Formamide, N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]-

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 the test item 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.

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 similar to the OECD Guideline 473, the test item was considered not to be clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

Mouse lymphoma assay

In a K1 in vitro gene mutation study in mouse lymphoma L5178Y cells, performed according to the OECD Guideline 490, the test item was considered to be mutagenic in the absence and in the presence of S9-mix under the test conditions described in the report.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-19 to 2005-11-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Non-GLP study performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with the following deviations: only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

1) only 3 strains were used: 2) no repeat experiment and 3) limited information provided on the test substance (i.e. purity was lacking) and materials and methods.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): T2078 (N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxy-phenyl)ethyl]formamide)
- Physical state: pale gray powder
- Lot/batch No.: 00464170
- Storage condition of test material: room temperature in the dark
- Other: received on 2005-08-31

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, and TA 102

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (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, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

untreated

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

Remarks:

untreated

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

Remarks:

untreated

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

Remarks:

untreated

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

Untreated negative controls:

yes

Remarks:

untreated

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

Remarks:

untreated

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: -in agar (plate incorporation)

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix of phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of the test material both with and without S9-mix.

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

Evaluation criteria:

no data

Statistics:

no data

Species / strain:

S. typhimurium, other: TA100, TA102, and TA98

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:
No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control plates gave counts of revertants colonies wihtin the normal range. All of the positive controls 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 efficacity of the S9-mix were validated. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test substance caused a visible reduction in the growth of the bacterial background lawn of strains TA100 and TA98 (with and without S9) at 5000 µg/plate. No reduction in bacterial background lawn growth was noted to TA102, however, a substantial reduction in the frequency of revertant colonies was noted both with and without S9 mix at the same concentration. Therefore, the test substance was tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains 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 absence and presence of metabolic activation. The test substance was considered to be non-mutagenic under the conditions of this test up to the limit concentration of 5000 µg/plate.

Reference 2

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:

2005-08-31 (date test substance received) to 2006-02-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Non-GLP study performed according to a method equivalent/similar to OECD Guideline 473 (In vi tro Mammalian Chromosome Aberration Test) with several data reporting deficiencies.

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.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name of test material (as cited in study report): T002078 (N-[(2-bromo-5hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxy-phenyl)ethyl]formamide)
Description : Pale grey powder
Purity : ca. 100%
Label : T2078 Code no. 89214 Lot: 00464170
Date received : 2005-08-31
Storage conditions : Room temperature in the dark
Other: received on 2005-08-31

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver homogenate metabolizing system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 14.85, 29.71, 59.41, 111.83, 237.65, 475.3, 950.6, 1901.2 and 3802.4 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, 59.41, 111.83, 237.65, 316.87, 396.07 and 475.3 μg/mL (0, 59.41, 111.83 and 237.65 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 59.41, 111.83, 237.65, 316.87, 396.07 and 475.3 μg/mL (0, 111.83, 237.65 and 316.87 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 29.71, 59.41, 89.12, 111.83, 178.25 and 237.65, μg/mL (0, 59.41, 89.12 and 111.83 μg/mL were selected for metaphase analysis)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: The test substance was soluble at the required concentration in Dimethyl sulfoxide (DMSO) therefore it was selected as the vehicle for this study.

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:

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:
-in medium

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

Statistics:

Statistics were included in the report. 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test substance was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: A precipitate of the test substance was observed at and above 950.6 µg/mL in all exposure groups in the preliminary toxicity test.

RANGE-FINDING/SCREENING STUDIES:
- A preliminary toxicity test was performed with the same experimental design described above for the chromosome aberration assays. The dose range for the Preliminary Toxicity Test was 14.85 to 3802.4 µg/mL and results of toxicity were used to set the concentration range for the chromosome aberration test. The molecular weight of the test substance was 380.24 and therefore the maximum recommended dose level was 3802.4 µg/mL, a 10 mM concentration. Microscopic assessment of the slides prepared from the cultures showed that metaphase cells were present at up to 237.65 µg/mL in the pulse exposure groups and at 118.83 µg/mL in the 24 hour continuous exposure group. The data showed clear evidence of dose-related test substance-induced toxicity in the three exposure groups. Therefore, the selection of the dose range for the chromosome aberration test was limited by toxicity in the three 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 controls 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 316.87 µg/mL in Groups 1 and 2 and at up to 118.83 µg/mL in Group 3. The test substance was seen to induce 34 % mitotic inhibition at 237.65 µg/mL and 83 % inhibition at 316.87 ug/mL (the latter dose level was considered too toxic for evaluation) in Group 1. In Group 2, 71 % mitotic inhibition was achieved at 316.87 µg/mL and 72 % mitotic inhibition at 118.83 µg/mL in Group 3. In all exposure groups the maximum dose level evaluated for chromosome aberrations was selected on the basis of toxicity.

