D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetraacetate, (1S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and in the Escherichia coli strain WP2uvrA, performed according to a method similar to OECD guideline 471, it was concluded that T003066 has no mutagenic properties towards the S. typhimurium and E. coli strains tested in the absence and in the presence of S9 -mix under the test conditions described in the report (Tsuji 2007).

In a supporting K2 bacterial reverse mutation assay in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and in the Escherichia coli strain WP2uvrA, performed according to OECD 471 and Council Regulation (EC) No. 440/2008 B13/14, T003066 was found to not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In vitro micronucleus assay

In a K2 in vitro micronucleus assay in cultured peripheral human lymphocytes, performed according to a method similiar to OECD Guideline 487, it was concluded that T003066 has to be considered as non-mutagenic in this in vitro test system when tested up to precipitating test item concentrations.

In vitro chromosome aberation study in mammalian

In a K1 in vitro chromosome aberration study in human lymphocytes assay, performed according to the OECD Guideline 473 and EU method B10, it was concluded that T003066 is not clastogenic in the human lymphocyte test system in the abscence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-06-23 to 2007-08-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Well documented study report, GLP compliant, and according to Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals. No deviations from the study protocol were recorded.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

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: M0331013A, Tanabe Seiyaku co., Ltd.
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Purity: 100%
- Appearance at ordinary temperature: white crytalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable at room temperature under light-shielded consitions
- Solubility and stability of the test substance in the solvent/vehicle: DMSO, 50 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

Target gene:

histidine locus (histidine-dependent S. typhimurium strains); tryptophan locus (tryptophan-dependent E.coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/5,6-benzoflavone induced rat liver (S9) from male Sprague-Dawley rats

Test concentrations with justification for top dose:

Dose-finding assay: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main assay: 0, 20, 39, 78, 156, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was selected based on the following reasons. As the information from sponsor, the test chemical was reported to be hydrolyzed by water, and its solubility in DMSO was found to be 50 mg/mL or more. The preliminary test on solvent selection in the testing facility indicated that the test chemical was dissolved at 5% in DMSO and there were no form or heat production during the dissolution.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix, all strains, 0.5 µg/plate (TA98), 1.0 µg/plate (TA100), 2.0 µg/plate (TA1535, TA1537), 10.0 µg/plate (WP2 uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

AF-2

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Without S9-mix, 0.01 µg/plate (TA100, WP2 uvrA), 0.1 µg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NaN3

Positive control substance:

sodium azide

Remarks:

Without S9-mix, 0.5 µg/plate (TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

ICR-191

Positive control substance:

other: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride

Remarks:

Without S9-mix, 1.0 µg/plate (TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation method

DURATION
- Preincubation period: 20 minutes (with shaking) - After preincubation, a 2.0 mL of top agar was added to the mixture and the resultant mixture was overlaid onto a minimum glucose agar plate. Agar soluion supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin (S. typhimurium) or with 0.5 mM L-tryptophan (E. coli) at a volume ratio of 10:1 was used as a top agar.
- Exposure duration: 48h incubation - After incubation, the growth inhibition of the tester bacterial strain was examined with a stereoscopic microscope, and the revertant colonies were counted.

SELECTION AGENT (mutation assays): histidine (S. typhimurium); tryptophan (E. coli)

NUMBER OF REPLICATIONS: duplicate at each dose level; triplicate for negative control

NUMBER OF CELLS EVALUATED: Revertant colonies within a circle with about 80 mm diameter on a plate with 86 mm diameter were counted with an automatic colony counted (and corrected based on the uncounted area by using a personal computer). When the number of revertant colonies on a plate was 1500 or more, the accuracy of the automatic colony counter was lowered and the colonies were counted manually at 5 different places on a plate with a stereoscopic microscope and the average number of colonies was applied to the area-based correction.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (dose-related)

OTHER:
- For all plates, the growth inhibition of bacterial strain was observed with a stereoscopic microscope (40-fold magnification) and the precipitation was observed by unaided eyes.

Evaluation criteria:

When the number of revertant colonies on test chemical treatment plates is remarkably increased compared to the spontaneous level (i.e. twice or more of the solvent control value), with a dose-dependency and reproducibility, it is judged to be positive for mutagenicity. Otherwise, it is judged as negative.

