Salicylonitrile

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxybenzonitrile

IUPAC Name:

2-hydroxybenzonitrile

Constituent 2

Reference substance name:

Salicylonitrile

EC Number:

210-259-3

EC Name:

Salicylonitrile

Cas Number:

611-20-1

Molecular formula:

C7H5NO

IUPAC Name:

2-hydroxybenzonitrile

Details on test material:

- Name of test material (as cited in study report): 2-hydroxybenzonitrile
- Substance type: pure test substance, recrystallised 2-cyanophenol
- Physical state: solid, yellowish (beige) crystalline powder
- Stability under test conditions: not determined
- Storage condition of test material: room temperature, stable

Method

Target gene:

his-D6610 (TA97a)
his-D3052 (TA98)
his-G46 (TA100, TA1535)
his-G428 (TA102)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: rfa, uvrB, pkM101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: rfa, uvrB, pkM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: rfa, uvrB, pkM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa, pkM101

Species / strain 5

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.

Controls

Controls 1

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

Controls 2

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Applicant's summary and conclusion

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.