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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Negative.

In vitro chromosome aberration test: positive without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2011-08-04 to 2011-08-12
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: 1176335
Purity: 99.8%
Target gene:
histidine locus and tryptophan-independent (Trp+) locus
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:
S9 mix
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since no relevant toxic effects were observed 5000 μg/plate was chosen as maximum test item concentration.
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1E+08-1E+09 cells/mL

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours at 37 °C

NUMBER OF REPLICATIONS: three plates

DETERMINATION OF CYTOTOXICITY
- Method: background growth

OTHER EXAMINATIONS:
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (version 1.21). The counter was connected to a printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not mandatory
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 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 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:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Remarks:
In experiment II, without metabolic activation, the number of colonies did not quite reach the lower limit of historical control data
Untreated negative controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.
A minor reduction in the number of revertants (below the indication factor of 0.5), was observed In strain WP2 uvrA at 5000 μg/plates in experiment I without S9 mix and in experiment II with S9 mix. No further toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level applied, neither in the presence nor in the absence of metabolic activation (S9 mix).
In experiment II, without metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 100 in the solvent control. Since the offset was rather small, this result is judged to be based upon fluctuations that may occur in biological systems but do not indicate an adverse effect of the test item.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
Conclusions:
The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation according to OECD471. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.

A minor reduction in the number of revertants (below the indication factor of 0.5), was observed In strain WP2 uvrA at 5000 μg/plates in experiment I without S9 mix and in experiment II with S9 mix. No further toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level applied, neither in the presence nor in the absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-02-12 to 2018-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch No.: 20170707
Purity: 99.13%
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.5, 1, 2.5, 5, 7.5, 10 mM both for without and with S9 mix
The dose group selection for microscopic analyses of chromosomal aberrations was based in accordance with the recommendations of the guidelines.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 2 days
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 17 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): at least 300 cells per concentration.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells is scored.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation:
No precipitation of the test item was noted without and with metabolic activation after the incubation in all dose groups evaluated.

Toxicity:
In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 10 mM (69 % RICC). With metabolic activation, no biologically relevant decrease of the RICC was noted at all concentrations tested.

Clastogenicity:
In experiment I without metabolic activation, the aberration rate of the negative control (1.3%) was within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). An increase of aberrant cells was noted in the highest tested concentration of 10 mM (5.3%).
With metabolic activation, the aberration rates of the negative control (2.0%) and all test concentrations of 5 mM (2.0%), 7.5 mM (0.7%) and 10 mM (0.7%) were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps).
The Fisher´s exact test was performed to verify the results in the experiment. A statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted for the test item concentration 10 mM in the experiment I without metabolic activation.
EMS (900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells:
A biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item in the highest evaluated concentration in experiment I without metabolic activation.
Conclusions:
The test item did induce structural chromosomal aberrations in the V79 Chinese hamster cell line without metabolic activation.
Therefore, the test item is considered to be clastogenic in this chromosome aberration test.
Executive summary:

The in vitro chromosomal aberration (CA) test is carried out using the Chinese hamster V79 cell line under without and with metabolic activation conditions based on OECD 473.

In experiment I without metabolic activation, an increase of aberrant cells was noted in the highest tested concentration of 10 mM. This induction was also statistically significant increased (p < 0.05) in the Fisher´s exact test. Also a dose-dependent increase was obvious, no statistically significant increase could be determined by the X² Test for trend.

In experiment I with metabolic activation, no induction in chromosome aberrations was observed in all test concentrations.

The test item did induce structural chromosomal aberrations in the V79 Chinese hamster cell line without metabolic activation.

Therefore, the test item is considered to be clastogenic in this chromosome aberration test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In vitro chromosome aberration test:

The in vitro chromosomal aberration (CA) test is carried out using the Chinese hamster V79 cell line under without and with metabolic activation conditions based on OECD 473.

The test item did induce structural chromosomal aberrations in the V79 Chinese hamster cell line without metabolic activation.

Therefore, the test item is considered to be clastogenic in this chromosome aberration test.

Due to negative result in Ames test and positive result in in vitro chromosome aberration test, the in vitro gene mutation test is waived, and an in vivo micronucleus test is proposed.

Justification for classification or non-classification

Ames test give negative result; In the in vitro chromosome aberration test, the test item did induce structural chromosomal aberrations in the V79 Chinese hamster cell line without metabolic activation, but also cytotoxocity was observed under this condition; the in vitro gene mutation test is waived; the conclusion of classification on the endpoint cannot be drawed, so according to ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a, section R.7.7.6.3, an in vivo micronucleus test is proposed.