4-[(4-HYDROXYPYRIMIDIN-2-YL)AMINO]BENZONITRILE

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 TA1535, TA1537 and TA102, performed according to OECD Guideline 471, it was concluded that T002487 has no mutagenic properties towards any of the bacterial strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

 

In vitro chromosome aberration study in Human Lymphocytes

In a non-GLP chromosome aberration study in human lymphocytes (K2), performed in accordance to a method similar to OECD Guideline 473, it was concluded that the test substance was non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells

In a K1 mouse lymphoma assay, performed according to the OECD Guideline 490, it was concluded that T002487 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:

2016-04-05 to 2016-05-31

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: M15GC2731
- Expiration date of the lot/batch: 2016-07-23 (retest date)
- Purity test date: 2015-03-29

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended or dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test or until the test item had completely dissolved in the mutagenicity tests.

OTHER SPECIFICS: correction factor 1.00

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) (2001).
- 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
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6
cells/ml.

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) heat inactivated horse serum (R5-medium)
Exposure medium 24h: 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 x10-5 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 ßnaphthoflavone)

Test concentrations with justification for top dose:

Dose range finding test 3h: 5.4, 17, 52, 164 and 512 μg/ml without and with S9-mix.
Dose range finding test 24h: 5.4, 17, 52, 164 and 512 μg/ml without S9-mix.
Mutation experiment 1: 0.054, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/ml without and with S9-mix.
Mutation experiment 2: 1.31, 2.63, 5.25, 10.5, 21, 41, 82 and 164 μg/ml without S9-mix (rejected: no appropriate levels of toxicity were obtained for the determination of the mutation frequency)
Mutation experiment 2A (repeat): 0.75, 1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, 25, 30, 40 and 50 μg/ml without S9-mix.

Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 512 μg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In DMSO, the test item formed an off-white suspension at concentrations of 51.2 mg/ml and above and a translucent solution at 5.2 and 16.4 mg/ml. Upon mixing with exposure medium the test item precipitated at concentrations
of 16.4 mg/ml (= 164 μg/ml) and above. Based on these solubility findings, DMSO was selected as vehicle and 512 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
- Exposure duration: 3 h or 24 h
- 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 (CEday2): 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 limitations: No solubility test was performed in water/exposure medium based on the information provided by the sponsor. Since the test item was poorly soluble and precipitated upon mixing with exposure medium, the highest tested concentration was 512 μg/ml exposure medium.
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).

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second experiment the relative total growth of the highest test item concentration was 6% compared to the total growth of the concurrent solvent controls

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Water solubility: No solubility test was performed in water based on the information provided by the sponsor
- Precipitation:
Dose range finding test 3h: at 164 and 512 µg/mL with and without S9-mix
Dose range finding test 24h: at 164 and 512 µg/mL with and without S9-mix
Mutation experiment 1: at 164 µg/mL
Mutation experiment 2A: no precipitation up to the highest concentration evaluated (30 µg/mL)

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 5.4 to 512 μg/mL in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
Both in the absence and presence of S9-mix, no toxicity in the relative 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.

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

OTHER: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 12 and 15 (3 hour treatment) and 143 and 153 (24 hour treatment)

Remarks on result:

other: 3 h 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 in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
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 TK mutation test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2004-06-10 to 2004-09-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare (MHW), Japanese Guidelines for Nonclinical Studies of Drugs Manual, Section 5. Reverse Mutation Test in Bacteria (1995).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FDA Toxicological Principles for the Safety Assessment of Direct Food Ingredients. Section IV.C1.a: Bacterial Reverse Mutation Test, “Redbook 2000.” U.S. FDA Washington DC, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Version / remarks:

S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61;18198-18202, April 24, 1996.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity : A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Version / remarks:

S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 61;16026-16030, November 21, 1997.

