(3R,3AS,6AR)-HEXAHYDROFURO[2,3-B]FURAN-3-OL

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 Salmonella typhimurium strains TA98, TA100 and TA1535 and TA1537 and in Escherichia coli strain WP2 uvrA, performed according to OECD Guideline 471, it was concluded that T002675 has no mutagenic properties towards the bacterial strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

Chromosome aberration study

In a K1 in vitro chromosome aberration study in human lymphocytes, performed according to OECD Guideline 473, T002675 was considered to be non-clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

Mammalian cell gene mutation test

In a K1 in vitro mammalian cell gene mutation test with L5178y mouse lymphoma cells performed according to OECD Guideline 490, it was concluded that T002675 is not mutagenic in the mouse lymphoma L5178Y test system under the test conditions described in the report.

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:

2007-02-08 to 2007-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D.

Version / remarks:

dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 603T-1
- Expiration date of the lot/batch: 2007-12-19
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicqted
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

Target gene:

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Napthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9;
Experiment 2: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9;

Since the test item was soluble in deionised water up to the stanard limit concentration recommended in the regulatory guidelines that this assay followed (5000 µg/plate), the highest tested concentration in the Pre-experiment was 5000 µg/plate.
The highest tested concentration in the mutation experiment 2 was selected based on the toxicity of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties; no precipitation of the test substance occurred up to the highest investigated dose.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation; at 10 µg/plate (TA100 and TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation; at 10 µg/plate (TA98), at 50 µg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation; at 3 µL/plate (WP2uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation; at 2.5 µg/plate (TA1535, TA1537, TA98 and TA100); at 10µg/plate (WP2uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I - in agar (plate incorporation);
- Experiment I - plate incorporation:
In the plate incorporation assay, the following materials were mixed in a test tube and poured onto the selective agar plates for each dose level: 100 µL test solution, 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), and 2000 µL overlay agar (molten at 45 °C)
- Experiment II - preincubation:
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 shaken 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. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 60 min (experiment II)
- Exposure duration: at least 48 hours (experiments I and II)
- Selection time (if incubation with a selection agent): at least 48 hours (experiments I and II; simultaneous with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): at least 48 hours

SELECTION AGENT (mutation assays): histidine (TA98, TA100, TA1535, TA1537); tryptophan (Wp2uvrA)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.

Rationale for test conditions:

Solubility limitations: Since the test item was fully soluble in deionised water, the highest tested concentration for the Mutation assay was 5000 µg/plate.

Evaluation criteria:

- The test substance was 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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls, such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data was not mandatory.
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both the individual and mean values of the plates for each concentration, together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Species / strain:

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

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:

E. coli WP2 uvr A

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: < 60g/L
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
- No dose range-finding test was performed. However, 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 and 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. Based on the results of experiment I, 5000 µg/plate was selected as the highest test item concentration for experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
- in both experiments, the data in negative control, solvent control and positive controls were within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in both the plate incorporation and pre-incubation experiments. No toxic effects, evident as a substantial reduction in the mean revertant colony counts or by a sparse or absent background bacterial lawn, occurred in the test groups either with or without activation.

Remarks on result:

other: Experiment I and II

Conclusions:

Interpretation of results: negative

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. Therefore, T002675 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06 to 2017-09-12

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: jsoontje-06-0456
- Expiration date of the lot/batch: 2018-01-31(retest date)
- Purity test date: 2017-02-17

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: correction factor is 1.05

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
(2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was kept below 1 x 10^6 cells/mL.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5- medium).
For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20- medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R 20-medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone)

Test concentrations with justification for top dose:

Dose range finding test (3h treatment): 63, 125, 250, 500, 1000, 1301 μg/mL with and without S9-mix;
Dose range finding test (24h treatment): 63, 125, 250, 500, 1000, 1301 μg/mL without S9-mix;
Mutagenicity assay I (3h treatment): 16, 31, 63, 125, 250, 500, 1000, 1301 μg/mL with and without S9-mix;
Mutagenicity assay I A (3h treatment): 16, 31, 63, 125, 250, 500, 1000, 1301 μg/mL without S9-mix;
Mutagenicity assay II (24h treatment): 16, 31, 63, 125, 250, 500, 1000, 1301 μg/mL without S9-mix;
Mutagenicity assay II A (24h treatment): 16, 31, 63, 125, 250, 500, 1000, 1301 μg/mL without S9-mix;

