Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-       Bacterial reverse mutation assay 

In a K2 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100 and TA102, performed according to a method similar to OECD Guideline 471, it was concluded that T002326 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of S9-mix under the test conditions described in the report, up to the maximum test concentration of 5000 μg/plate. An expert statement was also added to justify the fact that no further testing is necessary.

-       Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T002326 was considered to be non-clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

 -       In vitro gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T002326 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

 

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:

2016-05-23 to 2016-08-02

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:

A deviation to the study plan is reported in the results section, but does not consist in a deviation to the guideline.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mouse lymphoma assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15CB0987
- Expiration date of the lot/batch: 2016-07-29 (retest date)
- Purity test date: 2015-08-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

Target gene:

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)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Cell cycle length, doubling time or proliferation index: not indicated
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50U/mL and 50μg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium)
Exposure medium 3h exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium)
Exposure medium 24h exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, 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, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine (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.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

Dose-range finding test (3h treatment): 0, 5.4, 17, 52, 164, 512 µg/mL with and without S9
Dose-range finding test (24h treatment): 0, 4.95, 15.6, 47.7, 150, 512 µg/mL without S9

Mutation experiment I (3h treatment): 0, 0.18, 0.55, 1.7, 5.4, 17, 52, 164, 512 µg/mL with and without S9
Mutation experiment II (24h treatment): 0, 4, 8, 16, 32, 64, 128, 256, 512 µg/mL without S9

Justification for top dose: In all experiments, the highest concentration was determined by the solubility of the test item in the culture medium since the test item was not toxic and difficult to dissolve in aqueous solutions.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in culture medium and ethanol. In DMSO, the test item was soluble at 228 mg/ml after a few seconds of treatment with ultrasonic waves. Upon mixing with exposure medium the test item precipitated at concentrations of 51.2 mg/ml (= 512 μg/ml) and above.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; 15µg/mL (3h treatment), 5µg/mL (24h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9; 7.5 µg/mL (3h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hour treatment) per culture

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 48h (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): 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) to each well.

NUMBER OF REPLICATIONS:
Determination of cloning efficiency: 2 x 96-well microtiter plates/concentration
Determination of mutation frequency: 5 x 96-well microtiter plates/concentration (solvent controls and treatment groups); 10 x 96-well microtiter plates/concentration (positive controls)

NUMBER OF CELLS EVALUATED:
Determination of cloning efficiency: One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated (5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (solvent controls and treatment groups); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well con
taining 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Rationale for test conditions:

The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
Solubility limitation: the test item was poorly soluble in the exposure medium and precipitated at the dose of 512 µg/mL, which was the highest test concentration.
Toxicity: no severe toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix

Evaluation criteria:

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 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.

Statistics:

No statistical analysis

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects on pH and osmolality: not reported
- Precipitation: The test item precipitated in the exposure medium at the highest concentration tested (512 μg/ml), in all experiments.

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test item concentration range of 5.4 to 512 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period and with a test item concentration range of 4.95 to 512 μg/ml in the absence of S9-mix with a 24-hour treatment period. Cytotoxicity of the test item was calculated by the suspension growth and the relative suspension growth.
No toxicity in the suspension growth was observed up to and including the highest test item concentration of 512 μg/ml compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix, for the 3-hour and the 24-hour treatments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 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.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database, except in the second experiment, in which the mutation frequency of one of the solvent control cultures was just below the acceptability criteria range of this assay. Since the other solvent control culture was within the range, this deviation has no (adverse) effect on the results of the second mutation assay.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 19 and 21 (3 hour treatment) and 110 and 128 (24 hour treatment).