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

batch-no.: RT002487PFA021
Conversion factor =1.0000
Purity: 99.6%
Acceptance date: 2004-05-04
Retest date: 2006-03-24

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: see below for additional strain characteristics

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 (40 µL/mL for study 1 and 100 µL/mL for studies 2 and 3)

Test concentrations with justification for top dose:

Dose range finding study: 2.44, 4.88, 9.77, 19.53, 78.13, 156.25, 312.5, 625 and 1250 μg/plate.
The dose levels to be tested for the main experiments were selected based on the findings of the range finding experiment.
First, second and third study: 39.07, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was found to be not soluble in water at the concentration of 50 mg/mL. In DMSO, the test substance was found soluble at the concentration of 50 mg/mL (= 5000 µg/plate) after warming up. However, white flocculation was observed upon mixing with water down to the concentration of 2500 µg/plate. At the concentration of 1250 µg/plate, white precipitation in a colourless solution was obtained upon mixing the test substance with water. And at the concentration of 625 µg/plate, a completely colourless solution was found after mixing the test substance with water. The concentration of 1250 µg/plate was considered to be the highest applicable concentration for the range finding study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9; at 5 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9; at 1 µg/plate for T1535 and TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9; at 50 µg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9; at 5 µg/plate for TA102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9; at 1 µg/plate for TA1537 and 2.5 µg/plate for TA98, TA100, TA102 and TA1535

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

The following solutions were successively added to 2 ml histidine-biotine supplemented top agar:
- 0.1 ml of an overnight bacterial culture of the tester strain,
- 0.1 ml of a dilution of T002487 and
- either 0.5 ml of S9-mix containing 40 μl S9/ml (first study) or 0.5 ml of S9-mix containing 100 μl S9/ml (second and third study) for the activation portion, or 0.5 ml (first, second and third study) phosphate buffer for the non-activation portion.
The content of the tube was then mixed and poured onto minimal glucose agar petri dishes. The plates were incubated in the dark at 37°C for 48 to 72 hours.

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 to 72 hours
- Selection time: 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies, thinning of background lawn or occurrence of pinpoints

Evaluation criteria:

The mean number of the revertant colonies on the vehicle control plates was taken as the background level of the mutation test. Mean reversion values were considered as positive if:
- the test was valid;
- the test substance produced at least a two-fold increase in the mean number of revertants with one of the strains TA98, TA102 or TA100, or a threefold increase in the mean number of revertants with one of the strains TA1535, TA1537 at one or more concentration levels;
- a dose effect relationship was observed;
- these effects could be reproduced in an additional study.
When equivocal results are obtained, more test may be required, in order to evaluate the mutagenic potential of the test substance. If the test substance produces a positive response in a single test that cannot be reproduced in additional runs, the positive test data are discounted.
If the test substance produced a positive response in a single test that could not be reproduced in additional runs, the initial positive test data was discounted.

Statistics:

No data

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA102, TA98 and TA100

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
- Water solubility: The test substance was found to be not soluble in water at the concentration of 50 mg/mL.
- Precipitation: There was a dose related increase in precipitation of the test substance at 625 µg/plate and above in the dose range finding study and all studies. At the top dose of 2500 µg/plate, no bacterial background could be observed due to the heavy precipitation in study 1 and 3.


RANGE-FINDING/SCREENING STUDIES:
A range finding study was performed with strain TA100 in the absence and presence of S9-mix at concentrations ranging from 2.44 to 1250 µg/mL. At concentrations up to 1250 µg/plate the test substance did not reveal a biologically significant increase in the number of revertant colonies in the absence and in the presence (50 µL S9 homogenate) of S9-mix. No bacteriotoxic effects visualized by a decrease in the number of revertant colonies, thinning of the background lawn or occurrence of pinpoints were observed.
At the concentration of 625 µg/plate onwards in the absence and in the presence of rat liver S9-mix a dose related increase in precipitation was observed.
As in the range finding study, no bacteriotoxicity was observed, and the revertants could still be scored at the dose level of 1250 μg/plate, it was decided to select 2500 as top dose for the first study.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous and vehicle control revertant colonies for the strains in the absence and in the presence of S9-mix fell within the range of the laboratory's historical data in all studies. The positive controls showed a significant increase in the number of revertant colonies indicating their mutagenic activity.