Top dose justification: Based on the solubility test, DMSO was selected as vehicle and 1301 μg/mL was selected as the highest test item concentration in the dose range finding test. Based on the results of the dose range finding test, 1301 µg/mL was also selected as thee highest test item concentration in the mutagenicity assay I and II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in exposure medium. In DMSO, the test item was soluble at 1301 mg/mL.
The test item did not precipitate in the exposure medium up to and including the concentration of 1301 μg/ml (= 0.01 M). Based on these solubility findings, DMSO was selected as vehicle and 1301 μg/ml (= 0.01 M; the recommended top concentration in the guidelines) was selected as the highest test item concentration in the dose range finding test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix; at 15 µg/mL (3h treatment period), at 5 µg/mL (24h treatment period)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; at 7.5 µg/mL (3h treatment period)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 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: 3h or 24h
- Expression time (cells in growth medium): 48 (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays):
After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

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

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


DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG)

Rationale for test conditions:

Since the test item was insoluble in the exposure medium, the highest tested concentration for dose range finding test was 1301 μg/mL exposure medium.
The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/mL or 0.01 M (whichever is the lowest).

Evaluation criteria:

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

Statistics:

No statistical analysis

Key result

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: 7.46 compared to the concurrent solvent control 7.49
- Effects of osmolality: 0.446 Osm/kg compared to the concurrent solvent control 0.450 Osm/kg
- Water solubility: no data
- Precipitation: No precipitation was observed up to the concentration of 1301 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 63 to 1301 μg/ml in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period. Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1301 μg/ml compared to the suspension growth of the solvent control. Based on the results of the dose range finding test, 1301 μg/mL was selected as the highest test item concentration for the first and second mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database, except in the second experiment in which the mutation frequency of the solvent control cultures was just above the upper limit of the 95% control limits (151 an 152 per 106 survivors versus 95% upper control limit of 135 per 106 survivors). However, the observed mutation frequency of the solvent control cultures was still within the range of the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: 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 dose level

OTHER: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 16 and 28 (3 hour treatment) and 83 and 89 (24 hour 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 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-12 to 2007-07-18

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)

Version / remarks:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997

Deviations:

no

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:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 603T-1
- Expiration date of the lot/batch: 2007-12-019
- Purity: 97.76% (GC method)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water <60 g/L. Stability not indicated by the sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated by the sponsor


FORM AS APPLIED IN THE TEST (if different from that of starting material): The final concentration of deionised water in the culture medium was
10 % (v/v).

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Type and identity of media: The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) containing 10% FCS (fetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL), and HEPES (final concentration 10 mM).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used

Test concentrations with justification for top dose:

With respect to the molecular weight of the test item, 1300.0 μg/mL of TIC876 (T002675) (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 8.4 and 1300.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed before start and at the end of treatment. Since the cultures fulfilled the requirements
for cytogenetic evaluation, this preliminary test was designated Experiment I.

Range-finder/Experiment I (preparation interval: 22 hours):
• 4h exposure, with and without S9-mix: 0, 8.4, 14.8, 25.9, 45.3, 79.2, 138.6, 242.6, 424.5, 742.9 and 1300 µg/mL (0, 424.5, 742.9 and 1300 μg/mL were selected for metaphase analysis in the absence and presence of S9-mix.
• 22h continuous exposure, without S9-mix: 0, 8.4, 14.8, 25.9, 45.3, 79.2, 138.6, 242.6, 424.5, 742.9 and 1300 μg/mL (0, 424.5, 742.9 and 1300 μg/mL were selected for metaphase analysis in the absence of S9-mix).

Using reduced mitotic indices as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hrs treatment in the absence and presence of S9 mix. Therefore, the highest applicable test item concentration, 1300.0 μg/mL was chosen as top treatment concentration for Experiment II.

Experiment II (preparation interval: 46 hours):
• 46h continuous exposure without S9-mix: 0, 79.2, 138.6, 242.6, 424.5, 742.9 and 1300 μg/mL (0, 424.5, 742.9 and 1300 μg/mL were selected for metaphase analysis in the absence of S9-mix).
• 4h exposure with S9-mix: 0, 79.2, 138.6, 242.6, 424.5, 742.9 and 1300 μg/mL (0, 424.5, 742.9 and 1300 μg/mL were selected for metaphase analysis in the absence of S9-mix).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen due to 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 S9-mix: 37.5 μg/mL (0.133 mM) (22 hrs preparation interval) and 30.0 μg/mL (0.106 mM) (46 hrs preparation interval)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix: 825.0 μg/mL (6.65 mM) (22 hrs preparation interval); 660.0 μg/mL (5.32 mM) (46 hrs preparation interval); 495.0 μg/mL (3.99 mM) (22 hrs preparation interval)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Eposure time 4 hours:
About 50 - 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 (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany). 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 item. 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 item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once. After washing the cells were re-suspended in complete culture medium and cultured until
preparation.
- Exposure time 22 hours and 46 hours (without S9 mix)
About 50 - 80 hrs after seeding for each test group 2 blood cultures (10 mL each) are set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany). The culture medium was replaced with complete medium (with 10 % FCS) containing the test item 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: 4 and 22 hours (Experiment I); 4 and 46 hours (Experiment II)
- Expression time: 15 and 0 hours (Experiment I); 0 and 39 hours (Experiment II). Expression was halted three hours prior to cell harvest with the addition of the spindle inhibitor.
- Fixation time: 22 hours (Experiment I); 46 hours (Experiment II)

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 and 46 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 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).

