Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
arochlor induced rat liver S9 extract
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 or 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA100 and TA1535
Positive control substance:
9-aminoacridine
Remarks:
TA1537
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA1538
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
-Method: relative total growth
Evaluation criteria:
For a positive result, a dose related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence or absence of S9 at sub-toxic dose levels should be induced.
Statistics:
Dunnet's method of linear regression.
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 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:
not 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:
not 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:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed at and above 1500 μg/plate

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic in Salmonella strain TA100 up to 5000 μg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was exhibited to any strain used.

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains used, at any dose level with or without metabolic activation.

Conclusions:
Interpretation of results (migrated information):
negative

The test material was not genotoxic to Salmonella strains with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium were exposed to cashew nutshell liquid, at concentrations of 0, 50, 150, 500, 1500 and 5000 μg/plate in the presence and absence of mammalian metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background for the test substance. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP.
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
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
arochlor induced rat liver S9 extract
Test concentrations with justification for top dose:
0, 1.56, 3.125, 6.25, 12.5, 25 or 50 μg/ml without S9; 0, 0.78, 1.56, 3.125, 6.25, 12.5 or 25 μg/ml with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
DURATION
- Incubation time: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 16 and 40 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 44 hours
SPINDLE INHIBITOR (cytogenetic assays): demecolcine
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: first 100 metaphases
Evaluation criteria:
Statistically significant increase in the frequency of cells w ith aberrations, compared with controls.
Statistics:
Fisher's Exact test or Chi-squared test.
Key result
Species / strain:
lymphocytes: hyman lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

The test material did not induce a significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. It is therefore considered to be non-clastogenic to human lymphocytesin vitro.

Conclusions:
Interpretation of results (migrated information):
negative

The test material was not genotoxic under the conditions of this test.
Executive summary:

In a mammalian cell cytogenetics assay [chromosome aberration], human cell cultures were exposed to cashew nutshell liquid at concentrations of 0, 1.56, 3.125, 6.25, 25 and 50 μg/ml without S9 metabolic activation and 0, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 with S9 metabolic activation.

Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background with the test substance.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guideliens and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
arochlor induced rat liver S9 extract
Test concentrations with justification for top dose:
0, 0.75, 3, 6, or 12 μg/ml without S9; 0, 1.5, 3, 6, 12 or 18 μg/ml with S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2 x 10power5 cells/75cm squared flask

DETERMINATION OF CYTOTOXICITY
- cloning efficiency
Evaluation criteria:
The mutant frequency must meet or exceed 15 x 10E6 to compensate for random fluctuations to require statistical analysis. To be positive the following must be obtained.

A dose-related or toxicity related increase in mutant frequency, for at least 3 dose levels.
A mutagenic dose-response in one mutation assay should be confirmed in a second assay.
Treatments that reduce relative clonal survival to less than 5% may be included, but will not be used as sufficient evidence for mutagenicity.
A test article will be evaluated as non-mutagenic in 2 assays only if the minimum increase in mutant frequency is not observed and the dose range applied, should ideally, give toxicity w ith 10-15% survival. If the test article is relatively non-toxic, it should have been tested to the maximum recommended dose level of 5 mg/ml.
Statistics:
Frequency of mutants per clonable cells were analysed by the Cochran-Armitage test for trend at alpha level of 0.025 (one sided-) and Fisher-Irwin exact test for group comparisons for proportions as a significance level of 0.01 (one-sided)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance did not induce a significant or dose-related increase in mutant frequency per survivor in either the presence or absence of metabolic activation. Therefore, it was considered to be non-mutagenic to CHO cells at the HGPRT locus under the conditions of the test.
Remarks on result:
other: other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material w as not genotoxic under the conditions of this test.
Executive summary:

In a mammalian cell gene mutation assay [HGPRT], Chinese hamster ovary cell cultures were exposed to cashew nutshell liquid at concentrations of 0, 0.75, 1.5, 3, 6 and 12 μg/ml without S9 metabolic activation and 0, 1.5, 3, 6, 12 and 18 with S9 metabolic activation. Positive controls induced the appropriate response. There was no evidence of mutations induced over background with the test substance. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mammalian gene cell mutagenicity data.

Additional information

Gene mutation in bacteria:

A reverse gene mutation assay was performed according to OECD 471 (GLP study). S. typhimurium TA98, TA100, TA1535 and TA 1537 strains were exposed to CNSL at concentrations of 0, 50, 150, 500, 1500 and 5000 μg/plate in the presence and absence of mammalian metabolic activation. There was no evidence of induced mutant colonies over background for the test substance.

Chormosome aberrations:

A mammalian cell cytogenetics assay (chromosome aberration), was performed according to OECD 473 (GLP study). Human cell cultures (lymphocytes) were exposed to CNSL up to 50 μg/ml without S9 metabolic activation and up to 25 µg/ml with S9 metabolic activation. There was no evidence of chromosome aberrations induced over background with the test substance.

Gene mutation in mammalian cells:

A mammalian cell gene mutation assay (HGPRT) was performed according to OECD 476 (GLP study). Chinese hamster ovary cell cultures were exposed to CNSL at concentrations up to 12 μg/ml without S9 metabolic activation and 18 µg/ml with S9 metabolic activation. There was no evidence of mutations induced over background with the test substance.

Justification for selection of genetic toxicity endpoint

No study was selected, since all three in vitro studies were negative.

Short description of key information:

Key study: Test method OECD 471. GLP study. The substance was negative in a reverse gene mutation assay in bacteria.

Key study: Test method OECD 473. GLP study. The substance was negative in a mammalian cell cytogenetics assay.

Key study: Test method OECD 476. GLP study. The substance was negative in a gene mutation assay in mammalian cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results, the substance is not classified for mutagenicity in accordance with CLP Regulation (EU) no. 1272/2008.