cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

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 T001095 is not mutagenic in the Salmonella typhimurium reverse mutation assay in the absence and presence of metabolic activation.

 

- Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to the OECD Guideline 473, T001095 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 T001095 is not mutagenic in the mouse lymphoma L5178Y test system in the absence and presence of metabolic activation.

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:

2016-08-29 to 2016-10-11

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: I15DB1648
- Expiration date of the lot/batch: 2020-04-19
- Purity test date: 2016-04-27

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
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 1.03

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). 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: 7.82, 15.63, 31.25, 62.5, 125 without and with S9-mix.
Dose range finding test 24h: 7.82, 15.63, 31.25, 62.5, 125, 250 μg/ml without S9-mix.
Mutation experiment 1: Without S9-mix: 1.57, 3.13, 6.25, 12.5, 25, 50, 70, 85, 100, 115, 130 μg/mL; With S9-mix: 0.4, 0.79, 1.57, 3.13, 6.25, 12.5, 25, 30, 40, 50, 70, 85, 100 μg/mL
Mutation experiment 2: 3.13, 6.25, 12.5, 25, 50, 70, 85, 100, 115 and 130 μg/mL
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 125 and 250 μg/mL exposure medium for the 3 hour and 24 hour treatment, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
In DMSO, the test item formed a suspension at the concentration of 56 mg/mL and above and was soluble at 28 mg/mL. Upon mixing with exposure medium the test item precipitated directly at the concentration of 6.5 mg/mL (= 65 μg/mL) and above. After 3 hours, precipitation was observed at concentrations of 14 mg/mL (= 140 μg/mL) and above. Based on these solubility findings, DMSO was selected as vehicle and 125 and 250 μg/mL were selected as the maximum final concentration for the dose range finding test for the 3 and 24 hour treatment, respectively.

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-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:

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).
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 125 and 250 μg/ml exposure medium for the 3 hour and 24 hour treatment, respectively.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

No statistical analysis

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Precipitation: Dose range finding test 3h: 125 μg/mL with and without S9-mix
Dose range finding test 24h: at 250 μg/mL with and without S9-mix
Mutation experiment 1: at 115 and 130 μg/mL
Mutation experiment 2: at 115 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 7.82 to 125 μg/mL in the absence of S9-mix with 3 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period. In the 24 hour treatment, the cells were treated with a test item concentration range of 7.82 to 250 μg/mL in the absence of S9-mix.
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 115 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. Except in the first experiment, in which the mutation frequencies of one of the solvent control cultures both in the absence and presence of S9-mix were just above the upper control limits. However, the observed mutation frequencies of the solvent control cultures were within the acceptability criteria of this assay and these limits are 95% control limits and a slightly higher response is within the expected response ranges.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 21 and 25 (3 hour treatment) and 61 and 66 (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 after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-30 to 2005-12-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: 1) only three tester strains were used, and 2) no data on the purity of the test substance was provided.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

: 1) only three tester strains were used, and 2) no data on the purity of the test substance was provided.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00452023 ZT001095PAU121
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Keep away from direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle: no indicated

FORM AS APPLIED IN THE TEST (if different from that of starting material): white solid, dissolved in DMSO

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 ug/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 ug/plate

Justification for top dose: 5000 µg/plate was chosen as top dose for the Pre-experiment/Experiment I based on solubility findings. Since the test item precipitated at 5000 µg/plate, the top dose of 5000 µg/plate was chosen for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, with TA100 at 10 µg/plate

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

without metabolic activation, with TA 98 at 10 µg/plate

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, with TA 102 at 4.0 µL/plate

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 metabolic activation, with TA98 and TA100 at 2.5 µg/plate, with TA102 at 10.0 µg/plate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation
- The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test substance solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); 100 µL bacterial suspension (cf. test system, pre-culture of the strains); and 2000 µL overlay agar.
In the pre-incubation assay 50 uL test solution, solvent or 100 uL positive control, 500 uL S9 mix / S9 mix substitute buffer and 100 uL bacterial suspension were mixed in a test tube and incubated at 37 degrees C for 60 minutes. After pre-incubation, 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification, the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: Experiment I: not applicable; Experiment II: 60 minutes
- Exposure duration: at least 48 hours
- Expression time (cells in growth medium): at least 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): at least 48 hours