STAIN (for cytogenetic assays): At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique, respectively Giemsa or according to the fluorescent plus Giemsa technique

NUMBER OF REPLICATIONS: Two parallel cultures were treated per experiment.

NUMBER OF CELLS EVALUATED: The slides were evaluated using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates (10). 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: mitotic index (at least 1000 cells were counted per culture)

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

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:

- The test substance was classified as non-mutagenic if: 1) the number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the historical control data (0.0 - 4.0% aberrant cells, exclusive gaps); and 2) no significant increase of the number of structural chromosome aberrations was observed.
- The test substance was classified as mutagenic if: 1) the number of induced structural chromosome aberrations was not in the range of the testing facility's historical control data (0.0 - 4.0% aberrant cells, exclusive gaps); and 2) either a concentration-related or a significant increase of the number of structural chromosome aberrations was observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 –1.5 % % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Key result

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant increase was observed (Exp. I: solvent control: pH 7.2; 1300 µg/mL: pH 7.2)
- Effects of osmolality: No relevant increase in osmolarity was observed (Exp. I: solvent control: 280 mOsm; 1300 µg/mL: 298 mOsm)
- Evaporation from medium: no data
- Water solubility: < 60 g/L
- Precipitation: No visible precipitation of the test substance in the culture medium was observed at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES:
- Experiment I was performed as a toxicity pre-test; however, since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated as Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
The proliferation index of the lymphocytes in solvent control cultures in the 22 hrs preparation interval with and without S9 mix (4 hrs treatment; 1.00 and 1.16, respectively), in the 22 hours preparation interval without S9 mix (continuous treatment; 1.06), and in the 46 hrs preparation interval with and without S9 mix (1.66 and 2.71, 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 and about > 1.5 times at the late preparation interval (Table 4; page 24). This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No relevant cytotoxicity, indicated by reduced mitotic indices was observed in either experimental part, up to the highest applied concentration.

Remarks on result:

other: all strains/cell types tested

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.4 %).

Conclusions:

Interpretation of results:
negative with and without metabolic activation

In conclusion, it can be stated that 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 in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity: in vitro

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay performed according to OECD Guideline 471, A. Sokolowski investigated the potential of T002675 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

On the day of the experiment, the test item was dissolved in deionised water.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

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

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

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, T002675 was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Chromosome aberration study

In a K1 in vitro chromosome aberration study in human lymphocytes performed according to OECD Guideline 473, S. Kunz investigated the potential of T002675 to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.

The test item was dissolved in deionised water.

In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in the study (1300.0 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

In the first experiment, the exposure periods were 4 hours with and without S9 -mix, with a 20 hours recovery period, and a 22 hours continuous exposure period without S9 -mix. In the second experiment, the exposure period was 46 hours continuous exposure without S9 -mix.

Since no relevant toxicity was observed in the study, the test item was tested up to the highest required test item concentration. Both in the presence and absence of S9 -mix, 424.5, 742.9 and 1300 µg/mL were selected for scoring.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromosome aberrations.

T002675 was concluded to be non-clastogenic in the chromosome aberration test when tested up to the highest required concentration in the absence and presence of metabolic activation.

 -  Mammalian cell gene mutation test

In a K1 mammalian cell gene mutation test performed according to OECD Guideline 490, Gijsbrechts J evaluated the mutagenic activity of T002675 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test item was dissolved in dimethyl sulfoxide at a concentration of 1301 μg/mL.

In the first mutation experiment, the test item was tested with a concentration range of 16 to 1301 μg/mL (=0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The treatment period was 3 hours. No significant toxicity was observed 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 dose level. No test item precipitation was observed up to the concentration of 1301 μg/mL.

In the second mutation experiment, the test item was tested again with a concentration range of 16 to 1301 μg/mL in the absence of S9-mix. The treatment period was 24 hours. No significant toxicity was observed, the relative total growth (RTG) of the highest test item concentration was 47% compared to the total growth of the concurrent solvent groups. No test item precipitation was observed up to the concentration of 1301 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 was concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

 

Justification for classification or non-classification

Based on negative results in all in vitro genetic toxicity tests with T002675 and the criteria of the CLP Regulation (EC) 1272/2008, T002675 should not be classified for mutagenicity.