Conclusions:

Interpretation of results:
negative with and without metabolic activation

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-23 to 2003-07-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

1) only three strains were tested; 2) no repeat experiment; 3) limited information on the test substance and method ological details were presented

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Description: White powder
Date received: 24 April 2003
Storage conditions: Room temperature in the dark

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium, other: TA98, TA100 and TA102

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)

Remarks:

Without metabolic activation; for TA100 at 3 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation; for TA102 at 0.5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation;for TA98 at 0.2 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- Amino anthracene

Remarks:

With metabolic activation; for TA100 at 1 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone

Remarks:

With metabolic activation: for TA102 at 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation;for TA98 at 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control plates gave counts of revertant colonies within the normal range (no historical data control tables were provided). All of the positive control substances induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the Ames assay using S. typhimurium strains TA98, TA100 and TA102 in the absence and presence of metabolic activation. The test substance was considered to be non-mutagenic under the conditions of this test in all strains.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-04-23 (date of sample receipt) to 2004-09-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 metaphase spreads scored per concentration, 3) the solvent is not known.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Description : White powder
Chemical name : 4-[4,6-dihydroxy-2-pyrimidinyl)amino]benzonitrile
Purity : 100%
Label : T2326 CODE NO : 094265
Date received : 24 April 2003
Storage conditions : Room temperature in the dark

Species / strain / cell type:

other: Human lymhocytes

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2% rat liver homogenate metabolising system

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 8.92, 17.83, 35.66, 71.32, 142.63, 285.25, 570.5, 1141 and 2282 µg/mL
The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Group 1 (4-hour without S9 with 20 hour expression time): 0, 37.5, 75, 150, 300 and 600 µg/mL (0, 150, 300 and 600 µg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 37.5, 75, 150, 300 and 600 µg/mL (0, 150, 300 and 600 µg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 37.5, 75, 150, 300 and 600 µg/mL (0, 150, 300 and 600 µg/mL were selected for metaphase analysis)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; at 0.4 µg/mL for the 4(20) hour exposure and 0.2 µg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation; at 10 µg/mL for the 4(20) hour exposure (Group 2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: no data
DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor ; 0 hours (Group 3)
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS: Two cultures were tested per group. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations.

NUMBER OF CELLS EVALUATED: 100 cells per evaluated culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data

Rationale for test conditions:

No data

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

No data was provided on the tests performed. However, p<0.001 was considered to be statistically
significant.

Species / strain:

lymphocytes: Human

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:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: There was a precipitate of test material observed at and above 285.25 μg/mL in all exposure groups in the preliminary test. Precipitation was observed in all groups at 300 and 600 µg/mL in the main experiment.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the toxicity test was 8.92 to 2282 μg/mL, based on a 10 mM maximum dose level. There was a precipitate of test substance observed at and above 285.25 μg/mL in all exposure groups. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 2282 μg/mL in all the exposure groups. The data showed that there was no dose-related toxicity observed at up to the maximum dose level 2282 μg/mL in all exposure groups. Therefore, the selection of the dose range for the chromosome aberration test was based on the lowest precipitating dose level of 285.25 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
Vehicle control and positive control data were compared with historical control data. All of the vehicle and positive control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A microscopic assessment of the slides showed that metaphase cells were present at up to 600 μg/mL in all exposure groups. The test material did not induce a dose-related inhibition of mitosis at any dose level in any exposure group. Although there was some reduction in MI at 600 μg/mL in the 4(20)-hour without S9 exposure this was considered to be spurious as it was not observed at the same dose level in the 24-hour exposure group. Dose selection for metaphase analysis was therefore based on the precipitating dose levels.

Remarks on result:

other: all strains/cell types tested

For the positive controls in Group 1 and 3, only 50 cells were scored because approximately 50 % cells with aberrations had been observed.

For the positive control in Group 2, only 91 cells were scored because of the insufficient number of metaphases due to toxicity.

The test substance did not induce statistically significant increases in the frequency of cells with aberrations at any dose level in any of the three exposure conditions.

The test susbtance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Conclusions:

Interpretation of results:
negative

The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after 4(20)-hour exposure or a 24-hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro, under conditions of the assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay
A key study (K2, Bowles A, 2004) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Salmonella typhimurium strains TA98, TA100 and TA102 were treated with solutions of the test item using the Ames plate incorporation method at 9 dose levels, in triplicate, both with and without the addition of 10% liver S9 in standard co-factors. The dose range was 0.5 to 5000 µg/plate in the main experiment.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test item was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Expert statement in addition of Ames test

According to Annex VII, section 8.4, in vitro gene mutation study using bacteria (Ames test) is a standard information requirement at the present tonnage level. The registrant is aware of the version of the EU Test Method B.13/14/OECD TG 471 in force since 1997 which indicates that at least five strains of bacteria should be tested. The Ames test, currently included in the dossier, is performed following OECD 471 but included testing on three S. typhimurium strains (TA 98, TA 100, TA 102) only.