NUMBER OF REPLICATIONS:
-triplicate; 3 plates per strain per dose level, including the controls

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relative increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (with S9) with strains TA98 and TA100, and at 2500 and 5000 µg/plate (with S9) for TA102.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (with S9) with all strains.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 0.05 g/L
- Precipitation: Precipitation of the test substance in the overlay agar was observed at 5000 µg/plate in both experiments with and without S9 mix.
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity of the test substance, a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for Experiment I.
Toxicity of the test substance were evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment was reported as main experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I and II, the data in the negative and solvent control of strain TA102 were slightly above the historical control range. Since the deviation was rather small, the effect was considered to be based on biologically irrelevant fluctuations in the number of colonies.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- See below.

Remarks on result:

other: Experiment I

Table 2. Plates showing a reduced background growth at the following concentration (ug/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 98	/	5000	/	5000
TA 100	/	5000	/	5000
TA 102	/	2500 - 5000	/	5000

/ = No reduced background growth

Table 3. Toxic effects evident as a reduction in the number of revertants were observed at the following concentrations (ug/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 98	/	5000	/	/
TA 100	/	5000	/	/
TA 102	/	2500 - 5000	/	5000

/ = No toxic effect, evident as a reduction in the number of revertants

No substantial increase in revertant colony numbers of any of the three tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. In Experiments I and II, the data in the negative and solvent control of strain TA 102 were slightly above the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Positive controls: Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-18 to 2006-06-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Ninth Addendum, adopted July 21, 1997

Deviations:

yes

Remarks:

no data on the purity of the test substance was provided

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 4A: "Mutagenicity- In vitro Mammalian Chromosome Aberration Test" dated May 19, 2000

Deviations:

yes

Remarks:

no data on the purity of the test substance was provided

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00452023 ZT001095PUA121
- Code Number: 061260
- Expiration date of the lot/batch: not indicated by the sponsor
- Purity: not indicated by the sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from moisture. Keep away from direct sunlight
- Stability under test conditions: not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO

FORM AS APPLIED IN THE TEST: On the day of the experiment (immediately before treatment), the test item was suspended in DMSO. The final concentration of DMSO in the culture medium was 0.5 % (v/v)

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: no data
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

other: See below in "Details on test system".

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary cytotoxicity test / Toxicity Experiment I (4h exposure, with and without S9): 0, 18.3, 32, 56, 98, 171.5, 300.1, 525.2, 919.2, 1608.6 and 2815.0 μg/mL (0, 18.3, 32 and 56 μg/ml were selected for analysis of chromosome aberrations)

Toxicity Experiment II (22h continuous exposure, without S9): 0, 32, 56, 98, 171.5, 300.1, 525.2, 919.2, 1608.6 and 2815.0 μg/mL μg/mL (0, 32, 56, 98 and 171.5 μg/ml were selected for analysis of chromosome aberrations)

The highest treatment concentration in this study 2815 μg/mL (approx. 6.3 mM) was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item in an appropriate solvent (DMSO).
Using reduced mitotic indices as an indicator for toxicity in Experiment I, no clear toxic effects were observed in the absence and presence of S9 mix, up to the highest applied test item concentration.
Dose selection of experiment II was also based on test item toxicity and the occurrence of precipitation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; at 30.0 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; at 660 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Exposure time 4 hours (with and without S9 mix): About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix) containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once as described.
After washing, the cells were resuspended in complete culture medium and cultured until preparation.
- Exposure time 22 hours (without S9 mix only): About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) are set up in parallel in 25 square cm cell culture flasks. The culture medium was replaced with complete medium (with 10 % FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
- All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

DURATION
- Exposure duration: Experiment I: 4 hours; Experiment II: 22 hours
- Expression time: 15 hours (Experiment I); 19 hours (Experiment II) (Start of exposure until introduction of spindle inhibitor)
- Fixation time: Experiments I and II: 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 μg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.
STAIN (for cytogenetic assays): The cells used for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED:
- The slides were evaluated (according to the standard protocol of the "Arbeitsgruppe der Industrie Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromer regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was determined in a sample of 1000 cells per culture of each test group in %.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
- Determination of endoreplication: yes

- The positive control ethylmethane sulfonate was dissolved in a nutrient medium, and the positive control cyclophosphamide was dissolved in saline (0.9 % NaCl [w/v]).

