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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, RL1: not mutagenic with and without metabolic acitivation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Nov 2016 - 13 Jan 2017
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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
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
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
Metabolic activation system:
Rat liver S9 induced by ß-Naphthoflavone/phenobarbital
Test concentrations with justification for top dose:
1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 15.8, 158, 500, 1580, 2810 µg/plate (with and without S9 mix)
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

Evaluation criteria:
A lest material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if:
• a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
• "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.
Statistics:
Not performed as not mandatory for this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1580 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1580 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1580 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1580 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1580 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: 1580 µg/plate: precipitation at the beginning of experiment; 2810 µg/plate: precipitation until end of experiment

Ames test:
- Signs of toxicity : starting at >= 1580 µg/plate
- Individual plate counts : please refer to the attached background material
- Mean number of revertant colonies per plate and standard deviation : please refer to the attached background material

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: please refer to the attached background material
- Negative (solvent/vehicle) historical control data: please refer to the attached background material
Conclusions:
The test item is considered not mutagenic under the test conditions described.
Executive summary:

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/ß-Naphthotlavone pre-treated rats was used. In this study, two experimental series were performed.In the two series with S9 mix, 10 % S9 mix were used in the 1st and 20% S9 in the 2nd series, respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test item treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/ß-Naphthotlavone pre-treated rats was used. In this study, two experimental series were performed.In the two series with S9 mix, 10 % S9 mix were used in the 1st and 20% S9 in the 2nd series, respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test item treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.

Justification for classification or non-classification

The test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.