Statistics:

No statistical analysis is performed for data analysis.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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
- Water solubility: no information on solubility in water of the test substance. As the information from the sponsor, the test chemical was reported to be hydrolyzed by water.
- Precipitation: White precipitates of the test chemical were observed by unaided eyes at 4.9 μg/plate and higher in the absence of S9 and at 313 μg/plate and higher in the presence of S9 (Dose-finding assay); White precipitates of the test chemical were observed by unaided eyes at 20 μg/plate and higher in the absence of S9 and at 156 μg/plate and higher in the presence of S9 (Main assay).

RANGE-FINDING/SCREENING STUDIES: In the dose-finding assay, the highest dose was set at 5000 μg/plate, and a total of 7 doses were set with a common ratio of 4. Based on these results, the highest dose in the main assay was set at 313 μg/plate, and a total of 5 doses with a common ratio of 2, including at lease one dose producing precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA: Results were check with the laboratories historical control data.

Remarks on result:

other: Dose-finding assay

Validity of assays

- Positive control chemicals increased revertant colonies more than twice of the solvent control value of each strain, indicating the assays were performed properly.

- No bacterial growth due to contamination was observed in the sterility test in both the range-finding assay and the main assay.

- The test chemical did not increase the number of revertant colonies 2-fold or more compared to the solvent control value both in the presence and absence of metabolic activation in any of the strains.

Conclusions:

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

Based on the above results, it was concluded that Ac-GTB was not mutagenic under the test conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-22 to 2016-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: 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: I16CB1581
- Expiration date of the lot/batch: 2019-04-04 (retest date)
- 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 indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy adult, non-smoking volunteers
- Suitability of cells: recommended in international guideines (OECD, EC)
- Sex, age and number of blood donors if applicable: age 26 (Dose range finding assay/first cytogenetic assay); age 27 (second cytogenetic assay); age 30 (cytogenetic assay 2A)
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively).
- Methods for maintenance in cell culture if applicable: Immediately after blood collection lymphocyte cultures were started.
- Modal number of chromosomes: 46 +-2
- Normal (negative control) cell cycle time:
AGT (age 26): 12.8h
AGT (age 27): 13.5h
AGT (age 30): 12.9h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
Exopsure medium: Lymphocytes were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
- Properly maintained: yes (Immediately after blood collection lymphocyte cultures were started.)
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-/ß-naphthoflavone induced rat liver homogenate (S9-mix)

Test concentrations with justification for top dose:

Dose-range finding test: 0, 2, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL with and without S9-mix;
Cytogenetic assay 1: 0, 3.9, 7.8, 15.6 and 31.3 µg/mL with and without S9-miix; (7.8, 15.6 and 31.3 µg/mL were selected for metaphase analysis)
Cytogenetic assay 2: 0, 1, 2, 3.9 and 7.8 µg/mL without S9-mix; (dose selection based on the results of the dose range finding test; since no precipitate was observed at the highest dose level after 24h exposure, the test was aborted and the experiment was repeated as cytogenetic assay 2A)
Cytogenetic assay 2A: 0, 1, 2, 3.9, 7.8, 15.6, 31.3, 62.5 and 125 µg/mL with and without S9-mix; (3.9, 7.8 and 15.6 µg/mL were selected for metaphase analysis)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in culture medium and ethanol. In DMSO, the test item was dissolved. The test item precipitated in culture medium at the concentration of 3.13 mg/ml (= 31.3 μg/mL) and above. Based on these solubility findings, DMSO was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9; at 0.5 and 0.75 µg/mL (3h treatment); at 0.2 and 0.3 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; at 10 µg/mL (3h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A vehicle control was included at each exposure time.
After 3 h exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were treated for 24 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h fixation time).

DURATION
- Preincubation period: 48+-2h
- Exposure duration: 3h (cytogenetic assay 1) or 24h (dose range finding test; cytogenetic assay 2/2A)
- Expression time (cells in growth medium): 21-21.5h (cytogenetic assay 1) or 0h (dose range finding test; cytogenetic assay 2/2A)
- Fixation time (start of exposure up to fixation or harvest of cells): 27h (cytogenetic assay 1) or 24h (dose range finding test; cytogenetic assay 2/2A)

SPINDLE INHIBITOR (cytogenetic assays): colchicine
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed.