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 any statistically significant increase 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 continuous 24 hours exposure in the absence of metabolic activation. 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:

2017-04-18 to 2017-07-11

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: I15EB1917
- Expiration date: 2017-09-07

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A solubility test was performed. The test item was dissolved in dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany) up to a concentration of 380 mg/ml.
The test item concentrations were used within 2 hours of preparation.
The final concentration of the solvent in the exposure medium was 1.0% (v/v)

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)
- 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
- Horse serum: Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Exposure medium:
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 µg/ml trifluorothymidine (TFT) (Sigma).
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
- Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 62 – 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 – 37.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

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: 78, 156, 313, 625, 1250 μg/mL without and with S9-mix;
Dose range finding test 24h: 78, 156, 313, 625, 1250 μg/mL without and with S9-mix; the results have not been reported, since the test item already showed clear mutagenic responses after 3h treatment;
Mutation experiment 1: With S9-mix: 10, 20, 40, 80, 90, 100, 110, 120, 130, 140, 150, 160 and 170 μg/mL; Without S9-mix: 5, 10, 20, 40, 80, 90, 100, 110, 120, 130, 140, 150 and 160 μg/mL (rejected since no appropriate levels of toxicity were obtained);
Mutation experiment 1A: 12.5, 25, 50, 100, 120, 140, 160, 180, 200, 220, 240, 260 and 280 μg/mL, without S9-mix (rejected since no correct subculture was performed due to a technical error);
Mutation experiment 1B: 12.5, 25, 50, 100, 120, 140, 150, 160, 170, 180, 200, 220 and 240 μg/mL, without S9-mix (Rejected since the values of mutation frequency of both solvent control groups were outside of the acceptability criteria range);
Mutation experiment 1C: 12.5, 25, 50, 100, 120, 140, 150, 160, 170, 180, 200, 220 and 240 μg/mL, without S9-mix;

Since the test item was poorly soluble in the exposure medium, the test item was dissolved in DMSO as vehicle and the maximum final conentration for the dose range finding test was 1250 μg/mL exposure medium.
The highest tested concentration in the main mutation experiment was selected based on the toxicity of the test item.

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. In DMSO, the test item was soluble at 380 mg/ml. Upon mixing with exposure medium the test item precipitated at concentrations of 62.5 mg/ml (= 625 μg/ml) and above directly after the start of the treatment, whereas precipitation of the test item was only observed at 1250 µg/ml after the 3 hour treatment period. Based on these solubility findings, DMSO was selected as vehicle and 1250 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)

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:
- 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-diphenyltetrazoliumbromide (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:

Since the test item was poorly soluble in the exposure medium, the highest tested concentration for dose range finding test was 1250 μg/mL exposure medium.
The highest concentration tested 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 Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

up to 3.9- and 4.4-fold increases in the mutation frequency at the TK locus in the absence and presence of S9-mix, respectively

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG was 13% at 160 µg/mL (with S9-mix); RTG was 31% at 150 µg/mL and was below the acceptable limit of 10% above this dose level (without S9-mix)

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
- Precipitation:
Dose range finding test 3h: at 1250 μg/mL
Dose range finding test 24h: no data
Mutation experiment 1: no precipitation
Mutation experiment 1A: no precipitation
Mutation experiment 1B: no precipitation
Mutation experiment 1C: no precipitation

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 78 to 1250 µg/mL in the absence and presence of S9-mix with a 3 hour treatment period. The relative suspension growth was 5 and 15% at the test item concentration of 156 µg/mL compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. No cell survival was observed at test item concentrations of 313 μg/mL and above. The test item precipitated in the exposure medium at the test item concentration of 1250 µg/mL.
Based on the results of the dose range finding test, 160 and 170 μg/mL were selected as the highest test item concentrations for the mutation experiment 1 with and without S9-mix, respectively.

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.
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 induced an up to 3.9-fold increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (212 per 10^6 survivors) at the dose level of 100 µg/mL.
In the presence of S9-mix, the test item induced up to 4.4-fold dose related increases in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (204 per 10^6 survivors) at the dose levels of 90 µg/mL and above.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 14 and 19.

