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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, OECD 471 (Ames test: S. typhimurium TA97a,TA98, TA100, TA102, TA1535), Wolf 2008

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
Study period:
2008-03-03 to 2008-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-D6610 (TA97a)
his-D3052 (TA98)
his-G46 (TA100, TA1535)
his-G428 (TA102)
Species / strain / cell type:
S. typhimurium, other: TA97a
Additional strain / cell type characteristics:
other: rfa, uvrB, pkM101
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: rfa, uvrB, pkM101
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: rfa, uvrB, pkM101
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: rfa, pkM101
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: rfa, uvrB
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (post-mitochondrial supernatant, from livers of male Sprague-Dawley rats)
Test concentrations with justification for top dose:
Preliminary toxicity test (TA100 only): 5000, 1667, 556, 185, 62 microgram/plate
1st experiment: 5000, 1667, 556, 185, 62, 21 microgram/plate
2nd experiment: 1667, 556, 185, 62, 21, 7 microgram/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance is not soluble in water. DMSO is a common vehicle for the Ames test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 4-nitro-o-phenylenediamine, t-butyl-hydroperoxide
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: 2-aminoanthracene; 1,8-dihydroxy-anthraquinone
Remarks:
With metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st experiment: in agar (plate incorporation); 2nd experiment: preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 2 days at 37°C (plate incorporation and preincubation)

NUMBER OF REPLICATIONS:
- two independent experiments (plate incorporation and preincubation)
- triplicate repetitions per experiment

NUMBER OF CELLS EVALUATED: 2-3 x 10exp8 per plate (2-3 x 10exp9 per mL)

DETERMINATION OF CYTOTOXICITY
- Method: reduction or absence of bacterial background lawn, appearance of microcolonies instead of background lawn, clearly reduced numbers of revertant colonies

VALIDITY TESTS OF STRAINS (all performed in 2000-09, bacteria frozen since)
- ampicillin resistance (TA102: ampicillin/tetracycline resistance)
- UV sensitivity
- sensitivity against crystal violet
- spontaneous mutation frequencies
- sensitivities against positive controls

Evaluation criteria:
- Reproducible increase in number of revertants above strain-specific threshold, for at least one concentration
- Threshold for strains with low spontaneous revertant rate (TA98, TA1535): 2.5-fold the number of spontaneous revertants
- Threshold for strains with high spontaneous revertant rate (TA97a, TA100, TA102): 1.67-fold the number of spontaneous revertants
- Threshold values derived from variations in historical controls.
Statistics:
Mean and standard deviation
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
background lawn absent or reduced at 5000 microgram/plate, no background lawn without activation at 5000 microgram/plate (exp. 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
background lawn reduced without activation, microcolonies with activation, at 5000 microgram/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
background lawn absent without activation, reduced with activation, at 5000 microgram/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
background lawn absent without activation, microcolonies with activation, at 5000 microgram/plate, reduced lawn at 1667 microgram/plate (exp. 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
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:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Preliminary toxicity test with TA100: At 5000 micrograms/plate: no growth of background lawn, no revertants (without metabolic activation), microcolonies (with metabolic activation). Lower concentrations normal.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Number of spontaneous revertants comparable to historical controls

PRECIPITATION:
- none observed

Conclusions:
No increase of revertants (above threshold values) at any concentration, with or without metabolic activation. 2 -hydroxybenzonitrile is not mutagenic in the Ames test with the strains S. typhimurium TA97a, TA98, TA100, TA102, and TA1535. The study is considered relevant and reliable.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Ames test (bacterial reverse mutation in vitro): Negative

A key study conducted in 2002 according to guideline OECD 471 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labelling. Five S. typhimurium strains, TA97a, TA98, TA100, TA102, and TA1535, were investigated; TA102 is listed as a valid alternative to E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101), so that the requirements of the guideline are fully met. The substance did not elicit a mutagenic effect in any of the strains, with and without metabolic activation, and is therefore not considered a bacterial mutagen.


Justification for selection of genetic toxicity endpoint
GLP study according to OECD guideline, all bacterial strains required by the guideline were tested. S. typhimurium TA 102 is considered a valid alternative to E. coli strains.

Justification for classification or non-classification

The substance demonstrated negative results in an in vitro bacterial gene mutation test (Ames). As a result, and in accordance with Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5.2, the substance is not considered to be classified for germ cell mutagenicity.