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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 1000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation (LPT, 2016).

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:
2016-03-17 to 2016-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test item was completely dissolved in dimethylsulfoxide (DMSO) .
Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- obtained from Trinova Biochem according to Dr. Bruce N. AMES,
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem
Test concentrations with justification for top dose:
Plate incorporation test: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate;
Preincubation test: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate;
Vehicle / solvent:
The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle dimethylsulfoxide (DMSO) served as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.
Untreated negative controls:
yes
Remarks:
solvent test will be used as negative reference item
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg µg test item/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 1000 µg/plate. Hence, 1000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate;
* Preincubation test: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate;
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in highly purufied water for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in highly purifies water for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.
Rationale for test conditions:
The study was performed in compliance with:
- Regulation (EC) No. 440/2008 method B.13/14: Mutagenicity: Reverse Mutation Test Using Bacteria, adopted May 30, 2008;
- OECD Guideline for Testing of Chemicals, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997;
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 µg test item/plate in all test strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 µg test item/plate in all test strains.

see attchached document

Conclusions:
In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 1000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The purpose of this study was to evaluate the test substance for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Test item was completely dissolved indimethylsulfoxide (DMSO).The vehicle DMSO served as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 1000 µg/plate. Hence, 1000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 3.16 to 1000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn

and reduction of the number of revertants) was noted at the top concentration of 1000 µg test item/plate in all test strains.

 

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to acytotoxic concentration of 1000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

 

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 1000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the vitro genotoxicity study the test item must not be classifed according to the criteria of EC Directive 67/548/EECand EC Regulation 1272/2008.