3,4-dihydroxybenzonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests were performed: the Ames (gene mutation test in bacteria) and micronucleus test showed no indications for genetic toxicity, the mouse lymphoma test (gene mutation test with mammalian cells) indicated possible genotoxicity.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

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.

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.

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2016 - 20 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (male or female, aged 18 to 35 years).
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.
- Cell cycle length, doubling time or proliferation index: The Average Generation Time (AGT) of the cells
Dose range finding study: age 24, AGT = 13.7 h
First cytogenetic assay/Dose range finding 2: age 30, AGT = 12.9 h
Second cytogenetic assay: age 24, AGT = 13.7 h
- Sex, age and number of blood donors if applicable: age 18-35
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable: /
- Methods for maintenance in cell culture if applicable: /
- Modal number of chromosomes: /
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Life Technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

A concentration of 1351 µg/ml (= 0.01 M) showed no precipitation in the culture medium and was used as the highest concentration of CH02906.

In the dose range finding test blood cultures were treated with 63, 125, 250, 500, 1000 and 1351 µg CH02906/ml culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

Based on the results of the dose range finding test the following dose levels were selected for the first cytogenetic assay:
Without S9-mix: 10, 50, 100, 150, 200, 250 and 500 µg/ml culture medium (3 hours exposure time, 27 hours harvest time).
With S9-mix: 10, 100, 250, 500, 1000 and 1351 µg/ml culture medium (3 hours exposure time, 27 hours harvest time).

To obtain more information about the possible clastogenicity and aneugenicity of CH02906, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 1, 10, 15, 20, 25, 30 and 40 µg/ml culture medium (24 hours exposure time, 24 hours harvest time).

Untreated negative controls:

yes

Remarks:

same as vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

The test item was not soluble in RPMI culture medium. In addition no homogenous suspension could be obtained. Therefore dimethyl sulfoxide was selected as suitable vehicle.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): /

DURATION
- Preincubation period: 46 ± 2 hours
- Exposure duration: 3 hours and 24 hours
- Expression time (cells in growth medium): /
- Selection time (if incubation with a selection agent): /
- Fixation time (start of exposure up to fixation or harvest of cells):
3 hours exposure: 24 hours of incubation after exposure and before harvesting
24 hours exposure: harvesting and fixation immediately after exposure

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).

Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

NUMBER OF CELLS EVALUATED:
A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. In the second experiment, the highest concentration of MMC-C and CP was not examined for the presence of micronuclei since the lowest concentration resulted in a positive response. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: /
- Determination of endoreplication: /
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): /

- OTHER:

Rationale for test conditions:

In the dose range finding test blood cultures were treated with 63, 125, 250, 500, 1000 and 1351 µg CH02906/ml culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

Based on the results of the dose range finding test, the dose levels were selected for the first cytogenetic assay.

To obtain more information about the possible clastogenicity and aneugenicity of CH02906, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was 7.53 in dose range finding test compared to 7.98 in the solvent control
- Effects of osmolality: osmolality was 434 mOsm/kg compared to 438 mOsm/kg in the solvent control
- Evaporation from medium: /
- Water solubility: /
- Precipitation: A concentration of 1351 µg/ml (= 0.01 M) showed no precipitation in the culture medium
- Definition of acceptable cells for analysis: /
- Other confounding effects: /

RANGE-FINDING/SCREENING STUDIES:
Table 1 shows the cytokinesis-block proliferation index of cultures treated with various CH02906 concentrations or with the negative control item
Since extreme toxicity was observed after the 24 hours exposure, an additional dose range finding for this exposure time was performed.
In this additional dose range finding test blood cultures were treated with 1, 5, 10, 20, 40 and 80 µg CH02906/ml culture medium and exposed for 24 hours in the absence of S9-mix. Table 2 shows the cytokinesis-block proliferation index of cultures treated with various CH02906 concentrations or with the negative control item.

CYTOKINESIS BLOCK + NUMBER OF CELLS WITH MICRONUCLEI

Table 3 shows the cytokinesis-block proliferation index of cultures treated with various CH02906 concentrations or with the positive or negative control items.
The following dose levels were selected for scoring of micronuclei: Without and with S9-mix: 10, 100 and 250 µg/ml culture medium (3 hours exposure time, 27 hours harvest time).
Both in the absence and presence of S9-mix, CH02906 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (Table 4).

