guideline 471. Studies were conducted on the registered substance. In accordance with Annex VII of REACH, no further testing was performed since the in vitro gene mutation study in bacteria returned a negative result.

Justification for classification or non-classification

An in vitro gene mutation study in bacteria was conducted on the registered substance and returned a negative result. In accordance with Annex VII of REACH, no further testing was performed considering this result. Therefore, the registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.