STAIN (for cytogenetic assays): 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch Study number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED:
mitotic index/dose selection: the number of metaphases were counted for at least 1000 cells per culture (with a maximum deviation of 5%

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
analysis of slides: 150 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay.
The highest concentration examined for chromosome aberrations should be cultures that produce an inhibition of the mitotic index of 55 ± 5 %, whereas the mitotic index of the lowest concentration should have little or no cytotoxicity (approximately the same as vehicle control). Also cultures treated with an intermediate concentration should be examined. For poorly soluble test items, the highest concentration analysed should be the lowest insoluble concentration (determined at the end of treatment) irrespective of toxicity. The extent of precipitation may not interfere with the scoring of chromosome aberrations. If no precipitate or limiting toxicity is observed, the highest concentration examined should correspond to 2 mg/ml or 0.01 M, whichever is the lowest.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical vehicle control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the historical vehicle control data range.

Statistics:

Fisher’s exact test, one-sided, p < 0.05; Graphpad Prism version 4.03 was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 31.3 µg/mL and above (dose range finding test); at 62.5 µg/mL and above (cytogenetic assay 2A)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not indicated
- Precipitation:
Dose range finding test: at 3.9 μg/ml and above
Cytogenetic assay 1: at 31.3 μg/ml (top dose)
Cytogenetic assay 2: no precipitation observed
Cytogenetic assay 2A: at 5.6 μg/ml and above
- Definition of acceptable cells for analysis: Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed.

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. The test item was tested in the absence and in the presence of 1.8% (v/v) S9-mix, at the following dose levels: 0, 2, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL. Cytotoxicity was observed at 31.3 µg/mL and above, and the test item precipitated in culture medium at 3.9 µg/mL and above. Based on the results of the dose range finding test, the following dose levels were selected for the second cytogenetic assay: 1.0, 2.0, 3.9 and 7.8 μg test item/ml culture medium.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in all positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- Negative (solvent/vehicle) historical control data: The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

- Since no precipitate was observed at the highest dose level after the 24 hour exposure (cytogenetic assay 2), the test was aborted and the experiment was repeated (cytogenetic assay 2A).

- Polyploidy: No effects of the test item on the number of polyploid cells were observed both in the absence and presence of S9-mix.

- Endoreduplicated chromosomes: no effects on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Conclusions:

The test substance was considered not clastogenic under the test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-09-30 to 2008-11-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented study performed in accordance with the OECD Guideline 471 and Council Regulation (EC) No. 440/2008 B13/17. Although only three strains are tested, a rationale on the fact that the study is considered reliable is described in the section "Any other information on results incl. tables".

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 B13/14, dated May 30, 2008

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

The histidine dependant strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus.

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100 and TA102

Additional strain / cell type characteristics:

other: see Any other information on materials and methods incl. tables

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide (DMSO)
Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water deionised

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg/plate in TA 100 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

10 µg/plate in TA 98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water deionised

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

3.0 µg/plate in TA 102 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 and 10.0 µg/plate in TA98, TA100 and 102 with S9, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay
Experimental performance: The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL: Test 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: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL: Overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.

DURATION:
- Pre-incubation period: 60 minutes
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicates.
NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA102) 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:

No statistical evaluation of the data was not required.

Species / strain:

other: Salmonella typhimurium TA98, TA100 and TA102

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:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with T003066 at any dose 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.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitation of the test item was observed in the overlay agar on the incubated agar plates from 333 µg/plate up to 5000 µg/plate. The undissolved particles of the test item had no influence on the data recording.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed, 5000 µg/plate were chosen as maximal concentration for experiment II.

Appropriate reference mutagens were used as positive controls. They showed direct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) occurred in the test groups with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) occurred in strain TA 98 with S9 mix at 2500 µg/plate and 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

The Ames test described in this robust study summary 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.  

Conclusions:

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

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 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-10-14 to 2008-11-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented, non-GLP study performed according to a method equivalent to the draft proposal for OECD Guideline 487 "In vitro Mammalian Cell Micronucleus Test (Mnvit)", nonGLP.

