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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies conducted to recognised training guidelines.

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:
From Jun. 12, 1980 to Sep. 9, 1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
2-aminoanthracene was the only compound used to test the efficacy of the S9 fraction
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Plate incorporation (TA98, TA100, TA1535, TA1537 and TA1538) conc. range in tests: 0.4, 4.4, 44.3, 443.3, 4433 and 22167 µg/plate
Pre-incubation (WP2 uvrA) conc. range in tests: 0.3, 3.3, 32.8, 328.44, 3283.7 and 16420 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
methylmethanesulfonate
other: daunomycin, 4-nitr-1,2-phenylenediamine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 -aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
TA98, TA100, TA1535, TA1537 and TA1538: in agar (plate incorporation)
WP2 uvrA: preincubation

DURATION
- Preincubation period: 0.5 hours at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates per compound or concentration
-Independent repeat experiment was conducted.

Statistics:
Revertant colony numbers were calculated as average and standard deviation. No statistical analysis test was performed.

Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
22167 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxic effect was observed in all experiments macroscopically or during the routinely conducted microscopical examination of the plates, in some cases already at 4433 µg/plate, in all cases at 22167 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions of this study the test item did not show any mutagenic activity in the concentration range used with and without addition of a postmitochondrial liver homogenate (S-9) as metabolizing system.
Executive summary:

In this guideline (OECD 471) study conducted with GLP certification, the test material (EC xxx-xxx-x) was determined not to be mutagenic. The reverse mutagenicity study was conducted in S. typhimurium (TA98, TA 100, TA 1535, TA 1537, & TA1538) and E. coli (WP2 uvr A) with and without metabolic acitvation. Cytotoxicity was observed at the highest tested concentration (22167 µg/l). The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital i.p. and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
0 - 1700 μg/mL
Vehicle / solvent:
DMSO 1%
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 18h, 24h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): GIEMSA

NUMBER OF REPLICATIONS: in duplicate

NUMBER OF CELLS EVALUATED: 100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- pH value: yes
- osmolarity: yes
- solubility: yes
Evaluation criteria:
The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system. The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

In this guideline (OECD 473) study conducted with GLP certification, the test material (EC 231-272-0) was determined not to be genotoxic or cytoxic (up to the limit concentrations). The test was carried out over a range of concentrations (0 to 1700 µg/l) in Chinese hamster lung fibroblasts with and without metabolic activation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
0 - 1700 μg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylcholanthrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: with HAT-medium 24h
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 24-48h
- Selection time (if incubation with a selection agent):48 - 72h

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: in duplicate

NUMBER OF CELLS EVALUATED: all colonies per flask

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Other: pH, osmolarity, solubility, cell morphology
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent
negative control values and our historical negative control data range (see APPENDIX 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e.
15 mutants per 106 clonable cells) or isolated statistically significant increases without a
dose-response relationship may indicate a biological effect but are not regarded as sufficient
evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant
increased above the concurrent negative control and is within our historical negative
control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested in separate test
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no

RANGE-FINDING/SCREENING STUDIES: initial range-finding cytotoxicity test

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

(Test details requested)

Genetic toxicity in vivo

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

Additional information

Three in vitro assays were performed to evaluate the genotoxicity of the test substance. At first, the material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 0.4 μg - 22167 μg/plate (standard plate test) and 0.3 μg - 16420 μg/plate (pre-incubation test) in presence and absence of a metabolic activation system (S9 mix). A bacteriotoxic effect was observed at the highest concentrations. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

The objective of the second assay was to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster lung fibroblasts (V79) with and without exogenous metabolic activation. The test article was dissolved in DMSO. Three independent experiments were performed: experiment number one was performed without light protection, experiments two and three were performed under light protection. Cells were exposed for 4h with and without metabolic activation up to 1700 ul/ml or for 18 h without S9 up to 1700 μg/mL. No increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

At last, the test material was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced. Based on the solubility properties of the test substance test doses from 0 - 1700 µg/ml were tested in presence and absence of a metabolic activation system. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix

The test item did not induce mutations in bacteria or mammalian cells or chromosome aberrations in mammalian cells in vitro. Therefore, the substance is not considered to be genotoxic under the conditions of these tests.

Short description of key information:  An Ames tests, an HPRT assay and a chromosome aberration test in CHO cells was performed according or similar to OECD guidelines 471, 473 and 476 to evaluate the genotoxic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is not considered to be mutagenic under the conditions of these tests.  Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.