Table 5 shows the cytokinesis-block proliferation index of cultures treated with various CH02906 concentrations or with the positive or negative control items.
The following dose levels were selected for the scoring of micronuclei: Without S9-mix: 1, 15 and 25 µg/ml culture medium (24 hours exposure time, 24 hours harvest time).
CH02906 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (Table 6).


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See tables

ADDITIONAL INFORMATION ON CYTOTOXICITY:
/

Remarks on result:

other:

Remarks:

CH02906 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

STATISTICAL EVALUATION OF THE TEST RESULTS

Chi-square Test

TOTAL NUMBER OF CELLS WITH MICRONUCLEI; TREATMENT/CONTROL COMPARISON, (MONONUCLEATED/BINUCLEATED CELLS)¹⁾.

EXPOSURE         S9-MIX   NUCLEATED       P-VALUE        DECISION AT 95%

DOSE (mg/ml)                                                      one-sided          CONFIDENCE LEVEL

First cytogenetic assay

3 hours exposure time

Positive controls

MMC-C (0.38)          −                bi                       <0.0001              significant

Colch (0.1)                 −                mono                 <0.0001              significant

Colch (0.1)                 −                bi                       <0.0001              significant

CP (15)                      −                mono                 = 0.0292             significant

CP (17.5)                   −                mono                 = 0.0056             significant

CP (17.5)                   +                bi                       = 0.0002             significant

Second cytogenetic assay

24 hours exposure time

Positive controls

MMC-C (0.15)          -                 bi                       <0.0001              significant

Colch (0.05)               -                 mono                 <0.0001              significant

Colch (0.05)               -                 bi                       <0.0001              significant

 

¹⁾Only statistically significant results are presented.

able1          
Cytokinesis-block proliferation index of human lymphocyte cultures treated with CH02906 in the dose range finding test

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	131	290	79	1.90			0
63	218	253	29	1.62			31
125	276	211	13	1.47			47
250	319	169	12	1.39			57
500	366	133	1	1.27			70
1000	386	114	0	1.23			75
1351	434	66	0	1.13			85

 

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	228	249	23	1.59			0
63	485	15	0	1.03			95
125	500	0	0	1.00			100
250	500	0	0	1.00			100
500	498	2	0	1.00			99
1000	500	0	0	1.00			100
1351	500	0	0	1.00			100

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	Number of cells with ….nuclei			CBPI			% cytostasis
	1	2	3 or more
0	145	298	57	1.82			0
63	215	254	31	1.63			23
125	263	212	25	1.52			36
250	308	171	21	1.43			48
500	328	169	3	1.35			58
1000	335	165	0	1.33			60
1351	436	64	0	1.13			84

 

Table2          
Cytokinesis-block proliferation index of human lymphocyte cultures treated with CH02906 in the second dose range finding test

 

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/ml		Number of cells with ….nuclei				CBPI				% cytostasis
		1		2					3 or more
0		208		260		32		1.65				0
1		218		263		19		1.60				7
5		229		247		24		1.59				9
10		272		217		11		1.48				26
20		326		173		1		1.35				46
40		454		45		1		1.09				85
80		489		11		0		1.02				97

 

 

Table3          
Cytokinesis-block proliferation index of human lymphocytes cultures treated with CH02906 in the first cytogenetic assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	CBPI			Mean CBPI		% cytostasis
0	1.81	-	1.83	1.82		0
10	1.74	-	1.77	1.75		9
50	1.55	-	1.56	1.56		33
100	1.49	-	1.53	1.51		38
150	1.42	-	1.46	1.44		46
200	1.42	-	1.46	1.44		47
250	1.38	-	1.41	1.40		52
500	1.33	-	1.34	1.34		59
0.25 MMC-C	1.47	-	1.50	1.48		42
0.38 MMC-C	1.41	-	1.44	1.43		48
0.1 Colch	1.37	-	1.37	1.37		56
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml	CBPI			Mean CBPI		% cytostasis
0	1.81	-	1.82	1.82		0
10	1.81	-	1.81	1.81		2
100	1.51	-	1.53	1.53		36
250	1.39	-	1.40	1.40		51
500	1.33	-	1.36	1.34		58
1000	1.19	-	1.21	1.20		76
1351	1.06	-	1.08	1.07		92
15 CP	1.30	-	1.32	1.32		61
17.5 CP	1.25	-	1.25	1.25		70

