4-Hydroxy-3,5-dimethylbenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2003-09-11 to 2003-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dated May 19, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): T002251
- Physical state: Solid
- Appearance: Brown powder

Specific details on test material used for the study:

Batch No.: 00410155
Aggregate State at Room Temperature: solid
Colour: brown powder
Purity: 96 %
Stability in Solvent: Not indicated by the sponsor
Storage: Room temperature
Expiration Date: 2004-05-05

Method

Target gene:

The histidine dependant strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide (DMSO)
Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, at 10 μg/plate with TA1535 and TA100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation, at 10 μg/plate with TA 98, at 50 μg/plate with TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, at 4µL/plate with WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation, at 2.5 μg/plate with TA 1535, TA 1537, TA 98 and TA 100, at 10 μg/plate with WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay

Experimental performance:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL: Overlay agar

In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DURATION:
- Pre-incubation period: 60 minutes (experiment II)
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data was required.

Results and discussion

Test results

Test results 1

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:

see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I since relevant toxic effects were only observed at high concentrations and 5000 μg/plate were chosen as maximum concentration of the
main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
- Negative (solvent/vehicle) historical control data: In experiment I with metabolic activation, the data in the negative control of strain TA 98 and in the solvent control of strain TA 100 were slightly above the laboratory historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strong toxic effects reduced the background growth in the second experiment at 2500 and 5000 μg/plate in strain TA 98 without metabolic activation and in strain TA 100 with and without metabolic activation. Relevant toxic effects, evident as a reduction in the number of revertants below 0.5 times the corresponding control, occurred at the concentrations presented in the section "any additional information including tables".

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Strong toxic effects occurred at the following concentrations (μg/plate):

Strain	Experiment 1		Experiment 2
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA1535	2500-5000	2500-5000	2500-5000	1000-5000
TA1537	2500-5000	2500-5000	2500-5000	5000
TA98	2500-5000	5000	2500-5000	/
TA100	2500-5000	2500-5000	2500-5000	2500-5000
WP2uvrA	2500-5000	1000-5000	1000-5000	1000-5000

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.