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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, T41535, TA1537 and TA1538
Metabolic activation:
with and without
Metabolic activation system:
rat liver metabolic activation system
Test concentrations with justification for top dose:
Range Finder Experiment and Experiment 1: 1.6, 8, 40, 200, 1000 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: sterile purified water.
Untreated negative controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
EVALUATION AND COUNT
- Count: the top one or two dose treatment plates in the Range-Finder Experiment and Experiment 1 were counted manually due to the dark colour of the test article affecting automated counting.
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the top dose, in strain TA1535 only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
When data from the Range-Finder Experiment was analysed at the 1 % level using Dunnett's test, a statistically significant increase in revertant numbers was observed following TA100 treatments in the presence of S-9. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no treatments of any of the test strains, either in the absence or presence of S-9, induced statistically significant increases in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced on a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

PRECIPITATION
No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

TOXICITY
Evidence of toxicity, as shown by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9.

Summary of mean revertant colonies (-S9) - Experiment I

Substance Dose level (μg/plate) TA98
Mean ± SD
TA100
Mean ± SD
TA1535
Mean ± SD
TA1537
Mean ± SD
TA1538
Mean ± SD
Water 100 μl 51 ± 8 132 ± 13 29 ± 9 14 ± 3 21 ± 5
Test item 1.6 50 ± 3 115 ± 11 26 ± 5 17 ± 2 21 ± 6
8 49 ± 11 107 ± 11 29 ± 7 13 ± 5 20 ± 2
40 57 ± 7 118 ± 12 28 ± 3 17 ± 5 28 ± 5
200 62 ± 2 116 ± 14 19 ± 8 14 ± 3 29 ± 8
1000 43 ± 9 94 ± 32 11 ± 6 12 ± 2 22 ± 2
5000 50 ± 3 (M) 87 ± 4 (M) 14 ± 2 (M) 17 ± 4 (M) 21 ± 5 (M)

Positive controls




2-Nitrofluorene 5 μg 861 ± 85 - - - 364 ± 39
Sodium azide 2 μg - 674 ± 8 623 ± 48 - -
9-Aminoacridine 50 μg - - - 178 ± 15 -

SD: Standard deviation; (M): Plates counted manually

Summary of mean revertant colonies (+S9) - Experiment I

Substance Dose level (μg/plate) TA98
Mean ± SD
TA100
Mean ± SD
TA1535
Mean ± SD
TA1537
Mean ± SD
TA1538
Mean ± SD
Water 100 μl 55 ± 6 119 ± 11 20 ± 2 24 ± 2 32 ± 9
Test item 1.6 43 ± 20 128 ± 3 29 ± 4 15 ± 3 27 ± 6
8 61 ± 6 113 ± 12 24 ± 10 18 ± 2 29 ± 7
40 58 ± 5 117 ± 15 24 ± 1 15 ± 8 33 ± 6
200 64 ± 9 106 ± 28 21 ± 5 32 ± 8 36 ± 14
1000 49 ± 5 77 ± 4 18 ± 5 18 ± 8 33 ± 6
5000 52 ± 7 (M) 104 ± 11 (M) 13 ± 7 (M) 17 ± 3 (M) 23 ± 1 (M)

Positive controls




Benzo[a]pyrene 10 μg 253 ± 8 - - - -
2-Aminoanthracene 5 μg - 1783 ± 131 191 ± 11 331 ± 31 1329 ± 60

SD: Standard deviation; (M): Plates counted manually

Summary of mean revertant colonies Range-Finder Experiment

Substance Dose level (μg/plate)

TA100 (-S9)

Mean ± SD

TA100 (+S9) 

Mean ± SD

Water 100 μl 110 ± 7 99 ± 8
Test item 1.6 105 ± 1 108 ± 9
8 98 ± 11 107 ± 6
40 103 ± 8 116 ± 14
200 90 ± 5 119 ± 12
1000 106 ± 9 (M) 138 ± 13 (M)
5000 106 ± 26 (M) 103 ± 12 (M)

Positive controls

Sodium azide 2 μg 833 ± 9 -
2-Aminoanthracene 5 μg - 2286 ± 102

SD: Standard deviation; (M): Plates counted manually

Conclusions:
The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.
Executive summary:

The substance was tested for mutagenicity effects in vitro in strains of Salmonella typhimurium TA98, TA100, TA1535 TA1537 and TA1538, with and without metabolic activation. The test consists in two experiments, i.e. range-finding experiment and experiment 1, conducted at the following concentrions: 1.6, 8, 40, 200, 1000 and 5000 μg/plate.

No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

Evidence of toxicity, as shown by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9.

Analysed at the 1 % level using Dunnett's test a statistically showed a significant increase in revertant numbers in TA100 strain upon treatment in presence of S-9, in the range-finding experiment. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response. Following Experiment I treatments, no test item treatments of any of the test strains, either in absence or presence of S-9, induced statistically significant increases in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced on a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

Conclusion

The test item did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535 TA1537 and TA1538 under the conditions employed.

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

Additional information

The substance was tested for mutagenicity effects in vitro in strains of Salmonella typhimurium TA98, TA100, TA1535 TA1537 and TA1538, with and without metabolic activation. The test consists in two experiments, i.e. range-finding experiment and experiment 1, conducted at the following concentrions: 1.6, 8, 40, 200, 1000 and 5000 μg/plate.

No precipitation of test agent was observed following any of the treatments in either the presence or absence of S-9.

Evidence of toxicity, as manifest by a marked decrease in revertant numbers, was observed following the top dose treatment in strain TA1535 only in the presence and the absence of S-9. When the data from the Range-Finder Experiment was analysed at the 1 % level using Dunnett's test, a statistically significant increase in revertant numbers was observed following TA100 treatments in the presence of S-9. TA100 treatments were therefore repeated in Experiment I in order to investigate the reproducibility of this response.

In Experiment I, either in the absence or presence of S-9, no statistically significant increases in revertant numbers were seen, upon analysis at the 1 % level using Dunnett's test. As the increase in revertant numbers observed in TA100 in the presence of S-9 in the Range-Finder Experiment was very small in magnitude, showed little evidence of a dose-response and was not reproduced in a second experimental occasion, the increase was considered to be incidental and not to be indicative of test item mutagenic activity in this strain under the treatment condition.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern for genotoxicity.