 

Table4          
Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with CH02906 in the first cytogenetic assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	1	1	7		10	17
10	9	0	0	0	0		5	5
100	38	0	0	0	5		9	14
250	52	0	0	0	10		6	16
0.25 MMC-C	42	0	0	0	11		12	23
0.38 MMC-C	48	1	0	1	30		21	51^***
0.1 Colch	56	31	28	59^***	15		19	34^***

 

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	0	1	4		7	11
10	2	0	0	0	3		3	6
100	36	0	0	0	5		6	11
250	51	0	0	0	10		9	19
15 CP	61	3	3	6^*	9		11	20
17.5 CP	70	5	4	9^**	16		19	35^***

*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾ 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

Table5          
Cytokinesis-block proliferation index of human lymphocyte cultures treated with CH02906 in the second cytogenetic assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/ml	CBPI			Mean CBPI		% cytostasis
0	1.63	-	1.73	1.68		0
1	1.62	-	1.63	1.63		8
10	1.50	-	1.52	1.51		25
15	1.40	-	1.50	1.45		34
20	1.40	-	1.40	1.40		41
25	1.31	-	1.31	1.31		54
30	1.24	-	1.30	1.27		60
40	1.15	-	1.15	1.15		77
0.15 MMC-C	1.36	-	1.37	1.36		46
0.23 MMC-C	1.29	-	1.35	1.32		53
0.05 Colch	1.05	-	1.05	1.05		93

 

Table6          
Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with CH02906 in the second cytogenetic assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	4	5	3		4	7
1	8	1	1	2	4		6	10
15	34	2	2	4	3		3	6
25	54	1	0	1	1		1	2
0.15 MMC-C	46	2	1	3	22		19	41^***
0.05 Colch	93	80	65	145^***	23^2)		16^2)	39^***

*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾ 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾199 and 215 binucleated cells were scored for the presence of micronuclei, respectively.

STATISTICAL EVALUATION OF THE TEST RESULTS

Chi-square Test

TOTAL NUMBER OF CELLS WITH MICRONUCLEI; TREATMENT/CONTROL COMPARISON, (MONONUCLEATED/BINUCLEATED CELLS)¹⁾.

EXPOSURE         S9-MIX   NUCLEATED       P-VALUE        DECISION AT 95%

DOSE (mg/ml)                                                      one-sided          CONFIDENCE LEVEL

First cytogenetic assay

3 hours exposure time

Positive controls

MMC-C (0.38)          −                bi                       <0.0001              significant

Colch (0.1)                 −                mono                 <0.0001              significant

Colch (0.1)                 −                bi                       <0.0001              significant

CP (15)                      −                mono                 = 0.0292             significant

CP (17.5)                   −                mono                 = 0.0056             significant

CP (17.5)                   +                bi                       = 0.0002             significant

Second cytogenetic assay

24 hours exposure time

Positive controls

MMC-C (0.15)          -                 bi                       <0.0001              significant

Colch (0.05)               -                 mono                 <0.0001              significant

Colch (0.05)               -                 bi                       <0.0001              significant

 

¹⁾Only statistically significant results are presented.

HISTORICAL CONTROL DATA FORIN VITROMICRONUCLEUS STUDIES OF THE SOLVENT CONTROL

	Mononucleated			Binucleated
	+ S9-mix	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	0.93	1.07	0.87	3.57	3.45	4.14
SD	0.96	1.00	1.11	2.48	2.07	2.67
n	108	110	108	108	110	108
Upper control limit (95% control limits)	3.01	3.59	3.46	9.06	9.18	10.68
Lower control limit (95% control limits)	-1.16	-1.44	-1.71	-1.93	-2.28	-2.41

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between January 2014 and November 2016.

HISTORICAL CONTROL DATA FORIN VITROMICRONUCLEUS STUDIES OF THE POSITIVE CONTROL SUBSTANCES

	Mononucleated		Binucleated
	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	16.65	18.27	22.23	26.31	23.36
SD	18.38	20.91	9.15	20.99	19.41
n	224	222	116	224	222
Upper control limit (95% control limits)	60.92	68.40	40.44	63.59	63.09
Lower control limit (95% control limits)	-27.63	-31.87	4.02	-10.97	-16.37

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2014 and November 2016.

 

Conclusions:

The test is valid and CH02906 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Executive summary:

An in vitro micronucleus assay with CH02906 in cultured peripheral human lymphocytes.

