Registration Dossier

Administrative data

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 T001325 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of S9-mix under the test conditions described in the report, up to the maximum test concentration of 5000 μg/plate. An expert statement was also added to justify the fact that no further testing is necessary.

- Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T001325 was considered to be non-clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

- In vitro gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T001325 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Link to relevant study records

Referenceopen allclose all

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:
2003-07-30 to 2004-10-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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: Brown powder
Purity: 100%
Date received: 30 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
Additional strain / cell type characteristics:
not specified
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:
With and without metabolic activation: 0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl formamide
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation; 3 µg/plate for strain TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; 0.5 µg/plate for strain TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation; 0.2 µg/plate for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation; at 1 µg/plate for strain TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone
Remarks:
with metabolic activation; at 10 µg/plate for strain TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation; 5 µg/plate for strain 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, other: TA98, TA100 and TA102
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: A yellow color with an associated precipitate was observed at and above 1500 ug/plate. These observations did not prevent the scoring of revertant colonies.


RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (dimethyl formamide) 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.

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 material 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

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains (TA98, TA100 and TA102), with any dose of the test material, either with or without metabolic activation. The test substance was considered to be non-mutagenic under the conditions of this test.
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:
2003-04-30 (test substance received) to 2004-09-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
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 meta phase 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: Brown powder
Label: T 1325 Code No: 024666 NET 0.030 KG
Date received: 30 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% induced rat liver homogenate metabolising system (S9)
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 12.24, 24.48, 48.97, 97.94, 195.88, 391.75, 783.5, 1567 and 3134 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, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL (0, 25, 50 and 100 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL (0, 25, 50 and 100 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL (0, 25, 50 and 100 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:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle not identified
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
Remarks:
vehicle not identified
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: no data
Evaluation criteria:
Metaphase cells were analysed microscopically for the presence of chromosome aberrations. A positive response was recorded for a 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 provided on the tests performed. However, statistics were used in the study.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: A precipitate of test substance was observed at and above 12.24 µg/mL, with the test substance precipitating out of solution at and above 195.88 µg/mL in the preliminary toxicity test.


RANGE-FINDING/SCREENING STUDIES:
The dose range for the toxicity test was 12.24 to 3134 µg/mL, based on a 10 mM maximum dose level. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 3134 µg/mL in all the exposure groups. The data showed no dose-related test substance-induced toxicity. However, there was a 50% reduction in toxicity at the maximum dose level in the 24-hour exposure group that was considered to be due to a heavy precipitate obscuring the metaphases. Therefore, the selection of the dose range for the chromosome aberration test was based on precipitating dose levels and was 100 µg/mL for all exposure groups.

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 substances 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 up to the maximum dose level tested in all exposure groups. The test substance did not induce mitotic inhibition at any dose level tested in any exposure group. Dose selection for metaphase analysis was based on the maximum dose level tested, a precipitating dose level, 100 µg/mL.
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 4(20)-hour exposure or 24-hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-20 to 2016-08-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: I15FB2768
- Expiration date of the lot/batch: 2017-07-01 (retest date)
- Purity test date: 2015-09-04

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 indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated
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
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium:
For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone)
Test concentrations with justification for top dose:
Dose range finding test (3h treatment): 0, 3.75, 7.5, 15, 30 and 60 µg/mL with and without S9-mix;
Dose range finding test (24h treatment): 0, 3.75, 7.5, 15, 30 and 60 µg/mL without S9-mix;
Mutagenicity assay I (3h treatment): 0, 0.06, 0.12, 0.24, 0.47, 0.94, 1.88, 3.75, 7.5, 15 and 30 µg/mL with and without S9-mix;
Mutagenicity assay II (24h treatment): 00, 0.06, 0.12, 0.24, 0.47, 0.94, 1.88, 3.75, 7.5, 15 and 30 µg/mL without S9-mix;

Top dose justification: Since the test item precipitated upon mixing with exposure medium at 30 µg/mL, 60 µg/mL was selected as top dose for the dose range finding test. No toxicity was observed up to and including the precipitating dose level of 60 μg/ml in the dose range finding test. Therefore, the highest concentrations tested in the mutation experiments were determined by the solubility in the culture 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 non-homogenous suspension at the concentration of 25 mg/ml and a clear solution at 12.5 mg/ml after a few minutes of sonification. Upon mixing with exposure medium the test item precipitated at the concentration of 3 mg/ml (= 30 μg/ml). Based on these solubility findings, DMSO was selected as vehicle and 60 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-mix; at 15 µg/mL (3h treatment); at 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-mix, at 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)

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 48 (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 inclubation, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well.