Qualifier:

equivalent or similar to guideline

Guideline:

other: draft proposal OECD Guideline 487 "In vitro Mammalian Cell Micronucleus Test (Mnvit)" (3rd version)

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFA 171
- Expiration date of the lot/batch: 2010-03-26
- Purity test date: Not specified
- Purity: 99.3%

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

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. About 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) supplemented with 10% fetal calf serum. Additionally, the medium was supplemented with 1% 100x Penicilin/Streptomycin solution and 1% Amphotericin B. The cells were subcultured twice weekly. The cell cultures were incubated at 37°C in a humidified atmosphere with 4.5% carbon dioxide (95.5% air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I & Experiment II with and without S9 mix: 5.8, 11.6, 23.2, 46.3, 92.6, 185.3, 370.5, and 741.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: The solvent has been chosen to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5% THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 at 0.1 µg/mL in deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5% THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: griseofulvin

Remarks:

without S9 at 8.0 µg/mL in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5% THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 at 10 µg/mL in Saline (0.9% NaCl [w/v])

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (experiment I with and without S9 mix; experiment II with S9 mix), 24 hours (experiment II without S9 mix)
- Expression time (cells in growth medium): 20 hours in experiment I with and without S9 mix and in experiment II with S9 mix; 0 hours in experiment II without S9 mix
- For the micronucleus analysis, 24 hours after the start of the epxosure, the cells were treated on the slides in the chambers of quadriperm dishes with deionised water for 1 to 1,5 min at 37°C.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours: after incubation in deionised water the cells were fixed twice for > 1 min with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25% (v/v) formaldehyde.

STAIN (for cytogenetic assays): After preparation the cells were stained with May Grünwald and Giemsa.

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2-8 cells were scored per test group.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3-4 cells (c4), and 5-8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters respresented the cells that have divided 1, 2 or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

A test item can be classified as mutagenic if:
- The number of micronucleated cells is not in the range of the laboratory's historical control data and
- either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the laboratory's historical control data and/or
- no concentration-related increase in the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

when tested up to precipitating test item concentrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and somolarity: No influence of the test item on the pH value or osmolarity was observed
(Exp. I: solvent control 369 mOsm, pH 7.2 versus 348 mOsm and pH 7.3 at 741.0 µg/mL;
Exp. II: solvent control 340 mOsm, pH 7.6 versus 358 mOsm and pH 7.7 at 741.0 µg/mL)
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: In Experiment I in the absence of S9 mix test item precipation was observed at the end of treatment at a concentration of 46.3 µg/mL and above, and at 92.6 µg/mL and above in the presence of S9 mix. In Experiment II in the absence of S9 mix precipitation of the test item at the end of the treatment was observed at 185.3 µg/mL and above. In the presence of S9 mix no test item precipitation was observed at the end of treatment. However, test item precipitation at the start of treatment was observed at 185.3 µg/mL and above. Therefore, the highest applied concentration of 741.0 µg/mL and the first concentration (185.3 µg/mL), where test item precipitation was observed at the start of treatment as well as two non-precipitating concentrations (46.3 and 92.6 µg/mL) were evaluated for cytogenetic damage.

RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration (741.0 µg/mL; approx. 1.2 mM) was chosen with regard to the solubility properties of the test item in THF following the current Draft Proposal for a new Guideline No. 487. Dose selection was performed considering the toxicity data and the occurrence of precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments no statistically significant and biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration. The rates of micronucleated cells after treatment with the test item (0.25 - 1.35 %) were close to the solvent control values (0.25 - 1.25 %) and within the laboratories historical control data range.
Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) and CPA (10.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY: After test item treatment neither in the absence nor and in the presence of S9 mix the
proliferation index was reduced to values below 1.3 indicating no cytotxicity.

Conclusions:

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

In conclusion, it can be stated that under the experimental conditions reported, the test item RT003066 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.
Therefore, RT003066 has to be considered as non-mutagenic in this in vitro test system when tested up to precipitating test item concentrations.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-01 to 2017-04-10

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: I16CB1581
- Expiration date of the lot/batch: 2019-04-04 (retest date)
- 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 indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: L5178Y/TK+/--3.7.2C mouse lymphoma 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). Cell density was kept below 1 x 106 cells/ml.
- Normal cell cycle time (negative control): not indicated

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, 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).
Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 38 – 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.3 – 37.8°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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
- 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:

Since the test item precipitated upon mixing with exposure medium at 2000 µg/mL, this concentration was selected as top dose for the dose range finding test.
NEEDS ADAPTATION - SEARCH STUDY 514734

Dose range finding test (3h treatment): 0, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL with and without S9-mix;
Dose range finding test (24h treatment): 0, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL without S9-mix;

The highest concentrations tested in the first mutation experiment were determined by the solubility of the test item in the culture medium.
The highest concentrations tested in the second mutation experiment were based on the results of the dose range finding test.
Mutagenicity assay I (3h treatment): 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/ml with and without S9-mix;
Mutagenicity assay II (24h treatment): 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 μg/ml without S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility test NEEDS ADAPTATION - SEARCH STUDY 514734

Untreated negative controls:

no

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:

no

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

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : single, the solvent control was tested in duplicate
- Number of independent experiments : ?