ADDITIONAL INFORMATION ON CYTOTOXICITY: With all the strains, no bacteriotoxic effects visualised by a decrease in the number of revertant colonies, thinning of the background lawn or occurrence of pinpoints were observed.
In study 1 and 3, at the concentration of 625 μg/plate onwards, a dose related increase in precipitation was observed. At the top dose of 2500 μg/plate, no bacterial background could be observed due to the heavy precipitation.

Remarks on result:

other: all strains/cell types tested

First Study:

Sterility checks, genotypes of all the bacterial stains and bacterial titre of all the strains were according to the criteria. The tests with all the strains were considered acceptable for the evaluation of the mutagenic potential of the test substance.

Second Study:

As a bad lot of prepared bottom agar was used in the second study which did not allow the growth of the bacteria, the second study with the strains TA98, TA100, TA1537 and TA1535 was completely invalidated and repeated in study 3.

Sterility checks, genotypes and bacterial titre of the strain TA102 were according to the criteria. The tests with the strain TA102 were considered acceptable for the evaluation of the mutagenic potential of the test substance.

Third Study:

Sterility checks, genotypes and bacterial titre of TA98, TA100, TA1535 and TA1537 were according to the criteria. The tests with all the strains were considered acceptable for the evaluation of the mutagenic potential of the test substance.

Conclusions:

Interpretation of results:
negative with and wihtout metabolic activation

Based on the lack of a biologically significant increase in the reversion rate, it can be concluded that the test substance in the presence and in the absence of a rat liver metabolic activation system, has no mutagenic properties towards the Salmonella typhimurium strains under the test conditions described in the report.

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:

2004-09-09 (date test substance received) to 2005-02-04

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) only 100 cells per group were analyzed instead of the recommended 200 and 2) methodology was limited (no information provided on mitogen stimulation or spindle inhibitor); the sovent was 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
Purity : 100%
Label : TRT002487PFA05
Date received : 2004-09-09
Storage conditions : Room temperature in the dark

Species / strain / cell type:

other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver enzyme metabolising system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 8.29, 16.58, 33.16, 66.32, 132.63, 265.25, 530.5, 1061 and 2122 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, 4.15, 8.29, 16.58, 33.16, 66.32, 132.63,μg/mL (0, 16.58, 33.16 and 66.32 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 4.15, 8.29, 16.58, 33.16, 66.32, 132.63,μg/mL (0, 16.58, 33.16 and 66.32 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 4.15, 8.29, 16.58, 33.16, 66.32, 132.63,μg/mL (0, 33.16, 66.32 and 132.63 μg/mL were selected for metaphase analysis)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: not specified
- 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:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

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: yes

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 the aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Statistics were included in the study. However, no information was provided on the tests performed.

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the 24 hour continuous exposure group

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the preliminary and main study, a precipitate of the test substance was observed at the end of the exposure, at and above the 66.32 µg/mL, in the 4(20)-hour pulse exposure groups and at and above 132.63 µg/mL in the continuous exposure group.
In the main experiment precipitate observations were as those in the Preliminary Toxicity Test and therefore the lowest precipitating dose level was 66.32 μg/ml for the 4(20) hours pulse exposure groups and 132.65 μg/ml for the 24 hour continuous exposure group

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 8.29 to 2122 μg/ml. The maximum dose level was a 10 mM concentration. The test material induced no evidence of toxicity in any of the exposure groups. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 2122 μg/ml in the 4(20) hours exposure in the presence of S9 and in the 24 hours continuous exposure in the absence of metabolic activation (S9). There were no metaphases at 2122 μg/ml in the 4(20) hours without metabolic activation group but it was considered that heavy precipitate of test material on the slides was obscuring the metaphase cells and that there was in fact no toxicity. This was supported by the lack of toxicity observed at the same dose level in the 24 hours continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A microscopic assessment of the slides showed that metaphase cells were present at up to the maximum dose level tested, 132.63 µg/mL, in all exposure groups. The test substance was seen to induce no mitotic inhibition in the 4(20)-hour exposures both with and without metabolic activation (S9). In the 24-hour exposure group without S9 there was an approximate 50% mitotic inhibition at the lowest precipitating dose level, 132.65 µg/mL. This confirms that the maximum exposure of the test substance to cells was achieved at the lower precipitating dose levels.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolising system after a 4(20)-hour exposure or after a 24 hour continuous exposure without metabolic activation. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

