5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

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 typhimuriumstrainsTA98, TA100 and TA102, performed according a method similar to OECD Guideline 471, it was concluded that T000990 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of 10% liver S9 in standard co-factors. This well-documented study is used as key study for in vitro gene mutation in bacteria.

 - Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T000990 was considered to be non-clastogenic to human lymphocytes in vitro.

-  Gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T000990 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 bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-31 to 2003-09-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Non-GLP study conducted with methods similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay) with the following deviations: 1) only three strains were tested; 2) limited information on the test substance and methodological details were presented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

1) only three strains were tested; 2) limited information on the test substance and methodological details were presented.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Description: Pale beige powder
Purity: 100 %
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:

10% rat 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: 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:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9: 3 µg/plate for TA100

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 S9: 0.5 µg/plate for TA102

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 S9: 0.2 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 1 µg/plate for TA100

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 S9: 10 µg/plate for TA102

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 S9: 5 µg/plate for TA98

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 TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-04-24 (date test substance was received) to 2004-11-05

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 purity of the test substance was not provided, 4) 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: Pale beige powder
Date received: 24 April 2003
Storage conditions: Room temperature, in the dark

Species / strain / cell type:

other: human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver S9 homogenate

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 9.83, 19.67, 39.33, 78.66, 157.33, 314.65, 629.3, 1258.6 and 2517.2 µg/mL
- Group 1 (4-hour exposure, without S9): 0, 39.38, 78.75, 157.5, 315, 472.5 and 630 µg/mL (157.5, 315 and 472 µg/mL were selected for metaphase analysis.)
- Group 2 (4-hour exposure, with S9): 0, 39.38, 78.75, 157.5, 315, 630 and 945 µg/mL (78.75, 157.5 and 315 µg/mL were selected for metaphase analysis.)
- Group 3 (24-hour exposure, without S9): 0, 9.84, 19.69, 39.38, 78.75, 118.13 and 157.5 µg/mL (39.38, 78.75 and 118.13 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

Remarks:

No data was provided on the identity.

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation: 0.4 µg/mL (4 hour exposure), 0.2 µg/mL (24 hour exposure)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

No data was provided on the identity.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: 10 µg/mL

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:

other: human lymphocytes

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: There was no precipitation in the preliminary toxicity test.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed to determine the concentration range for the chromosome aberration test. The test substance was evaluated at a dose range of 9.83-1258.6 µg/mL using the experimental design described above for the chromosome aberration tests. The data showed clear evidence of dose-related test substance induced toxicity in all three of the exposure groups. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 314.65 µg/mL in the 4 hour exposure without metabolic activation, at up to 629.3 µg/mL in the 4 hour exposure with metabolic activation and at up to 157.33 µg/mL in the continuous exposure group without metabolic activation. A precipitate of the test substance was not observed at the end of the exposure period in any of the exposure groups. Therefore, the selection of the dose range for the chromosome aberration test was limited by toxicity for all three of the exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range (historical data control range). The positive control substances induced statistically significant increases in the frequency of cells with aberrations (all within historical data control range). 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:
The test substance was seen to induce 31% mitotic inhibition at 472.5 µg/mL in Group 1 and 68% mitotic inhibition at 118.13 µg/mL in Group 3. Therefore, acceptable levels of toxicity were achieved in both of these cases. In Group 2, a modest dose-related increase in mitotic index was observed in dose levels up to 315 µg/mL. This could be due to test substance induced toxicity synchronizing the cells. A microscopic assessment of the slides showed that metaphase cells were present at up to 472.5 µg/mL in Group 1, at up to 315 µg/mL in Group 2, and at up to 157.5 µg/mL in Group 3. However, in all exposure groups, the maximum dose level evaluated for chromosome aberrations was selected on the basis of toxicity.

Remarks on result:

other: Groups 1, 2 and 3

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for induction of chromosome aberrations in human lymphocytes. The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolizing system. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-31 to 2016-08-09

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)

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: A15KB3783
- Expiration date of the lot/batch: 2016-10-31 (retest date)
- Purity test date: 2016-05-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available
- Solubility and stability of the test substance in the solvent/vehicle: Not available

OTHER SPECIFICS
correction factor: 1

Target gene:

TK locus

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
- 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: basic medium supplemented with 5% (v/v) heatinactivated horse serum (R5-medium)
Exposure medium 24h: basic medium supplemented with 10% (v/v) heatinactivated horse serum (R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heatinactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heatinactivated 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, 17, 52, 164, 512, 1600 μg/mL with and without S9
Dose-range finding test (24h treatment): 0, 17, 52, 164, 512, 1600 μg/mL without S9

