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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In a mutagenicity test, performed equivalent to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance both with and without metabolic activation. According to the results obtained, the test substance is not mutagenic under these test conditions.
Link to relevant study records
Reference
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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, limitations in design and reporting but adequate for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, no replication experiment performed and only one positive control used to test efficacy of the S9-mix
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: His-locus
- E. coli: Trp-locus
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:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix (Aroclor 1254)
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg ( E .coli WP2 uvrA: 60 µg)
Positive control substance:
other: 2-Aminoanthracene (2-AA)
Remarks:
all strains with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5.0 µg
Positive control substance:
other: N-methyl-N'- nitro-N-nitrosoguanidine (MNNG)
Remarks:
TA 1535, TA 100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
100 µg
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
TA 98 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E .coli WP2 uvrA without metabolic activation
Details on test system and experimental conditions:
- METHOD: Standard plate test
- METHOD OF APPLICATION: in agar (plate incorporation)
- NUMBER OF REPLICATIONS: 3 test plates per dose or per control
- DETERMINATION OF CYTOTOXICITY: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in the number of reverant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
slight decrease in the number of reverant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

According to the results obtained, the test substance is not mutagenic under these test conditions.
Executive summary:

In a mutagenicity test, performed equivalent to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance both with and without metabolic activation. In the standard plate test strains were exposed to 0, 20, 100, 500, 2500, and 5000 µg/plate. No precipitation of the test substance was found. A slight bacteriotoxic effect was observed. An increase in the number of his+ or trp+ revertants was not observed either without S9 mix or after the addition of a metabolizing system. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.

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

Additional information

Additional information from genetic toxicity in vitro:

In a mutagenicity test, performed equivalent to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance both with and without metabolic activation (BASF 1999). In the standard plate test strains were exposed to 0, 20, 100, 500, 2500, and 5000 µg/plate. No precipitation of the test substance was found. A slight bacteriotoxic effect was observed. An increase in the number of his+ or trp+ revertants was not observed either without S9 mix or after the addition of a metabolizing system. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.

In addition, four publications were available investigating the effect of the inhibition of poly(ADP-Rib) polymerase by the test substance and the subsequent effect on sister chromatid exchange and unscheduled DNA synthesis. These studies have however major limitations in design and reporting and therefore could not be used for the assessment of the genotoxicity of the test substance.

In a sister chromatid exchange assay in a Chinese hamster Ovary K1 cell line the test substance was examined for its SCE inducing properties (Oikawa 1980). In addition, the inhibition of poly(ADP-Rib) polymerase by the test substance was evaluated. Significant induction in numbers of SCEs was observed and the test substance was observed to be a moderately strong inhibitor of poly(ADP-Rib) polymerase. It is hypothesized that the test substance induce SCE by inhibiting poly(ADP-Rib) polymerase.

Two studies were identified in which the test substance was evaluated for the effects of inhibiting poly(ADP-Rib) polymerase leading to unscheduled DNA synthesis after ultraviolet irradiation in human peripheral lymphocytes (Miwa 1981 and Sims 1982). Exposure of the test substance to human peripheral lymphocytes resulted in a 2.4 fold increase in [3H]dThd incorporation compared to the control after UV irradiation (Miwa 1981). Without UV irradiation a 1.5 fold increase in [3H]dThd incorporation compared to the control was observed. In the study by Sims (1982) an inhibition of poly-(ADP-ribose) polymerase (75% compared to control) and a stimulation of Unscheduled DNA synthesis (116% compared to control) after UV irradiation was observed.

In another study (Althaus 1982), the effect of the test substance on ADP-ribosyltransferase activity and Unscheduled DNA synthesis in response to the direct-acting carcinogen methyl methanesulfonat was determined hepatocytes. Unscheduled DNA synthesis was quantitated in cultured hepatocytes as the amount of [methyl-3H]thyimidine incorporated into nuclear DNA. Results showed an inhibition of ADP-ribosyltransferase activity with the test substance (ca 43% compared to control at 10 mM test substance) accompanied by an reduced DNA repair synthesis (ca 90% of the control value at 10 mM test substance) in response to the direct-acting carcinogen, methyl methanesulfonate.

Additionally, in the Opinion of the Scientific Committee on Food on the 16th additional list of monomers and additives for food contact materials (2001), it is written that teh test substance produced negative results in the gene mutation assay in bacteria, a chromosomal aberration assay in cultured mammalian cells, a gene mutation assay in cultured mammalian cells and in a mouse bone marrow assay. However, no original study reports are available for these statements.

Based on the curent information and available literature, a mutagenic potential of the test substance cannot be derived.


Justification for selection of genetic toxicity endpoint
The only GLP-compliant OECD guideline study available.

Justification for classification or non-classification

Based on the available information on the genetic toxicity (OECD 471 guideline study), the test substance does not need to be classified in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.