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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The in vitro bacterial reverse mutation assay was negative with and without metabolic activation.
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:
9 March - 28 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Protocol for the study was developed in accordance with the OECD guideline on the bacterial reverse mutation test. Study is performed by a GLP accredited laboratory.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Bacterial strains were exposed to the test system at 0.06, 0.19, 0.56, 1.67, and 5 µl/plate. The lower concentrations were prepared by 1:3 serial
dilutions of the highest concentration.
Vehicle:
96% ethanol
Negative controls:
yes
Remarks:
vehicle of test substance
Solvent controls:
yes
Remarks:
ethanol 96%
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details of test system and conditions.
Details on test system and conditions:
The bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia) in nutrient broth supplemented with the corresponding antibiotics when required.
Inoculums were liquid grown overnight until the late exponential-early stationary phase of growth was reached (approximately 1.2-1.4 OD at 660nm). This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approximately 1-2x109 CFU/mL).
Evaluation criteria:
The Samonella typhimurium and the Escherichia coli reverse mutation assays are considered acceptable if:
1) the vehicle control is within historical range in each strain.
2) the responses of the positive controls are in historical range.
Statistics:
The test substance was considered mutagenic if for any strain the total number of revertants in tester strain TA98, TA100 ,WP2(pKM101) is greater than two times the concurrent control and the total number of revertants in tester strains TA1535, TA1537 is greater than three times the concurrent control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No cytotoxicity was observed at the maximum test substance concentration (Table 2).
None of the concentrations showed an increase in the colony count (R value) either with or without S9 metabolic activation.
No dose-response for the test substance was observed in any of the bacterial strains tested.

Table 2 Mean results of the test substance cytotoxicity assay with S. typhimurium TA100. The test substance was dissolved in 96% ethanol.

µl/plate revertants
/plate
s.d. R
0 (solvent) 73 7.8
0.06 78 9.3 1.1
0.19 71 2.3 1.0
0.56 69 2.0 0.9
1.67 64 2.6 0.9
5.00 64 6.1 0.9

Table 2a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - direct incorporation.

TA98 TA100 TA1535 TA1537 WP2uvrA
revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R
solvent control 20 2.6 76 7.5 20 2.1 7 0.6 260 19.2
positive control 390 31.3 19.5 761 71.2 10.0 761 45.5 37.4 154 9.7 23.2 2024 27.2 7.8
5.00 20 3.0 1.0 71 17.9 0.9 19 4.0 1.0 3 1.5 0.5 215 9.7 0.8
1.67 19 2.6 1.0 73 15.6 1.0 20 7.5 1.0 5 2.6 0.8 215 31.6 0.8
0.56 15 1.2 0.8 66 7.5 0.9 17 1.2 0.9 5 0.6 0.8 291 20.5 1.1
0.19 24 1.5 1.2 71 10.7 0.9 17 2.3 0.8 6 1.0 0.9 312 9.3 1.2
0.06 18 3.6 0.9 90 2.3 1.2 16 6.0 0.8 4 1.2 0.7 272 35.8 1.0

Table 2b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - pre-incubation.

TA98 TA100 TA1535 TA1537 WP2uvrA
revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R
solvent control 21 3.2 76 2.3 20 3.6 8 1.5 245 26.9
positive control 421 37.8 20.4 835 11.8 10.9 937 72.5 46.9 167 17.6 21.8 2221 48.1 9.1
5.00 16 1.0 0.8 0 0.0 0.0 16 3.8 0.8 4 2.0 0.5 191 5.9 0.8
1.67 17 0.0 0.8 11 12.7 0.1 16 8.1 0.8 4 2.6 0.5 168 20.4 0.7
0.56 17 2.6 0.8 48 10.7 0.6 15 4.7 0.8 4 4.0 0.6 168 15.9 0.7
0.19 16 3.5 0.8 67 6.7 0.9 18 7.0 0.9 6 2.1 0.7 281 18.2 1.1
0.06 18 4.0 0.9 71 8.5 0.9 18 4.0 0.9 6 1.5 0.7 269 8.1 1.1

Table 3a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) with metabolic activation - direct incorporation.

TA98 TA100 TA1535 TA1537 WP2uvrA
revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R
solvent control 19 6.1 71 9.1 21 3.1 7 2.6 271 33.3
positive control 679 32.3 35.1 1606 61.9 22.5 372 13.5 18.0 183 9.2 26.1 1866 64.3 6.9
5.00 14 1.2 0.7 85 9.3 1.2 14 2.1 0.7 5 3.1 0.7 109 2.6 0.4
1.67 13 0.6 0.7 68 4.4 1.0 21 6.7 1.0 4 1.7 0.6 166 19.1 0.6
0.56 17 2.9 0.9 84 19.3 1.2 19 6.5 0.9 4 0.6 0.5 303 35.5 1.1
0.19 16 4.2 0.8 80 7.8 1.1 19 4.9 0.9 6 1.5 0.8 264 7.0 1.0
0.06 17 3.1 0.9 93 12.5 1.3 18 7.5 0.9 6 2.1 0.9 214 27.6 0.8

Table 3b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - pre-incubation.

TA98 TA100 TA1535 TA1537 WP2uvrA
revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R revertants
/ plate
SD R
solvent control 22 2.1 74 1.5 18 6.1 6 1.5 263 10.6
positive control 809 67.4 37.3 1518 92.3 20.6 399 92.9 22.1 187 18.7 33.1 2009 94.6 7.6
5 17 4.7 0.8 0 0.0 0.0 16 7.0 0.9 5 1.5 0.9 113 14.5 0.4
1.67 20 4.4 0.9 0 0.0 0.0 18 1.7 1.0 3 1.5 0.5 127 19.0 0.5
0.56 22 5.0 1.0 0 0.0 0.0 17 6.1 1.0 4 2.1 0.8 190 22.5 0.7
0.19 20 6.1 0.9 11 2.1 0.1 23 5.0 1.3 5 1.5 0.8 293 22.0 1.1
0.06 16 3.5 0.8 31 9.6 0.4 14 1.5 0.8 6 2.0 1.1 300 52.1 1.1
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Based on the results, it is concluded that the test substance is not mutagenic in the Salmonella typhimurium and the Escherichia coli reverse mutation assay.
Executive summary:

The bacterial reverse mutation test (Ames test) assesses the mutagenic or promutagenic potential of the test substance in the several bacterial strains. The test was performed in accordance with OECD Guideline 471. No cytotoxic activity was observed at a test item concentration of 5μL/plate. Five test substance doses ranging from 5.00 and 0.06 μL/plate were assayed. None of the concentrations showed an increase in the colony counting either with or without S9 metabolic activation regardless of the exposure procedure. No dose response for the test substance was observed in any of the tested bacterial strains. Based on the results obtained in this study, it can be concluded that the test item does not induce point mutations or frame-shifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure. Therefore, the test substance is considered to be NON MUTAGENIC / NON PRO-MUTAGENIC under the experimental conditions assayed.

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

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Protocol for the study was developed in accordance with the OECD guideline on the bacterial reverse mutation test. Study is performed on the substance by a GLP accredited laboratory.

Justification for classification or non-classification

The in vitro bacetrial reverse mutation test showed a negative result when the test substance was exposed either by direct incorporation or through the pre-incubation procedure in the presence and absence of metabolic activation.