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

 In a non-GLP study (K2) bacterial reverse mutation assay (Vanparys, 2004), performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay), T002487 was tested in five strains of Salmonella typhimurium strains TA98, TA100 TA1535, TA1537 and TA102.

T002487, dissolved in DMSO, was used at seven concentrations in the absence and in the presence of S9- mix: 39.07, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate in the main study.

In the absence and in the presence (20 and 50 μl S9 homogenate/plate) of a metabolic activation system, T002487 did not cause any biologically significant increase in the number of revertant colonies above the vehicle control incidence with all of the strains. No bacteriotoxic effects were observed. At concentration of 625 μg/plate onwards, a dose related increase in precipitation was observed. At the top dose level of 2500 μg/plate, no bacterial background could be observed due to heavy precipitation. The vehicle and positive control counts of all strains fell within the required norms and the genotypes could be confirmed for all the strains validating the results from this study.

It was concluded that T002487 has no mutagenic properties towards the various S. typhimurium strains under the test conditions.

 

In vitro chromosome aberration study in Human Lymphocytes

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material in a K2 study (Wright, 2005), performed according to a method similar to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test). A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Three exposure groups were used in the study: a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period; a 24-hour continuous exposure in the absence of S9. The dose range for the Preliminary Toxicity Test was 8.29 to 2122 μg/ml. The maximum dose level was a 10 mM concentration.

Based on the results of the Preliminary Toxicity Test, 132.63 µg/mL was selected as the maximum dose in the cytogenetic assay.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to the maximum dose level tested, 132.63 µg/mL, in all exposure groups. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 66.32 μg/ml, in the 4(20)-hour pulse exposure groups and at and above 132.63 μg/ml in the continuous exposure group. The test material induced no mitotic inhibition in the 4(20)-hour exposures both with and without metabolic activation. In the 24 -hour exposure group without S9 there was an approximate 50% mitotic inhibition at the lowest precipitating dose level, 132.65 µg/mL. This confirms that the maximum exposure of the test material to cells was achieved at the lower precipitating dose levels.

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.

The test material did not induce statistically significant increases in the frequency of cells with aberrations in the absence or presence of metabolic activation (S9) after a 4(20) hr exposure or after 24 hours continuous exposure in the absence of S9. The test material did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after a 4(20)-hour exposure or after a 24-hour continuous exposure without metabolic activation. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

 

In vitro gene mutation study in mammalian cells

In a K1 study, Verspeek-Rip C (2016) investigated the effect of T002487 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, according to the OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene). The test was performed in the presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone) with a 3 hour treatment period and in the absence of S9-mix with a 3 and 24-hour treatment period. The vehicle of the test item was dimethyl sulfoxide.

In the first mutation experiment, the test item was tested up to concentrations of 164 μg/ml in the absence and presence of S9-mix. The treatment period was 3 hours. No toxicity was observed up to the concentration of 164 μg/ml in the absence and presence of S9-mix. The test item precipitated in the culture medium at the concentration of 164 μg/ml.

In the second mutation experiment, the test item was tested up to concentrations of 30 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The Relative Total growth (RTG) was 6% at the concentration of 30 μg/ml. No precipitation was observed up to the concentration of 30 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 solvent 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 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 was concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Justification for classification or non-classification

Based on the negative results in all in vitro genetic toxicity tests with T002487 and the criteria of the CLP Regulation (EC) 1272/2008, T002487 should not be classified for mutagenicity.