3,4-dihydroxybenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test material form:

solid: particulate/powder

Remarks:

white to brownish

Details on test material:

Batch 151222

Method

Target gene:

The thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Eight doses of the test item were tested in the mutation assay (0.5, 1, 5, 10, 100, 225, 500 and 650 mg/L), based on a dose-range finder test. The test item was tested in the absence and presence of S9-mix with a 3 hour treatment period. An additional mutation experiment was performed in the absence of S9-mix with a 3 hour treatment period.

The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (Merck Darmstadt, Germany).
Reasoning: Guideline vehicle.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control: dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Cleansing
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine (Sigma), 2 x 10^-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Dose-range Finding Test
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The test item was tested in the absence and presence of S9-mix.
The highest tested concentration was 1351 μg/ml exposure medium. This concentration was equal to 0.01 M.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 ml growth medium (R10-medium).
To obtain more information about the cytotoxicity of the test item, an additional experiment was performed. Cytotoxicity data were obtained by treating 4 x 10^6 cells (10^6 cells/ml for 3 hour treatment) with a number of test item concentrations increasing by approximately half log steps.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. After 24 hours of subculturing, the cells were counted. If less than
1.25 x 10^5 cells/ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests. The results of the dose range finding study with the 24 hours treatment period have not been reported, since the test item already showed clear mutagenic responses after the 3 hours treatment period.

Mutagenicity Test
Eight doses of the test item were tested in the mutation assay. The test item was tested in the absence and presence of S9-mix with a 3 hour treatment period. An additional mutation experiment was performed in the absence of S9-mix with a 3 hour treatment period.
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of the Cells
Per culture 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) were used. The cell cultures were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to CH02906 in exposure medium in the absence and presence of S9-mix. In the additional experiment, cell cultures were exposed to CH02906 in exposure medium for 3 hours to CH02906 in exposure medium in the absence of S9-mix.
After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 ml growth medium (R10-medium).

Expression Period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the Mutation Frequency
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First mutagenecity test
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 10 to 500 μg/ml showed similar cytotoxicity. Therefore, the dose levels of 50 and 375 μg/ml were not regarded relevant for mutation frequency measurement. The dose level of 800 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
In the presence of S9-mix, the dose levels of 225 to 500 μg/ml showed similar cytotoxicity. Therefore, the dose level of 225 μg/ml was not regarded relevant for mutation frequency measurement. The dose level of 650 and 800 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.5, 1, 5, 10, 100, 225, 500 and 650 μg/ml exposure medium. With S9-mix: 0.5, 1, 5, 10, 50, 100, 375 and 500 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 8% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 12% compared to the total growth of the solvent controls.
Evaluation of the mutagenicity
In the presence of S9-mix, CH02906 did not induce a biologically relevant increase in the mutation frequency at the TK locus. Increases above the 95% control limit were observed at several dose levels in the presence of S9-mix. Although these increases were above the historical control data range, the increases in the mutation frequency (with a maximum of 181 per 10^6 survivors) were below the MF(controls) + 126 (253 per 106 survivors) and not more than a 1.4-fold increase was observed. Therefore these increases were considered not biologically relevant.
In the absence of S9-mix, CH02906 induced a maximum 3.7-fold increase in the mutation frequency at the TK locus. CH02906 showed a maximum 3.1- and 3.9-fold increase in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls. Furthermore, this maximum increase was above the positive threshold of MF(controls) + 126 (GEF= 224 x 10^-6). Increases above MF(controls) + 126 were however only observed for the dose levels of 10 and 225 μg/ml, not at 100 or 500 μg/ml. In addition, a statistical significant dose related trend (p<0.001) was observed.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of CH02906, a second mutation experiment was performed in the absence of S9-mix with a 3 hour treatment period.
Based on the results of the first experiment, the following dose levels were selected for mutagenicity testing: 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 650 and 750 μg/ml exposure medium.
Evaluation of toxicity
The dose levels of 100 to 650 μg/ml showed similar cytotoxicity. Therefore, the dose levels of 200, 400 and 650 μg/ml were not regarded relevant for mutation frequency measurement. The dose level of 750 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.5, 1, 5, 10, 50, 100, 300 and 500 μg/ml exposure medium.
The relative total growth of the highest test item was 11% compared to the total growth of the solvent controls.
Evaluation of mutagenicity
CH02906 induced a maximum 2.8-fold increase in the mutation frequency at the TK locus. CH02906 showed a maximum 2.3- and 4.8-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls. Furthermore, this increase was above the positive threshold of MF(controls) + 126 (GEF= 219 x 10^-6). Again, increases in mutation frequency were highest at intermediate doses (10 and 50 μg/ml) and no increases above the MF(controls) + 126 treshold were observed for the highest doses. In addition, a statistical significant dose related trend (p<0.001) was observed.

Aceeptability criteria
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 9 and 18.

Applicant's summary and conclusion

Conclusions:

CH02906 is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of CH02906 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence and presence of S9-mix with 3 hour treatment period. The study procedures described in this report were based on the most recent OECD guideline (OECD 490, 29 July 2016).

Batch 151222 of CH02906 was a white to brownish powder with a purity of 99.9%. The test item was dissolved in dimethyl sulfoxide.

In the first experiment, CH02906 was tested up to concentrations of 650 and 500 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 8 and 12% in the absence and presence of S9-mix, respectively. The test item did not precipitate in the culture medium at these dose levels.

In the absence of S9-mix, increases above the positive threshold of MF(controls)+ 126 (GEF= 224 x 10-6) were observed only at intermediate dose levels (10 and 225 μg/ml).

In the presence of S9-mix, none of the tested concentrations reached a mutation frequency ofMF(controls)+ 126.

Since the meaningful responses were not seen in the intermediate dose level of 100 μg/ml or at the higher dose level of 500 μg/ml with the same RTG, an additional experiment in the absence of S9-mix was performed to verify this mutagenic response.

In the additional experiment, CH02906 was tested up to concentrations of 500 μg/ml in the absence of S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was 11%. The test item did not precipitate in the culture medium at this dose level.

Increases above the positive threshold of MF(controls)+ 126 (GEF= 219 x 10-6) were observed again only at intermediate dose levels (10 and 50 μg/ml)..

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the presence of S9-mix, CH02906 did thus not induce a significant increase in the mutation frequency.

In the absence of S9-mix, however, the test item induced increases above the historical solvent control data range and also above the GEF + MF(controls)in two independent experiments. The test item was cytotoxic to the test system (RTG from 20 to 8% and 35 to 11% in the first and additional experiment, respectively) at dose levels from 10 to 650 μg/ml. The mutagenic effects were seen at intermediate dose levels with toxicity, where at the highest dose levels with the same toxicity only increases in the mutation frequency were observed above the historical solvent control data range.

Since the increases were seen in two independently repeated experiments and the increases were above the GEF + MF(controls), the test item is considered mutagenic in this test system in the absence of S9-mix. In addition, a statistical significant dose related trend (p<0.001) was observed in both experiments.

In conclusion, CH02906 is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.