Mutation experiment I (3h treatment): 0, 1, 100, 150, 200, 250, 300, 325, 350 μg/mL without S9; 0, 100, 150, 200, 250, 300, 325, 350, 375 µg/mL with S9
Mutation experiment II (24h treatment): 0, 20, 50, 60, 70, 75, 80, 85 μg/mL without S9

Justification for top dose: Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 1600 μg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in exposure medium and ethanol. In DMSO, the test item formed a suspension at the concentration of 160 mg/ml and a solution at 51.2 mg/ml. Upon mixing with exposure medium the test item precipitated at the concentration of 160 mg/ml (= 1600 μg/ml). Based on these solubility findings, DMSO was selected as vehicle and 1600 μg/ml was selected as the maximum final concentration for the dose range finding test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; 15 μg/mL (3h treatment), 5 μg/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) were used.

DURATION
- Preincubation period: not applicable
- 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) (Sigma) 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 containing 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Rationale for test conditions:

Solubility limitation: The test item proved to be insoluble in exposure medium and ethanol. In DMSO, upon mixing with exposure medium, the test item precipitated at the concentration of 160mg/mL (=1600 µg/mL), which was the highest test concentration

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:

cytotoxicity

Remarks:

relative total growth of highest test item concentration was 7% without metabolic activation and 13% with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

relative total growth of highest test item concentration was 13%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in pH and osmolality were observed at the highest, non-precipitating concentration of 512 μg/ml (7.48 and 0.502 Osm/kg, respectively) compared to the concurrent solvent control (7.37 and 0.440 Osm/kg, respectively)
- Water solubility: not indicated
- Precipitation: In the dose range finding, precipitation of the test item concentration of 1600 μg/ml was observed during the first 5 minutes of exposure. However after 3 and 24 hour treatment, no precipitation of the test item was observed anymore. The highest non-precipitating concentration tested was 512 µg/mL.

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test item concentration range of 17 to 1600 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period and with a test item concentration range of 17 to 1600 μg/ml in the absence of S9-mix with a 24-hour treatment period. In the absence of S9-mix, the relative suspension growth was 13% at the test item concentration of 512 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1600 μg/ml.
3h dose range finding experiment: In the presence of S9-mix, the relative suspension growth was 62% at the test item concentration of 52 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/ml and above.
24h dose range finding experiment: The relative suspension growth was 92% at the test item concentration of 52 μg/ml compared to the relative suspension growth of the solvent control No cell survival was observed at the test item concentration of 164 μg/ml and above.

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.
- Negative (solvent/vehicle) historical control data: 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 solvent control database.

The suspension growth over the two-day expression period for cultures treated with DMSO was between 10 and 25 (3 hour treatment) and 127 and 130 (24 hour treatment).

Remarks on result:

other: 3h treatment

Conclusions:

Intepretation of the 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.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

A key study (K2, Thompson P and 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 material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material 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 on internal 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 strainsthat 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 convinced that the proposed 3 strains are sufficient for registration of intermediates in the production of active pharmaceutical ingredients. It should be noted however that, as TA102 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 TA102 detects cross-linking agents such as bleomycin and mitomycin C. Therefore, no additional test is performed to evaluate five strains.  

 

Chromosome aberration:

Durward (2004) performed an in vitro chromosome aberration study in human lymphocytes (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 S9 mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item, ranging from 39.8 to 630 µg/mL in the first experiment and from 9.84 to 157.5 µg/mL in the second experiment. 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 the exposure period was 24 hours without S9 mix.

The test item induced mitotic inhibition of 31% at 472.5 μg/mL in the 4(20) hour pulse exposure group without S9. In the 24-hour exposure without S9 68% mitotic inhibition was reached at 118.13 μg/mL. In the 4(20)-hour exposure group in the presence of metabolic activation a modest dose-related increase in mitotic index was observed in dose levels up to 315 μg/ml. This could be due to test material induced toxicity synchronising the cells. Dose selection for metaphase analysis was based on toxicity for all exposure groups.

100 metaphases were scored per concentration for the chromosome aberrations.

The test material did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.

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

T000990 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 L5178Y mouse lymphoma cells.

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. The test was performed in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3 and 24-hour treatment period.

In the first mutation experiment, the test item was tested up to concentrations of 350 and 375 μg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. The Relative Total growth (RTG) was 7 and 13% in the absence and presence of S9-mix, respectively. No test item precipitation was observed up to the concentration of 375 μg/ml.

In the second mutation experiment, the test item was tested up to concentrations of 85 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The Relative Total growth (RTG) was 13% at the concentration of 85 μg/ml. No test item precipitation was observed up to the concentration of 85 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 available in vitro genetic toxicity tests with T000990 and the criteria of the CLP Regulation (EC) 1272/2008, the test item should not be classified for mutagenicity.