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Genetic toxicity in vitro

Description of key information
No evidence of genotoxicity was observed in a bacterial reverse mutation assay, an in vitro chromosomal aberration assay and an in vitro mammalian cell mutation assay.
Link to relevant study records
Reference
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:
From August 25, 2010 to June 01, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471 and OPPTS 870.5100, in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidelines S2A and S2B (ICH, 1995, 1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
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:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Initial mutagenicity assay: 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5,000 µg/plate with and without S9
Confirmatory assay: 10.0, 50.0, 100, 500, 1000, and 5,000 µg/plate with and without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9 (TA98)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 (TA100, TA1535)
Negative solvent / vehicle controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9 (TA1537)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 (WP2uvrA)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 (TA98)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 (TA100, TA1535, TA1537, WP2uvrA)
Details on test system and experimental conditions:
TEST SYSTEM RATIONALE:
The bacterial reverse mutation assay has been shown to be a sensitive, rapid, and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his–) or tryptophan (trp–) dependent cells are exposed to the test substance and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his– or trp– cells.

SOURCE OF TESTER STRAINS:
Salmonella tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California. E. coli tester strain WP2uvrA was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

PLATING PROCEDURES:
Each plate was labelled with a code that identified the test substance, test phase, tester strain, activation condition, and dose level. Dilutions of the test substance were prepared immediately prior to their use. Treatments were performed by adding 100 µL tester strain and 50 µL of test or control substance to 2.5 mL of molten selective top agar (maintained at 45±2°C). After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal bottom agar in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52±4 h at 37±2°C. Cultures were treated in the presence of S9 in an identical manner, except using 2.0 mL undiluted molten selective top agar and adding 500 µL S9 mix.

- In an initial mutagenicity assay, all doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in duplicate plates. In an independent confirmatory assay, all test and control substances were evaluated in triplicate plates.

SCORING THE PLATES:
Plates which were not evaluated immediately following the incubation period were held at >0 to 10°C until such time that colony counting and bacterial background lawn evaluation could take place.

BACTERIAL BACKGROUND LAWN EVALUATION:
The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted as applicable.

COUNTING REVERTANT COLONIES:
Revertant colonies were counted by automated colony counter and/or by hand.
Evaluation criteria:
ASSAY EVALUATION CRITERIA:

Criteria for a Positive Response:
A test substance is considered to have produced a positive response if it induces a dose dependent increase in revertant frequency that is ≥2.0 fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥3.0 fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.

Criteria for a Negative Response:
A test substance is considered to have produced a negative response if no dose dependent, ≥2.0 fold or ≥3.0 fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

Criteria for an Equivocal Response:
Even after repeated trials, a test substance may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test substance may be considered to have produced an equivocal response.

- Other criteria also may be used in reaching a conclusion about the study results (e.g. comparison to historical control values, biological significance, etc.).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY ASSAYS
In the initial mutagenicity assay, normal growth was observed in all five tester strains at all doses evaluated with and without S9. However, the test substance was incompletely soluble in the aqueous top agar at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, approximated or were less than those observed in the concurrent vehicle control cultures.

In the confirmatory mutagenicity assay, all doses of the test substance, normal growth again was observed in all five tester strains at all doses evaluated with and without S9. In addition, the test substance again was incompletely soluble at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, again approximated or were less than control values. All positive and vehicle control values were within acceptable ranges. The positive control response of TA98 with S9 in initial mutagenicity assay was above 2009 historical positive control range. Since the high response confirmed the sensitivity of the test system in this study, the response above 2009 historical positive control range was accepted and will be used for 2010 historical positive control. There was, therefore, no impact on the study. All criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The substance was not mutagenic with or without metabolic activation under the conditions of this bacterial reverse mutation assay.
Executive summary:

The test substance was investigated for the ability to induce reverse mutations at the histidine locus in several strains of Salmonella (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of E. coli strain WP2uvrA according to OECD Guideline 471 and OPPTS 870.5100, in compliance with GLP. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial mutagenicity assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The studies were performed in the presence and absence of Aroclor-induced rat liver S9. In the confirmatory mutagenicity assay, normal growth was observed in all five tester strains at all doses evaluated with and without S9. In addition, the test substance was incompletely soluble at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, approximated or were less than control values. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. The substance was not mutagenic with or without metabolic activation under the conditions of this bacterial reverse mutation assay (Xu Y, 2011).

