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Genetic toxicity in vitro

Description of key information

OECD 476: not mutagenic in mammalian cells with and without S9 mix

OECD 471: not mutagenic in bacterial cells with and without S9 mix

Link to relevant study records

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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:
Sep 16-26, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
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:
Aroclor-induced rat liver S-9mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 5, 15.8, 50, 158, 1580, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 158, 500, 1580, 5000 µg/plate
Vehicle / solvent:
Solvent: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies.
The following criteria, based upon the historical controls of the laboratory and statistical considerations,
are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak increases".
Interpretations:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
Evaluation criteria:
see datails on test system
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:
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:
no cytotoxicity
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 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Precipitation of the test material on the agar plates occurred at a concentration of 5000 µg/plate. Toxicity to the bacteria was not observed.

Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 mix were used in the 1st and 2nd series, respectively.

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Study Design

The GLP compliant study was performed in accordance with OECD Guideline 471 (adopted 1997). The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254 -pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1stand 2ndseries, respectively.

Results

The test item  was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at a concentration of 5000 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N`-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2 -Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain
Thus, the test item was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 07 - Jul 19, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase (TK +/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 with 10 % heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- S9 mix: 15.8 , 50.0, 158, 500, 1580 and 5000 µg per mL medium
+ S9 mix: 15.8 , 50.0, 158, 500, 889 and 1580 µg per mL medium
Vehicle / solvent:
Dimethyl sulfoxide (DMSO), routinely used, fits purpose
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline N-oxide (NQO) without S9; 7, 12-Dimethylbenz [ a]anthracene (DMBA) with S9
Details on test system and experimental conditions:
The test material was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time in the first experimental series was 3 hours in the presence and 24 hours in the absence of S9 mix. In the analysis, colony sizing was performed in order to discriminate between large and small colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected.

Evaluation criteria:
The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.
Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>= 50 µg/mL or <= 500 µg/mL depending on exp. conditions)
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
- Effects of pH: no effects
- Effects of osmolality: no effects
- Evaporation from medium: no
- Water solubility: soluble
- Precipitation: >=889 µg/mL
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: negative controls are within the historical range
Conclusions:
From the results of this study it is concluded that the test item is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

Study Design

The test material was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time in the first experimental series was 3 hours in the presence and 24 hours in the absence of S9 mix.
In the analysis, colony sizing was performed in order to discriminate between large and small colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected.
The following concentrations have been investigated: without S9 mix: 15.8 , 50.0, 158, 500, 1580, and 5000 µg per mL medium and with S9 mix: 15.8 , 50.0, 158, 500, 889, and 1580 µg per mL medium.

Results

Precipitation of the test material in the incubation medium was observed at concentrations >=889 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations >= 50 µg/mL, or <500 µg/mL, depending upon the experimental conditions. The doses tested were selected to determine viability and mutagenicity (5 -trifluorothymidine (TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4 -nitroquinoline N-oxide (without S9 mix) and 7,12 -dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test material in the two experimental series in the absence and presence of S9 mix.

It is therefore concluded that the test material is non-mutagenic in this test system under conditions  where the positive controls exerted potent mutagenic effects.

No relevant increases in mutant frequency were observed following treatment with the test material in the two experimental series in the absence and presence of S9 mix.

Conclusion

It is therefore concluded that the test material is non-mutagenic in this test system under conditions  where the positive controls exerted potent mutagenic effects.

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 data provided which are considered to be reliable and suitable, the test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.