Rationale for test conditions:

The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.

Evaluation criteria:

A test substance was classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test substance is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the historical control data (0.0 - 0.8% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

clear cytotoxic effects were observed in the highest evaluated concentration 171.5 µg/mL (45.2 % of control)

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 relevant increase in pH value was observed (e.g., Exp. I: solvent control pH 7.4 versus pH 7.6 at 2815 µg/mL).
- Effects of osmolality: No relevant increase in the osmolarity was observed (e.g., Exp. I: solvent control: 395 mOsm versus 375 mOsm at 2815 µg/mL).
- Evaporation from medium: no data
- Water solubility: <0.05 g/L
- Precipitation: In experiment I, visible precipitation of the test substance in the culture medium was observed at 56 µg/mL and above in the absence of S9 mix and at 32 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in experiment II in the absence of S9 mix at 56 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity was characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described for the mutagenicity assay.
The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times were 4 hours (with and without S9 mix). The preparation interval was 22 hours after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.
With respect to the ability to formulate a homogeneous suspension of the test substance, 2815 µg/mL of the test substance were applied as top concentration for treatment of the cultures in the pre-test. Doses over 2815 µg/mL led to an inhomogeneous suspension in DMSO that was not applicable. Test substance concentrations between 18.3 and 2815 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test substance before start of treatment was observed at 67.2 µg/mL and above in the absence of S9 mix, and at 38.4 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0 - 1.5 % aberrant cells, exclusive gaps) were close to the solvent control value (1.5 5 aberrant cells, exclusive gaps) and clearly within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
- In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 - 0.4 %) as compared to the rates of the solvent controls (0.0 - 0.2 %)
- In both experiments, EMS (660.0 μg/mL) and CPA (30.0 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiment I, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity, as indicated by clearly reduced mitotic indices could be observed. In contrast, in Experiment II, after continuous exposure in the absence of S9 mix, clear cytotoxic effects were observed in the highest evaluated concentration 171.5 µg/mL (45.2 % of control), being far in the range of test substance precipitation.

Remarks on result:

other: all strains/cell types tested

Remarks:

4-hour exposure period

The proliferation index of the lymphocytes in solvent control cultures in the 22 hrs preparation interval with and without S9 mix (4 hrs treatment; 1.01 and 1.18, respectively), was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. The proliferation index in Experiment II was not evaluable due to poor metaphase quality.

The low proliferation index in Experiment I and the non-evaluable proliferation index in Experiment II, have no detrimental impact on the validity of the study because the proliferation is proven by a sufficient number of mitotic cells, and by a clear clastogenicity observed after treatment with the positive control substances.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration.

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, Wolf T, 2005) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). T001095 was tested in the Salmonella typhimurium reverse mutation assay with three histidine-requiring strains (TA98, TA100 and TA102). The test item was dissolved in dimethyl sulfoxide.

In a dose range finding test, the test item was tested at a concentration range of 3 to 5000 µg/plate in the absence and presence of 10% liver S9 -mix in standard co-factors. Results of the dose range finding test were reported as the first mutation experiment. The test item precipitated at the top dose of 5000 µg/plate. Cytotoxicity, as evidences by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn, was observed at 5000µg/plate.

In the second mutation experiment, the test item was tested at a concentration range of 33 to 5000 µg/plate in the absence and presence of 10% liver S9 -mix in standard co-factors. The test item precipitated at the top dose of 5000 µg/plate. Cytotoxicity, as evidences by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn, was observed at 5000 µg/plate.