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

Additional information

Additional information from genetic toxicity in vitro:

Ames test

The test substance was investigated for the ability to induce reverse mutations at the histidine locus in several strains of Salmonella (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of E. coli strain WP2uvrA according to OECD Guideline 471 and OPPTS 870.5100, in compliance with GLP. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial mutagenicity assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The studies were performed in the presence and absence of Aroclor-induced rat liver S9. In the confirmatory mutagenicity assay, normal growth was observed in all five tester strains at all doses evaluated with and without S9. In addition, the test substance was incompletely soluble at the highest three or four doses evaluated with and without S9. Revertant frequencies for all doses of test substance, in all tester strains with and without S9, again approximated or were less than control values. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.The substance was not mutagenic with or without metabolic activation under the conditions of this bacterial reverse mutation assay (Xu Y, 2011).

Chromosomal aberrations in vitro

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in cultured human lymphocytes according to OECD Guideline 473 and OPPTS 870.5375. The highest concentration of test substance tested in the assay was 700 µg/mL, which was above the solubility limit of the formulated test substance after dosing into culture medium.In the initial chromosomal aberrations assay, the highest concentrations analysed (168 and 240 µg/mL, respectively) exhibited >50% reduction in mitotic index (MI) and a precipitate at end of treatment period. In the confirmatory chromosomal aberrations assay, the highest concentrations analysed (122 and 175 µg/mL, respectively) exhibited 13 to 15% reduction in MI and a precipitate at end of treatment period. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analysed.The vehicle control cultures were in the historical control range for cells with chromosomal aberrations and the positive control cultures had significant increase in cells with chromosomal aberrations as compared with the vehicle control cultures. The high doses selected for analysis in the assay had a precipitate at the end of the treatment period and/or ≥50% reduction in mitotic index as recommended for this assay by the OECD testing guidelines.Under the test conditions, the test substance was considered negative for inducing chromosomal aberrations in cultured human lymphocytes without and with metabolic activation system (Xu Y, 2011b).

Mammalian cell mutation in vitro

An in vitro mammalian cell mutation assay was performed in Chinese hamster ovary (CHO) cells, at the hprt locus to test the potential of test substance to cause gene mutation as per OECD Guideline 476 and OPPTS 870.5300.Treatments were carried out for 5 h in the presence and absence of an exogenous metabolic activation system (S9). Positive and vehicle controls were evaluated concurrently, and all test and control substance concentrations were evaluated in duplicate cultures.In a preliminary dose range-finding assay, clear evidence of cytotoxicity was observed in the presence and absence of S9. Adjusted relative survival values at concentrations of 10.9 µg/mL with S9 and 1,400 µg/mL without S9 were 2.19 and 61.37%, respectively. In addition, the test substance precipitated from solution in the aqueous treatment media at concentrations ≥43.8 µg/mL with S9 and ≥21.9 µg/mL without S9. There were no significant changes noted in the pH of the media.In an initial assay, average mutant frequencies of the cultures treated with test substance ranged from 2.7 to 6.6 TG^r mutants/10^6 clonable cells with S9, and 2.9 to 8.5 TG^r mutants/10^6 clonable cells without S9. In confirmatory assay, average mutant frequencies of the cultures treated with test substance ranged from 1.7 to 7.3 TG^r mutants/10^6 clonable cells with S9, and 1.4 to 8.6 TG^r mutants/10^6 clonable cells without S9.The criteria for a positive response were not met as there were no statistically significant or dose-dependent increases in average mutant frequency observed with or without S9 (p > 0.05), and none of the observed values represented a net increase of ≥15 TG^r mutants/10^6 clonable cells over the concurrent vehicle controls.

Under the study condition, test substance was found to be negative in the CHO/HPRT forward mutation assay in the presence or absence of metabolic activation system (Xu Y, 2011c).


Justification for selection of genetic toxicity endpoint
The studies followed internationally accepted guidelines and were conducted in compliance with GLP.

Justification for classification or non-classification

The test substance was evaluated for its genotoxic potential in a bacterial reverse mutation assay, an in vitro chromosomal aberration assay and an in vivo micronucleus assay. No evidence of genotoxicity was observed in any of the above assays, hence the test substance does not require classification for mutagenicity according to CLP criteria (EC 1272/2008) or Directive 67/548/EEC.