This report describes the effect of CH02906 on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of CH02906 was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

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 cytogenetic assay, CH02906 was tested up to 250 µg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, CH02906 was tested up to 25 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The mean number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the mean number of mono- and binucleated cells with micronuclei 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.

CH02906 did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that CH02906 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8-23 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21,1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other:

Remarks:

S. typhimurium strains are histidine-requiring, E. coli strain is tryptophan-requiring

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

Vehicle control used as negative control

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle of the test item was dimethyl sulfoxide (Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (Sigma) 2-aminoanthracene

Details on test system and experimental conditions:

CH02906 was tested in the Salmonella typhimurium reverse mutation assay with four histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9- mix (rat liver S9-mix induced Aroclor 1254).

Cell culture
Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates:
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar:
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions:
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 33.2 – 39.1°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Study design
1) Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
2) First experiment: direct plate assay
The above mentioned dose range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
3) Second experiment: pre-incubation assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
4) Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) Incasearepeatexperimentisperformedwhenapositiveresponseisobservedinoneofthe tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

/

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

both in presence and absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

both in the absence and presence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Remarks:

Not valid in the absence of S9-mix

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

both in the absence and presence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
Based on the results of this study it is concluded that CH02906 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative control values were within the laboratory historical control data ranges.

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix in the first experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Based on the results of this study it is concluded that CH02906 is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo mammalian alkaline comet assay test showed that the substance was not genotoxic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

Temporary deviations from the maximum level of target humidity occurred. 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:

mammalian comet assay

Species:

rat

Strain:

Wistar

Remarks:

Strain: Crl: WI(Han) Condition: Outbred, SPF-Quality

Details on species / strain selection:

The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Sex: dose finding was done with both sexes, the main test with males.
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before last dosing (approximately 7 g/rat) until maximum 4 hours after administration of the test item.
- Housing:
Caging: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group were housed together.
The animals were housed in room number A0.03.
Cage Identification: Colour-coded cage card indicating Test Facility Study No., group, animal number(s).
- Diet (e.g. ad libitum):
SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany (pellets).
Ad libitum, except during designated procedures.
- Water (e.g. ad libitum):
Municipal tap water.
Freely available to each animal via water bottles.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 24 Aug 2021 (study initiation) To: 28 Oct 2021

Route of administration:

oral: gavage

Vehicle:

Propylene glycol.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item.
The test item was dissolved in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of PG is 1.04 g/mL. Test item concentrations were treated with ultra-sonic waves to obtain a colourless solution. Test item concentrations were dosed within 3 hours after preparation.
Any residual volumes were discarded.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Daily: oral gavage on day 1 and 2

Post exposure period:

None: Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS Liver, Glandular Stomach and Duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

375 mg/kg bw/day (nominal)

Dose / conc.:

187.5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

No. of animals per sex per dose:

5-8 (3 additional animals in highest doses may be used to compensate for possible deaths)

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany)
- Justification for choice of positive control(s): Guideline positive control
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg body weight dissolved in physiological saline

Tissues and cell types examined:

Approximately 3-4 hours after the last treatment with the test item, vehicle or EMS Liver, Glandular Stomach and Duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Details of tissue and slide preparation:

Liver
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.
Isolation of glandular stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study (Ref 4).
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The fore- stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.
Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study (Ref 4).
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodeunum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded.
The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Preparation of slides:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 12-35 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides:
The cells on the slides were overnight (16-17.5 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 (glandular stomach and duodenum) or 30 (liver) minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 (glandular stomach and duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4.0-5.7°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5-6 minutes. The slides were subsequently immersed for 5-7 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 4-6 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of the liver, stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

Evaluation criteria:

To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control. There was no effect of the test item on Hedgehogs, no hedgehogs were found in any of the slides

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical observations were made in the groups treated with 500 mg test item /kg body weight. No mortality was observed.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In the highest dose group, unexpected deaths were observed (6 out of 8 animals), and therefore this groups could not be analyzed. An additional study was performed at 500 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Results of the main study 1

Additional information on results:

Dose-range finding study:
In the dose-range finding test, male and female animals dosed with up to 1000 and 750 mg test item per kilogram body weight showed treatment related clinical signs or mortality. Male animals were more affected than females. Based on the results of the dose-range finding study dose levels of 187.5, 375 and 750 mg/kg body weight were selected as appropriate doses for male animals for the main test.