Remarks on result:

other: 3h treatment

Evalutation of mutagenicity

In the absence of S9-mix, the test item induced an up to 3.9-fold increase in the mutation frequency at the TK locus.The test item showed up to 5.1- and 2.3-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

In the presence of S9-mix, the test item induced an up to 4.4-fold increase in the mutation frequency at the TK locus. The test item showed up to 4.3- and 9.7-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

Conclusions:

Interpretation of results: Positive in the absence and presence of S9-mix
The responses observed in the absence and presence of S9-mix fulfilled the criteria for a positive response, therefore the test item is considered to be mutagenic. In addition, a statistical significant dose related trend (p<0.001) was observed in the absence and presence of S9-mix.
In conclusion, the test item is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro studies were selected as key studies: a bacterial reverse mutation assay (Ames test), an in vitro chromosome aberration test and an in vitro mouse lymphoma test.

Bacterial reverse mutation assay:

An Ames-test was performed according to a method equivalent to OECD Guideline 471 with the following Salmonella typhimurium strains: TA 98, TA 100 and TA102 (Thompson, 2005).

T002078 was tested with and without a metabolic activation system, according to the plate incorporation method. Bacteria were exposed to T002078 at nine dose-levels selected from a preliminary toxicity test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. After 72 hours of incubation, the revertant colonies were scored.

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strains TA100 and TA98 (with and without S9 -mix) at 5000 µg/plate. No reduction in bacterial background lawn growth wsa noted to TA102, however, a substantial reduction in the frequency of revertant colonies was noted both with and without S9 -mix at the same concentration. The test material was 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 the absence of S9 -mix.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

 

Expert statement in addition to 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 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 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 TA 102 is also tested, the potential to detect certain specific types of mutagens.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.  

In addition, as the test substance showed a positive result in thein vitromammalian gene mutation test (mouselymphoma[ assay), the substance is classified as mutagenic (Cat2). An additional Ames test, covering all five strains will not impact this result

In vitro mammalian chromosome aberration test:

An in vitro chromosome aberration test was conducted according to a method equivalent to OECD Guideline 473 (Pickard, 2006).

The test material was evaluated in human lymphocytes with and without metabolic activation, at the following dose levels: 59.41, 118.83, 237.65, 316.87, 396.07, 475.3 µg/mL.

The preliminary toxicity test was performed in the dose range 4.85 -3802.4 µg/plate. A precipitate of the test material was observed at the end of the exposure period at and above 950.6 µg/mL in all three exposure groups. The data show clear evidence of dose-related test material-induced toxicity in the three exposure groups.

The test substance did not induce any statistically significant increase 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 continuous 24 hours exposure in the absence of metabolic activation. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro. 

 

In vitro mammalian gene mutation test:

In a K1 in vitro mouse lymphoma assay (Verspeek-Rip, 2017), performed according to the OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene), the effects of the test item on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells was investigated. The test was performed in the absence and presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone) with a 3 hour treatment period. The test item was dissolved in DMSO.

In the mutation experiment, the test item was tested up to concentrations of 10 to 170 and 12.5 to 240 μg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. The Relative Total Growth (RTG) was 31 and 13%, at the concentrations of 150 and 160 μg/ml in the absence and presence of S9-mix, respectively. No test item precipitation was observed up to the concentration of 160 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 induced an up to 3.9-fold increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the Global Evaluation Factor (GEF) + MF(controls) (212 per 106 survivors) at the dose level of 100 μg/ml. In the presence of S9-mix, the test item induced up to 4.4-fold dose related increases in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (204 per 106 survivors) at the dose levels of 90 μg/ml and above.

Taken together, the responses observed in the absence and presence of S9-mix fulfilled the criteria for a positive response, therefore the test item is considered to be mutagenic. In addition, a statistical significant dose related trend (p<0.001) was observed in the absence and presence of S9-mix. It is concluded that the test item is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Positive results were observed in the in vitro mouse lymphoma assay, and negative results were observed in the in vitro bacterial reverse mutation assay and the chromosome aberration assay. In order to implement appropriate risk management measures for substances potentially meeting the criteria for the highest risk categories 1A, 1B or 2 for genotoxicity, the test item is classified according to a worst case scenario without doing any further in vivo testing.

Based on the available data, the criteria laid down in the CLP Regulation and the precautionary principle, the test item T002078 should be classified as Muta 2 - H341 Suspected of causing genetic defects.