The selection of these three strains is based oninternal historical data gathered during more than 25 years of testing. This is not a specific observation but is widely accepted in pharmaceutical companies. In general, most compound screening strategies are based on this limited set of 2 strains (TA 98 and TA 100).Moreover, the introduction of plasmid pKM101 in strains TA1535 and TA1538 resulted in the corresponding isogenic strains TA100 and TA98. Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone recombinational DNA repair pathway. Thus TA100 and TA98 are believed to be more sensitive than their plasmid-free counterparts. Therefore the number of compounds that is exclusively positive in TA1535 and/or TA1537 (the 2 strains that are not tested) is extremely small (less than 2.3% or 90 on 3083 compounds tested). The relationship and historical aspects are clearly described in Mortelmans et al, Mutation Research 455 (2000). We therefore are convincedthat the proposed 3 strains aresufficient for registration of intermediates in the production ofactive pharmaceutical ingredients. It should be noted however that, as TA 102 is also tested, the potential to detect certain specific types of mutagens has been elaborated. This mutation is also reverted by mutagens that cause oxidative damage. In addition, this DNA repair proficient strain TA 102 detects cross-linking agents such as bleomycin and mitomycin C. Therefore, no additional test is performed to evaluate five strains.  

 

Chromosome aberration:

Wright N (2004) performed a in vitro chromosome aberration study in human lymphocytes key study (K2, method equivalent/similar to OECD Guideline 473).

The test item, dissolved in vehicle, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and the presence of metabolic activation by 2% S9 mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item, ranging from 37.5 to 600 µg/mL in the two main experiments. A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Except where there was the need to clarify an equivocal response only one of the duplicate cultures was assessed for the presence chromosome aberrations. Two independent experiments were performed. In the first experiment (Groups 1 and 2) the exposure periods were 4 hours with and without S9 mix, with a 20 hour recovery period. In the second experiment (Group 3) the exposure period was 24 hours without S9 mix.

A microscopic assessment of the slides showed that metaphase cells were present at up to 600 μg/ml in all exposure groups. The test item did not induce a dose-related inhibition of mitosis at any dose level in any exposure group. Although there was some reduction in MI at 600 μg/ml in the 4(20)-hour without S9 exposure this was considered to be spurious as it was not observed at the same dose level in the 24-hour exposure group. Dose selection for metaphase analysis was therefore based on the precipitating dose levels. 100 metaphases were scored per concentration for the chromosome aberrations.

The test item did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.
The test item did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.
T002326 was therefore considered to be non-clastogenic to human lymphocytes in vitro.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

In vitro gene mutation study in mammalian cells:

Verspeek-Rip (2016) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178 mouse lymphoma cells (K1, method according to OECD Guideline 490).

The test item, dissolved in dimethyl sulfoxide, was assessed for its potential to induce forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. Two independent experiments were performed. In the first experiment the exposure periods were 3 hours with and without S9 mix, with a 21 hour recovery period. In the second experiment the exposure period was 24 hours without S9 mix. Cell cultures were exposed to a series of concentrations of the test item, ranging from 0.18 to 512 µg/mL in the two main experiments.

In both experiments, no significant toxicity was observed up to the concentration 512 μg/ml in the absence and presence of S9-mix. The test item precipitated in the culture medium at the concentration of 512 μg/ml.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.

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

It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Based on the results of positive control chemicals the test conditions were considered adequate and it was concluded that the metabolic activation system (S9 -mix) functioned properly. In addition, the acceptability criteria for the negative control substance were met and the mutation frequency was situated within the 95% control limits of the distribution of the historical solvent control database.

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T002326 and the criteria of the CLP Regulation (EC) 1272/2008, T002326 should not be classified for mutagenicity.