METHOD OF TREATMENT/ EXPOSURE:
- 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)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 15 days
- Method used: microwell plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 5 μg/ml trifluorothymidine (TFT), incubated for 11 or 12 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 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)
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
relative suspension growth (RSG) (see calculations in section "any other information on materials and methods incl. tables")
- Any supplementary information relevant to cytotoxicity:
Mutation Experiment 1
No toxicity was observed up to and including the highest tested dose level. To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 50 μg/ml was not selected for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 μg/ml exposure medium.
In the absence and presence of S9-mix, the relative total growth (RTG) was not decreased compared to the total growth of the concurrent solvent control group up to and including the highest precipitating dose level.

Mutation Experiment 2
No toxicity was observed up to and including the highest tested dose level. To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 25 μg/ml was not selected for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
0.2, 0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 μg/ml exposure medium.
The relative total growth (RTG) was not decreased compared to the total growth of the concurrent solvent control group up to and including the highest precipitating dose level

METHODS FOR MEASUREMENTS OF GENOTOXICIY ?

- OTHER: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 13 and 16 (3 hour treatment) and 61 and 71 (24 hour treatment)

Rationale for test conditions:

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).
In the absence and presence of S9-mix, the relative total growth (RTG) was not decreased compared to the total growth of the concurrent solvent control group up to and including the highest precipitating dose level

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH / osmolality: The pH and the osmolality of the culture medium containing the highest, non precipitating concentration were not determined. The highest non-precipitating dose level was 6.25 μg/ml.
- Possibility of evaporation from medium: no data (NOT POSSIBLE?)
- Water solubility: no data
- Precipitation and time of the determination: The test item precipitated in the culture medium at the concentration of 25 μg/ml
- Definition of acceptable cells for analysis: NOT APPLICABLE?
- Other confounding effects: /

RANGE-FINDING/SCREENING STUDIES (if applicable):
L5178Y mouse lymphoma cells were treated with a test item concentration range of 3.13 to 100 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: The relative suspension growth was 108 and 146 at the test item concentration of 25 μg/ml compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. The test item precipitated in the exposure medium at 25, 50 and 100 μg/ml. No toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 μg/ml compared to the suspension growth of the solvent control.
24h dose range finding experiment: The relative suspension growth was 96 at the test item concentration of 12.5 μg/ml compared to the relative suspension growth of the solvent control. The test item precipitated in the exposure medium at 12.5 μg/ml and upwards. No toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 μg/ml compared to the suspension growth of the solvent control.

STUDY RESULTS

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: In the absence and presence of S9-mix, the relative total growth (RTG) was not
decreased compared to the total growth of the concurrent solvent control group up to and including the highest precipitating dose level

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of 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.
- 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

Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Deviation to the study plan

In the first mutation experiment in the presence of S9-mix, the value of the cloning efficiency of one of the solvent controls was not within the acceptability criteria range of 65 and 120%.

Evaluation: The value of the cloning efficiency (125%) was above the upper limit of the range (120%). Since the value was just outside of the range and the value of the cloning efficiency (111%) of the other solvent control was within the range, this

deviation had no effect on the results of the first mutation experiment in the presence of S9-mix.

The study integrity was not adversely affected by the deviation.

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.
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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Bacterial reverse mutation assay

In a K1 key study (Tsuji, 2007), performed according to a method similar to OECD guideline 471, T003066 was evaluated for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains (TA98, TA100, TA1535 and TA1537) and in a gene of tryptophan-requiring Escherichia coli bacterial strain (WP2uvrA) in the presence and abscence of metabolic activation system (S9). The test item was dissolved in dimethyl sulfoxide.

In a dose range finding test, the test item was tested at a concentration range of 1.2 to 5000 µg/plate with and without S9 in triplicate. A total of 7 doses wer set with a common ratio of 4. In the main assay, the highest dose was set at 313 µg/plate, and a total of 5 doses were set with a common ratio of 2.

In the dose-finding and main assays, the test chemical did not increase the number of revertant colonies 2 -fold or more compared to the solvent control value both in the presence and absence of metabolic activation in any of the strains.

