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Administrative data

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:
2016-04-06 to 2016-05-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test item was completely dissolved in highly purified water.
Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
Vehicle / solvent:
The test item was completely dissolved in highly purified water. The vehicle highly purified water was employed 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:
Highly purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
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 test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate (see tables 1 and 2). Hence, 5000 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: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
* Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 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
* 4-nitroquinoline-N-oxide in DMSO for E.coli WP2, 5 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100, TA 1535 and WP2, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, and TA 1537, 10 µg/plate
- negative control: the vehicle highly purified water 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: Overnight cultures were grown in a water bath for 15 h at 37°C in Oxoid 2 nutrient broth. The final cell density was approximately 10E8 - 10E9 cells/mL.

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- in addition, a significant (pBiological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine or tryptophan-free agar plates.

A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
Salmonella typhimurium TA98: 20 - 60
TA100: 100 - 200
TA1535: 10 - 35
TA1537: 3 - 20
Escherichia coli WP2 uvrA: 25 - 65
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
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.

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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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:
cytotoxicity
Remarks:
with metabolic activation, cytotoxicity was noted at the top concentration of 5000 µg/plate
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:
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in the Salmonella typhimurium strains and in the Escherichia coli test strain in the experiments without S9 mix.
In the experiments with metabolic activation, cytotoxicity (reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in the Escherichia coli strain WP2 uvrA [pKM101].

see attchached document

Conclusions:
In conclusion, under the present test conditions, test item tested up to the concentration of 5000 µg/plate (cytotoxic in the Escherichia coli strain WP2 uvrA [pKM101] in the presence of metabolic activation), caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in the Escherichia coli strain WP2 uvrA [pKM101], 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).

Test item was examined in the 4 Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in one Escherichia coli strain WP2 uvrA [pKM101] in respective 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 in highly purified water. The vehicle highly purified water 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 the Salmonella typhimurium test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 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 31.6 to 5000 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

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in the Salmonella typhimurium strains and in the Escherichia coli test strain in the experiments without S9 mix. In the experiments with metabolic activation, cytotoxicity (reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in the Escherichia coli strain WP2 uvrA [pKM101].

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to the concentration of 5000 µg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation 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, test item tested up to the concentration of 5000 µg/plate (cytotoxic in the Escherichia coli strain WP2 uvrA [pKM101] in the presence of metabolic activation), caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in the Escherichia coli strain WP2 uvrA [pKM101], 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 in vitro genotoxicity study the test item must not be classifed according to the criteria of EC Directive 67/548/EECand EC Regulation 1272/2008.