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

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

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG) (see calculations in section "any other information on materials and methods incl. tables")
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
No statistical analysis
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation:
Dose range finding test: at 30 and 60 µg/mL directly after adding; at 7.5 µg/mL and above after 3h (3h treatment) or after 24h (24h treatment)
Mutagenicity assay I: at 15 and 30 µg/mL (3h treatment)
Mutagenicity assay II: at 7.5 and above (24h treatment)

RANGE-FINDING/SCREENING STUDIES:
L5178Y mouse lymphoma cells were treated with a test item concentration range of 3.75 to 60 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 24-hour treatment period.
3h dose range finding experiment: 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 60 μg/ml compared to the suspension growth of the solvent control.
24h dose range finding experiment: No toxicity in the relative suspension growth was observed up to test item concentrations of 60 μg/ml compared to the solvent control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database.


OTHER:
Suspension growth: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 15 and 19 (3 hour treatment) and 114 and 116 (24 hour treatment)

Evaluation of the mutagenicity (first experiment):

An increase above the 95% upper control limit was observed at the mid dose of 0.47 μg/ml in the presence of S9-mix. Although this increase is above the 95% upper control limit, no dose-related response is observed and the increase in the mutation frequency (147 per 10^6 survivors) is below the MF(controls) + 126 (208 per 10^6 survivors), therefore this increase is considered to be not biologically relevant.

Conclusions:
Interpretation of results:
negative with and without metabolic activation

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y mutation 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 and Bowles, 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 formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test item was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour with an associated precipitate was observed at and above 1500 μg/plate. These observations did not prevent the scoring of revertant colonies.

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

Expert statement in addition of Ames test

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

 

The selection of these three strains is based oninternal historical data gathered during more than 25 years of testing. This is not a specific observation but is widely accepted in pharmaceutical companies. In general, most compound screening strategies are based on this limited set of 2 strains (TA 98 and TA 100). Moreover, the introduction of plasmid pKM101 in strains TA1535 and TA1538 resulted in the corresponding isogenic strains TA100 and TA98. Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone recombinational DNA repair pathway. Thus TA100 and TA98 are believed to be more sensitive than their plasmid-free counterparts. Therefore the number of compounds that is exclusively positive in TA1535 and/or TA1537 (the 2 strains that are not tested) is extremely small (less than 2.3% or 90 on 3083 compounds tested). The relationship and historical aspects are clearly described in Mortelmans et al, Mutation Research 455 (2000). We therefore are 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:

Wright (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 3.125 to 100 µg/mL in the two main experiments. A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Except where there was the need to clarify an equivocal response only one of the duplicate cultures was assessed for the presence of chromosome aberrations.

Two independent experiments were performed. In the first experiment (Groups 1 and 2) the exposure periods were 4 hours with and without S9 mix, with a 20 hour recovery period. In the second experiment (Group 3) the exposure period was 24 hours without S9 mix.

A microscopic assessment of the slides showed that metaphase cells were present at up to 100 μg/mL in all exposure groups. The test item did not induce a dose-related inhibition of mitosis at any dose level in any exposure group. Dose selection for metaphase analysis was therefore based on the precipitating dose levels. 100 metaphases were scored per evaluated culture for the chromosome aberrations.

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

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

T001325 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 15 µg/mL in the absence and presence of S9-mix. The treatment period was 3 hours. No significant toxicity was observed up to the concentration 15 µg/mL in the absence and presence of S9-mix. The test item precipitated in the culture medium at the concentration of 15 µg/mL.

In the second mutation experiment, the test item was tested up to concentrations of 7.5 µg/mL in the absence of S9-mix. The treatment period was 24 hours. No significant toxicity was observed up to the concentration 7.5 µg/mL. The test item precipitated in the culture medium at the concentration of 7.5 µg/mL.

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

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

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


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

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T001325 and the criteria of the CLP Regulation (EC) 1272/2008, T001325 should not be classified for mutagenicity.