Main study 1:
Mortality: The animals of the groups treated with 187.5 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Clinical observations were made in the groups treated with 375 and 750 mg test item /kg body weight. In the highest dose group, unexpected deaths were observed (6 out of 8 animals), and therefore this groups could not be analyzed. An additional study was performed at 500 mg/kg, which was determined to be the MTD for both males and females.
Comet Slide Analysis:
No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male treated animals (up to 375 mg/kg bw) compared to the vehicle treated animals. The slides of the 2 surviving animals at 750 mg/kg bw were not analyzed as this dose was considered above the MTD.
The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 3.77 ± 0.45% (mean ± SD), 5.88 ± 0.93% (mean ± SD) and 9.78 ± 4.35% (mean ± SD), respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 73.67 ± 1.38% (mean ± SD; p<0.001 Students t test;), 49.40 ± 12.0% (mean ± SD; p<0.001 Students t test;) and 61.96 ± 3.16% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) were analyzed. However, the highest test dose was higher than the MTD, due to the observed deaths. Therefore, an additional study was performed with 500 mg/kg bw as the MTD.

Main Study 2:
Mortality and Toxic Signs: The animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Clinical observations (lethargy and ataxia) were made in the groups treated with 500 mg test item /kg body weight.
Comet Slide Analysis in the Additional Study: No statistically significant increase in the mean Tail Intensity (%) was observed in Liver and Duodenum cells of test item treated male treated animals compared to the vehicle treated animals.
The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 4.13 ± 0.49% (mean ± SD), 6.73 ± 1.33% (mean ± SD) and 5.76 ± 1.46% (mean ± SD), respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 82.24 ± 3.69% (mean ± SD; p<0.001 Students t test;), 59.73 ± 2.25% (mean ± SD; p<0.001 Students t test;) and 61.98 ± 2.76% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Conclusions:

The comet assay is valid and 3,4-dihydroxybenzonitrile is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of 3,4-dihydroxybenzonitrile when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in Liver, Duodenum and Stomach.

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

The study procedures described in this report were based on the most recent OECD guideline.

Batch CH02906 / CQ lot 019233401 of the test item was a white powder. The test item was dissolved in propylene glycol.

Based on the results of the dose-range finding test, initially a study with one sex (males) was performed. In the main study male animals were dosed with vehicle (propylene glycol), test item (at 187.5, 375 and 750 mg/kg body weight) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

No statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male animals compared to the vehicle treated animals.

The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 3.77 ± 0.45% (mean ± SD), 5.88 ± 0.93% (mean ± SD) and 9.78 ± 4.35% (mean ± SD), respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 73.67 ± 1.38% (mean ± SD; p<0.001 Students t test;),

49.40 ± 12.0% (mean ± SD; p<0.001 Students t test;) and 61.96 ± 3.16% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

Adequate numbers of cells and doses were analysed. However, unexpected deaths were observed at the highest dose level (6 out of 8 animals). Therefore, the Maximum Tolerated Dose was lowered from 750 mg/kg body weight to 500 mg/kg body weight, and this dose group was analyzed in an additional study in males. This was considered the MTD for both males and females and consequently only one sex was used.

In the additional study, no statistically significant increase in the mean Tail Intensity (%) was observed in Liver, Duodenum and Stomach cells of test item treated male animals compared to the vehicle treated animals.

The mean Tail Intensity in Liver, Duodenum and Stomach cells of vehicle-treated rats was 4.13 ± 0.49% (mean ± SD), 6.73 ± 1.33% (mean ± SD) and 5.76 ± 1.46% (mean ± SD), respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 82.24 ± 3.69% (mean ± SD; p<0.001 Students t test;), 59.73 ± 2.25% (mean ± SD; p<0.001 Students t test;) and 61.98 ± 2.76% (mean ± SD; p<0.001 Students t test;) in Liver, Duodenum and Stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

In conclusion, the test is valid and 3,4-dihydroxybenzonitrile is not genotoxic in the Comet assay in Liver and Glandular Stomach and Duodenum cells when sampled approximately
3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Following indications for genetic toxicity in an in vitro test (mouse lymphoma L5178Y test system, OECD 490), an in vivo test (in vivo mammalian alkaline comet assay, OECD 489) was performed. The in vivo test showed that the test substance was not genotoxic.