1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

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 TA1535, TA 1537, TA98 and TA 100 and in Escherichia coli strain WP2 uvrA performed according to OECD guideline 471, it was concluded that the test item was not mutagenic in the absence and in the presence of S9 -mix.

In vitro micronucleus assay

An in vitro micronucleus assay in cultured peripheral human lymphocytes according to OECD guideline 487, it was concluded that the test item induces the micronuclei frequency in the absence of S9 -mix in binucleated cells only and is thus considered a clastogenic compound.

In vitro gene mutation study in mammalian cells

An in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro micronucleus assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro micronucleus study

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-11 to 2018-11-05

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:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

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: I18IB2522
- Expiration date of the lot/batch: 2019-03-19 (retest date)
- Purity test date: 2018-10-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: not indicated
- Solubility in vehicle: Acetone 150 g/L
- Stability in vehicle: Acetone Stable for 6 hours

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study. Test item concentrations were used within 1.5 hours of preparation.

Target gene:

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

The maximum final concentration for the dose range finding test was selected based on the solubility of the test item in acetone (highest concentration recommended in OECD test guideline).
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Mutation experiment 1: 17, 52, 164, 512, 1000 and 2500 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix
Mutation experiment 2: 154, 275, 492, 1000, 1400 and 2000 µg/plate in all tester strains with and without 10% (v/v) S9-mix

Since in the second mutation experiment in tester strain WP2uvrA no dose level with precipitation and/or cytotoxicity was observed in the presence of S9-mix an additional experiment was performed with this tester strain at the following dose levels:
Mutation experiment 2A: 2000, 3500 and 5000 μg/plate in the presence of 10% (v/v) S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in ethanol and THF at 50 and 100 mg/ml, respectively. In acetone, the test item was soluble at 100 mg/ml (= 5000 μg/plate). Based on these solubility findings and the stability findings of Test Facility Study No. 20161739, acetone was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; 5 μg/plate (TA1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix; 10 μg/plate (TA98)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 μg/plate (TA100)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9-mix; 10 μg/plate (WP2uvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5 μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2 μg/plate (TA100 with 10% S9-mix), 15 μg/ plate (WP2uvrA with 5 and 10% S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C.
The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.5 ml of a dilution of the test item in acetone and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation:
Mutation experiment 1: not observed at the start or at the end of the incubation period in any tester strain
Mutation experiment 2: not observed at the start of the incubation period but was observed at the end of the incubation period in tester strains TA1535 and TA1537 at the dose levels of 1000 and 1400 μg/plate in the absence of S9-mix and at 1400 μg/plate in the presence of S9-mix
Mutation experiment 2A: not observed at the start or at the end of the incubation period

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The dose range finding test results are reported as a part of mutation experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

- Cytoxicity:
Mutation experiment 1: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix at the two highest dose levels tested, except in tester strain TA98 in the presence of S9-mix, where toxicity was only observed at 2500 μg/plate.
Mutation experiment 2: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix at 1000, 1400 and/or 2000 μg/plate, except in tester strain WP2uvrA in the presence of S9-mix.
Mutation experiment 2A: Cytotoxicity was observed at all concentrations tested.

- Mutagenicity:
Mutation experiment 1: In tester strain TA1537 in the presence of S9-mix, the test item induced a 4.3-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was only observed at the mid dose level tested (512 μg/plate). Furthermore, this increase was within the historical control data range. Therefore, this increase is considered to be not biologically relevant. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested in any of the tester strains.
Mutation experiment 2: In tester strain TA1537 in the absence of S9-mix, the test item induced a 3.2-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was only observed at the mid dose level tested (492 μg/plate). Furthermore, this increase was within the historical control data range. Therefore, this increase is considered to be not biologically relevant. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested in any of the tester strains.
Mutation experiment 2A: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Conclusions:

Interpretation of the results: negative with and without metabolic activation
All bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase in the number of revertants in any of the experiments.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-10-17 to 2018-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: I18IB2522
- Expiration date of the lot/batch: 19 March 2019 (retest date)
- Purity: 100.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: No specific handling conditions required
- Solubility and stability of the test substance in the solvent/vehicle:
Solubility in vehicle: Acetone: 150 g/L
Stability in vehicle: Acetone: Stable for 6 hours

- OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study. The test item was dissolved in acetone. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. The final concentration of the solvent in the culture medium was 0.25% (v/v).

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy adult, non-smoking volunteers (aged 18-35)
- Suitability of cells: stimulated human lymphocytes were used because they are sensitive indicators of clastogenic and aneugenic activity of a broad range of chemicals
- Sex, age and number of blood donors if applicable: ages 26 and 32
- Whether whole blood or separated lymphocytes were used if applicable: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Methods for maintenance in cell culture if applicable:
Lymphocyte cultures: 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). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: AGT: 14.1h (age 32; dose-range finding study); AGT 13.8h (age 26; first cytogenetic assay); AGT: 14.2h (age 26; second cytogenetic assay).

