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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of three different reliable in vitro genotoxicity studies (bacterial mutation, chromosome aberration, mammalian celll mutation), conducted in both in the presence and absence of a metabolic activation system, demonstrate that the substance is non-mutagenic and non-genotoxic. The substance was also negative for mutagenicity in an reliable (with restrictions given that it did not follow any guidelines and not in accordance with GLP, but the study was conducted to nationally acceptable standards) in vitro bacterial mutation assay both in the presence and absence of a metabolic activation system.

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:
22 March 2007 to 23 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
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:
S9 fraction was derived from rats induced with phenobarbitone/beta-napthoflavone mixture.
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested and/or reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. A test material will be considered non-mutagenic (negative) if the above criteria aare not met.
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
Positive controls validity:
valid
Remarks on result:
other: not-mutagenic
Conclusions:
The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The substance underwent a test to evaluate the mutagenic potential in the Ames test according to the OECD Guideline 471 employing Salmonella typhimurium strains TA1535, TA1537, TA100, TA98, and Eschericia coli WP2, with and without S9 (metabolic activation). Under the conditions of these tests the substance is considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
Saccharomyces cerevisiae
Remarks:
D4
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.001µl to 10 µl based on quantitative or qualitative evidence of some chemically induced physiological effect at the highest dose level. The low dose was below a concentration that demonstrated a toxic effect.
Vehicle / solvent:
Deionised water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
ethylmethanesulphonate
other: QM for TA-1537; anthracene; ,4-dihydroxy-2-napthoic acid for D4.
Evaluation criteria:
Strains TA1535, TA1537 and TA1538: if the solvent control value is within the normal range, a chemical that produced a positive response over three concentrations with the lowest equal to twice the solvent control is considered mutagenic.

Strains TA98, TA100 and D4: If the solvent control value is within the normalk range, a chemical that produces a positive response over three concentrations with the highest increase equal to twice the solvent control value for TA100 and 2-3 times the solvent control value for strains TA98 and D4 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.
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 applicable
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 applicable
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:
not applicable
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Saccharomyces cerevisiae
Remarks:
D4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The substance showed no evidence of mutagenic activity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, and in Saccharomyces cerevisiae strain D4 in the presence and in the absence of a metabolic activation system.
Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, and in Saccharomyces cerevisiae strain D4 in the presence and in the absence of a metabolic activation system. Concentrations of up to 10 μl/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in either bacterial strain. It was concluded that the substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2007 to 18 April 2007
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)
Version / remarks:
1997
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: Chromosome aberration test in human lymphocytes in vitro
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Beta naphthoflavone and phenobarbitone mixture induced rat liver homogenate metabolising system (S9).
Vehicle / solvent:
Minimum Essential Medium (MEM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Experiment 1 - 4-hour exposure to test material with/without S9 followed by 20-hour culture treatment-free media prior to cell harvest.
Experiment 2 - 24-hour continuous exposure to the test material with/without S9 followed by 20-hour culture treatment-free media prior to cell harvest.

Evaluation criteria:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: not clastogenic
Conclusions:
The substance is considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The substance underwent genotoxicity testing according to OECD Guideline 473 employing human lymphocytes, with and without metabolic activation (S9 fraction). The substance was considered to non-genotoxic under the conditions of the study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 to 16 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with Phenobarbital/beta naphthaflavone.
Test concentrations with justification for top dose:
62.5, 125, 250, 500, 1000, 2000 μg/mL.
The molecular weight of the test item was 216.23 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test (Relative Suspension Growth). This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).
Vehicle / solvent:
R0 medium.
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Evaluation criteria:
A substance is considered to be clearly positive if the increase in mutation frequency above the the concurrent background exceeds the Global Evaluation Factor of 126x10-6 and the increase is concentration-related.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

In an in vitro cell transformation assay to evaluate the mutagenic and clastogenic potential L5178Y mouse lymphoma cells were exposed to the substance at 62.5, 125, 250, 500, 1000, 2000 μg/mL. the substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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

Genetic toxicity in vivo

Description of key information

An in vivo genotoxicity assay was conducted on the substance according to OECD Guideline 474. The results of the test indicates that the substance is non-genotoxic in vivo under the conditions of the study.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2013 to 16 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
On occassions the relative humidity was outside the target range of 30-70%. The animals in the main test, with the exception of the positive control group were housed in groups of seven.
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Species:
mouse
Strain:
ICR
Details on species / strain selection:
Sufficient albino Hsd: ICR (CD-1)
Sex:
male/female
Route of administration:
intraperitoneal
Vehicle:
Phoshate Buffered Saline (PBS)
Duration of treatment / exposure:
Once.
Frequency of treatment:
Once.
Post exposure period:
24 hours (500, 1000 or 2000 mg/kg bw) and 48 hours (2000 mg/kg bw group) post-treatment.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Seven/dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg bw)
Tissues and cell types examined:
Bone marrow smears prepared and polychromatic erythrocytes per animal was scored. The number of normochromatic erythrocytes were also counted and scored for incidence of micronuclei.
Evaluation criteria:
Micronuclei are cicular, but occassionally may be oval or half-moon shaped and have a sharp contour with even staining.
Statistics:
The ratio of polychromatic to normochromtic erythrocytes was calculated together with appropriate group of mean values and standard deviations.
A positive mutagenic response was demoinstrated when a statistically significant, dose-responsive, toixicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for iether the 24 or 48-hour kill times compared to their corresponding control group.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No premature deaths or clinical signs of toxicity observed in any of the animals dosed.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test item was considered to be non-genotoxic under the conditions of the test.
Executive summary:

An in vivo gentoxicity assay was conducted on the substance according to OECD Guideline 474. The results of the test indicates that the substance is non-genotoxic in vivo under the conditions of the study.

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

Additional information

Justification for classification or non-classification

Based on the findings of several reliable genetic toxicity studies (in vitro and in vivo) conducted on the substance, classification is not justified.