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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

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:
21 October 2015 to 23 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD and EC guidelines and in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Test 1: 5, 15, 50, 150, 1500, 5000 µg/plate
Test 2: 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle used: Ethanol
- Justification for choice: The solubility of DAILUBE IS-30 was assessed at 50 mg/mL in DMSO and ethanol. It was found to be insoluble in DMSO, however in ethanol it formed a colourless solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
In the absence of S9 mix, 2 μg/plate for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In the absence of S9 mix, 50 μg/plate for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In the absence of S9 mix, 2 μg/plate for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
In the absence of S9 mix, 2 μg/plate for strain WP2 uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9 mix, 5 μg/plate for strains TA100 and TA1535 10 μg/plate for strain WP2 uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
In the presence of S9 mix, 5 μg/plate for strains TA98 and TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
other: Ethanol
Details on test system and experimental conditions:
First test

Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was ethanol. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37C for ca 72 hours. After
this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Some plates were scored manually because of toxicity.

Second test

As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for ethanol, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.
Evaluation criteria:
Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be included in the second test, unless otherwise justified by the Study Director.
Statistics:
None stated.
Key result
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
First test

Toxicity (observed as thinning of the background lawn of non-revertant colonies) was seen in strains TA1535 following exposure to DAILUBE IS-30 at 5000 μg/plate in the absence of S9 mix in the first mutation test. Toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers) was seen in strains TA100, TA1535 and TA1537 following exposure to DAILUBE IS-30 at 5000 μg/plate in the presence of S9 mix in the first mutation test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DAILUBE IS-30 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second test

No evidence of toxicity was obtained following exposure to DAILUBE IS-30.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DAILUBE IS-30 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed and was not classified according to the CLP regulation.