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-in activated (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.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 53-97 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.4 -37.3°C).
- Properly maintained: not applicable
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically 'cleansed' against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

The highest tested concentration was determined by the solubility of the test item in the vehicle (highest achievable concentration).
Dose-range finding test: 23.4, 46.9, 93.8, 187.5 and 375 µg test item/ml culture medium (3 and 24 hours exposure time in the absence of S9-mix and for 3 hours in the presence of S9-mix).

The highest test concentration (375 μg/mL) was considered to be the limit of solubility. Based on the results of the dose range finding test, the following dose levels were selected for the cytogenetic assay:
First cytogenetic assay:
With S9-mix: 5, 25, 50, 175 and 375 µg/ml culture medium (3 hour exposure time, 27 hour harvest time).
Without S9-mix: 5, 25, 50, 175 and 375 µg/ml culture medium (3 hour exposure time, 27 hour harvest time).

Second cytogenetic assay:
Without S9-mix: 5, 175 and 375 µg/ml culture medium (3 hour exposure time, 27 hour harvest time).
With S9-mix : 5, 175 and 375 µg/ml culture medium (3 hour exposure time, 27 hour harvest time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Based on a stability study (Charles River Laboratories Study No. 20161739), the vehicle of the test item was acetone. In acetone, the test item was soluble at 150 mg/ml. A concentration of 200 mg/ml resulted in a non-homogeneous suspension. Based on these solubility findings, acetone was selected as vehicle and 375 µg/ml was selected as maximum concentration for the dose range finding test and is considered as the highest achievable concentration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.25% (v/v) acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9-mix; 0.25 and 0.38 µg/ml for a 3 hour exposure period and 0.15 and 0.23 µg/ml for a 24 hour exposure period

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.25 % (v/v) acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

Without S9 mix; 0.1 µg/ml for a 3 hour exposure period and 0.05 µg/ml for a 24 hour exposure period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.25 % (v/v) acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix; 15 and 17.5 µg/ml for a 3 hour exposure period.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 2 (A, B)
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium with Cytochalasin B (5 µg/ml) and incubated for another 24 hours.

Second cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item with cytochalasin B (5 µg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

TREATMENT AND HARVEST SCHEDULE: First cytogenetic assay: 3h exposure; Second cytogenetic assay: 24h exposure
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times): First cytogenetic assay: 27 hour harvest time; Second cytogenetic assay: 24 hour harvest time

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B (5 µg/ml). First cytogenetic assay: cell exposure during 24 hours after exposure to the test item; Second cytogenetic assay: during 24h simultaneously with exposure to the test item

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed.
Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately thereafter, ethanol / acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol / acetic acid fixative (3:1 v/v).
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 Test Facility 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 spread and analysed per concentration (number of replicate cultures and total number of cells scored): Three analysable concentrations were scored for micronuclei (2 cultures per concnetration).
At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per
culture were scored for micronuclei separately. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus

- Determination of polyploidy: not specified
- Determination of endoreplication: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium with Cytochalasin B (5 µg/ml) and incubated for another 24 hours.

Second cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item with cytochalasin B (5 µg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

DURATION
- Exposure duration: 3h (dose range finding test; first cytogenetic experiment) or 24h (dose range finding test; second cytogenetic experiment)
- Expression time (cells in growth medium): 24h (dose ranging test; first cytogenetic experiment) or no expression time (dose range finding test; second cytogenetic experiment)
- Fixation time (start of exposure up to fixation or harvest of cells): 27h (dose range finding test; first cytogenetic experiment) or 24h (dose range finding test; second cytogenetic experiment)

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

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68 ). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately thereafter, ethanol / acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol / acetic acid fixative (3:1 v/v).

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 Test Facility 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:
Three analysable concentrations were scored for micronuclei. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. The number of micronuclei per cell was not recorded.
Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number ofexamined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle
control.
b) The increase is dose-related in at least one experimental condition 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 or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher 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 exact test (one-sided, p < 0.05) was used to identify results significantly different from control group.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Possibility of evaporation from medium: no data
- Water solubility: no data
- Precipitation and time of the determination: No test item precipitation in the culture medium was observed up to the highest tested concentration.