Positive control chemicals increased revertant colonies more than twice of the solvent control value of each strain, indicating the assays were performed properly. Based on these results, it was concluded that the test substance was not mutagenic under the test conditions of this study.

In the dose-finding and main assays, the growth inhibition of bacteria was not induced by the test chemical in the absence of the presence of metabolic activation.

White precipitates of the test chemical were observed by unaided eyes at:

4.9 µg/plate and higher in the absence of S9 and at 313 µg/plate and higher in the presence of S9 in the dose finding assay

20µg/plate and higher in the abscence of S9 and 156 µg/plate and higher in the presence of S9 in the main assay

Based on the results T003066 was found to be not mutagenic.

In a K2 supporting study (Sokolowski, 2009), performed according to a method similar to the OECD Guideline 471, T003066 was evaluated for its ability to induce reverse mutations at the histidine locus in four strains of Salmonella typhimurium (TA98, TA100, and TA102), in the presence and absence of a metabolic activation system (S9), according to the plate incorportion test (experiment I) and the pre-incubation test (experiment II).The test item was dissolved in dimethyl sulfoxide.

Each concentration and the controls were tested in triplicate. Due to irregular background growth in strains TA100 and TA102 in experiment I, this part was repeated under identical conditions (reported as part of experiment I). The test item was tested at the following concentrations:

- pre-experiment/experiment I: 3 -10 -33 -100 -333 -1000 -2500 -5000 µg/plate

- experiment II: 33 -100 -333 -1000 -2500 -5000 µg/plate

The plates incubated with the test item showed normal backgorund growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic events, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) occurred in strain TA98 with S9 mix at 2500 µg/plate and 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the three tester strains was observed following treatment with the test substance at any dose 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 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.

Based on the results T003066 was found to be not mutagenic.

In vitro micronucleus assay

A K2 supporting study equivalent to the OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test), was performed, non GLP compliant (Bohnenberger,2009). The test item dissolved in THF was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment II, the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.

In each experimental group two parallel cultures were set up. 1000 cells per culture were scored for micronuclei.

No influence of the test item on the pH value or osmolarity was observed.

In Experiment I in the absence of S9 mix test item precipitation was observed at the end of treatment at a concentration of 46.3 µg/mL and above and at 92.6 µg/mL and above in the presence of S9 mix. In Experiment II in the absence of S9 mix precipitation of the test item at the end of the treatment was observed at 185.3 µg/mL and above. In the presence of S9 mix no test item precipitation was observed at the end of treatment. However, test item precipitation at the start of treatment was observed at 185.3 µg/mL and above. Therefore, the highest applied concentration of 741.0 µg/mL and the first concentration (185.3 µg/mL), where test item precipitation was observed at the start of treatment as well as two non-precipitating concentrations (46.3 and 92.6 µg/mL) were evaluated for cytogenetic damage. In this study, no cytotoxicity was observed at the evaluated concentrations.

In both experiments no statistically significant and biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration. The rates of micronucleated cells after treatment with the test item (0.25 -1.35%) were close to the solvent control values (0.25 -1.25%) and within the laboratories historical control data range.

Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) and CPA (10.0 µg/mL) were used as positive controls and showed a distinct increase in the pêrcentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells in vitro in the absence and in the presence of metabolic activation.

The test substance is considered as non-mutagenic in this in vitro test system when tewsted up to precipitating test item concentrations.

Chromosome aberration:

A key study (K1, Eurlings, 2016) performed an in vitro chromosome aberration study in human lymphocytes according to OECD Guideline 473 and EU B.10.

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 S9-mix.

 

In the first cytogenetic assay, the test item was tested up to 31.3 μg/mL for a 3 hour exposure period with a 24 hour fixation time in the absence and presence of 1.8% S9-mix. Precipitation occured at 31.3 µg/mL.

In the second cytogenetic assay, the test item was tested up to 7.8 μg/mL for a 24 hour continuous exposure period with a 24 hour fixation time in the absence of S9-mix. Since no precipitate was observed at the highest dose level after 24h exposure, the test was aborted and the experiment was repeated as cytogenetic assay 2A. In the cytogenetic assay 2A, the test item was tested up to 125 μg/mL and precipitation occured at 5.6 µg/mL and above.

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.

under the conditions of the test, the test substance is considered not clastogenic.

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T003066 and according to the criteria of the CLP Regulation (EC) 1272/2008, T003066 should not be classified for mutagenicity.