The test item did not induce a significant dose-related increase in the number of revertant colonies in each of the three tester strains (TA98, TA100 and TA102).

Based on the results of this study, it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Expert statement:

According to Annex VII, section 8.4,in vitrogene 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 threeS. Typhimuriumstrains (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 TA 1535 and TA 1538 resulted in the corresponding isogenic strains TA 100 and TA 98. Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone recombinational DNA repair pathway. Thus TA 100 and TA 98 are believed to be more sensitive than their plasmid-free counterparts. Therefore the number of compounds that is exclusively positive in TA 1535 and/or TA 1537 (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 has been elaborated. This mutation is also reverted by mutagens that cause oxidative damage. In addition, this DNA repair proficient strain TA 102 detects cross-linking agents such as bleomycin and mitomycin C. Therefore, no additional test is performed to evaluate five strains.

Chromosome aberration:

A key study (K2, Kunz S, 2006) performed an in vitro chromosome aberration study in human lymphocytes according to OECD Guideline 473.

The test item, dissolved in dimethyl sulfoxide, was assessed for its potential to induce chromosomal aberrations in cultured peripheral human lymphocytes in the presence and absence of 0.5% (v/v) S9-mix. 

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item, ranging from 18.3 to 2815 µg/mL with and without metabolic activation in the first experiment (4 hour exposure period), and from 32 to 2815 µg/mL without metabolic activation in the second experiment (22 hour exposure period). A minimum of three concentrations 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.

In the first cytogenetic assay, appropriate toxicity (minimal reduction of the mitotic index below 50%) was reached at 56 µg/mL ,and 18.3, 32 and 56 µg/mL were selected for analysis of chromosome aberrations. In the second cytogenetic assay, appropriate toxicity (minimal reduction of the mitotic index to 45 ± 5%) was reached at 171.5 µg/mL, and 32, 56, 98 and 171.5 µg/mL were selected for analysis of chromosome aberrations.

The number of cells with chromosome aberrations found in the vehicle control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the number of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations 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 Experiment I, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In contrast, in Experiment II, after continuous exposure in the absence of S9 mix, clear cytotoxic effects were observed in the highest evaluated concentration 171.5 μg/mL (45.2 % of control) being far in the range of test item precipitation.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.0 - 1.5 % aberrant cells, exclusive gaps) were close to the solvent control value (1.5 % aberrant cells, exclusive gaps) and clearly within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

It is concluded that this test is valid and that the test item is not clastogenic in human lymphocytes under the experimental conditions described in this report.

In vitro gene mutation study in mammalian cell

Verspeek-Rip C (2017) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (key study, K1).

The test item 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 9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3- and 24-hour treatment period.

A correction factor of 1.03 was used to correct for the purity/composition of the test item. The test item was suspended at concentrations of 56 mg/mL and higher in dimethyl sulfoxide. At concentrations of 28 mg/mL and lower the test item was dissolved in dimethyl sulfoxide.

In the first mutation experiment, the test item was tested up to concentrations of 115 and 85 μg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. The relative total growth (RTG) was 35 and 12% in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at the concentration of 115 μg/ml.

In the second mutation experiment, the test item was tested up to concentrations of 115 μg/ml in the absence of S9-mix. The treatment period was 24 hours. The relative total growth (RTG) was 11% at the concentration of 115 μg/ml. No precipitation of the test item in the culture medium was observed up to the concentration of 115 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 this report.

Based on the results of the 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 mutation frequency found in the solvent control cultures was within the acceptability criteria. Although the mutation frequencies of one of the solvent control cultures in the first experiment both in the absence and presence of S9-mix were just above the 95% upper control limits of the distribution of the historical solvent control database, these limits are 95% control limits and a slightly higher response is within the expected response ranges.

Justification for classification or non-classification

Based on negative results in all in vitro  genetic toxicity tests with T001095 and the criteria of the CLP Regulation (EC) 1272/2008, T001095 should not be classified for mutagenicity.