RANGE-FINDING/SCREENING STUDIES (if applicable): Dose-related cytotoxicity was observed after 3 hours of exposure at 23.4 and 93.8 μg/ml culture medium and above in the absence and presence of S9-mix,
respectively and after 24 hours of exposure at 46.9 μg/ml culture medium and above.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The number of mono- and binucleated cells with micronuclei found in the solvent and negative control 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 binucleated cells with micronuclei.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the second cytogenetic assay. Furthermore, the number of mono- and binucleated cells with micronuclei 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.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:

In the first cytogenetic assay, the test item was tested up to 375 μg/ml for a 3-hour exposure period with a 27-hour harvest time in the absence and presence of S9-mix. No appropriate toxicity was reached at 375 μg/ml culture medium in the absence of S9-mix (cytostasis/cytotoxicity of 23% compared to concurrent vehicle control) or in the presence of S9-mix (cytostasis/cytotoxicity of 21% compared to concurrent vehicle control). No test item precipitation in the culture medium was observed up to the highest tested concentration. The highest test concentration (375 μg/mL) was considered to be the limit of solubility
The following dose levels were selected for scoring of micronuclei:
Without S9-mix: 5, 175 and 375 μg/ml culture medium (3 hour exposure time, 27 hour harvest time).
With S9-mix : 5, 175 and 375 μg/ml culture medium (3 hour exposure time, 27 hour harvest time).

In the second cytogenetic assay, the test item was tested up to 375 μg/ml for a 24-hour exposure period with a 24-hour harvest time in the absence of S9-mix. The highest test concentration was considered to be the limit of solubility.
No appropriate toxicity was reached at 375 μg/ml culture medium in the absence of S9-mix (cytostasis/cytotoxicity of 47% compared to concurrent vehicle control). No test item precipitation in the culture medium was observed up to the highest tested concentration. The highest test concentration (375 μg/mL) was considered to be the limit of solubility.
The following dose levels were selected for the scoring of micronuclei: 50, 175 and 375 μg test item/ml culture medium.

- Genotoxicity results
In the first cytogenetic assay, in the absence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In the presence of S9-mix, the test item induced dose-related and statistically significant increases in the number of binucleated cells with micronuclei. However, these increases were within the 95% control limits of the distribution of the historical vehicle control database. Since it was not possible to test up to concentrations showing the required cytotoxicity or test item precipitation at the end of treatment, the dose-related and statistically significant increases could be biologically relevant. Therefore, the results are considered equivocal.
In the second cytogenetic assay, the test item induced a dose-related and statistically significant increase in the number of binucleated cells with micronuclei. The statistically significant increase was above the 95% control limits of the distribution of the historical vehicle control database. This increase was considered biologically relevant.

Conclusions:

it is concluded that this test is valid and that the test item induces the formation of micronuclei in human lymphocytes in the absence of S9-mix after 24hour exposure to the test item. In the presence of S9-mix, the test item is considered equivocal for its ability to induce the formation of micronuclei. Since the test item induces the micronuclei frequency in the absence of S9-mix in binucleated cells only, it may be considered a clastogenic compound.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor 1254 induced rat liver metabolic activation system). The test item was dissolved in acetone at a concentration of 100 mg/ml.  In a dose range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The test item did not precipitate on the plates up to the concentration of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of  S9-mix at concentrations of 1600 and 5000 μg/plate. Results of the dose range finding test were reported as part of the first mutation experiment. Based on the results of the dose range finding test, the test item was tested in the first mutation experiment at a concentration range of 17 to 2500 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates up to the concentration of 2500 μg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix at concentrations of 1000 and 2500 μg/plate, except in tester strain TA98 in the presence of S9-mix, where toxicity was only observed at 2500 μg/plate.  In a second mutation experiment, the test item was tested at a concentration range of 154 to 2000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates in tester strains TA1535 and TA1537 at the dose levels of 1000 and  1400 μg/plate in the absence of S9-mix and at 1400 μg/plate in the presence of  S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix. Since in tester strain WP2uvrA no dose level with precipitation and/or cytotoxicity was observed in the presence of S9-mix an additional experiment was performed with this tester strain at the dose levels of 2000, 3500 and 5000 μg/plate in the presence of  10% (v/v) S9-mix. The test item did not precipitate on the plates up to the concentration of 5000 μg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed at all concentrations tested. The test item did not induce a biologically relevant and/or dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation in any of the experiments. The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro micronucleus study

An in vitro micronucleus assay was performed with the test item in cultured peripheral human lymphocytes (OECD 487; Buskens, 2019). The effect of the test item on the induction of micronuclei formed in cultured peripheral human lymphocytes in the presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and  ß-naphthoflavone) with a 3 hour exposure period and in the absence of S9 -mix with a 3 and 24 hour exposure period was investigated. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. The test item was dissolved in acetone. In the first cytogenetic assay, the test item was tested up to 375 μg/ml for a 3-hour exposure period with a 27-hour harvest time in the absence and presence of S9-mix. The highest test concentration was considered to be the limit of solubility. In the second cytogenetic assay, the test item was tested up to 375 μg/ml for a 24-hour exposure period with a 24-hour harvest time in the absence of S9-mix. The highest test concentration was considered to be the limit of solubility. The number of mono- and binucleated cells with micronuclei found in the solvent 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 binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system  (S9-mix) functioned properly. In the first cytogenetic assay, in the absence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.  In the presence of S9-mix, the test item induced dose-related and statistically significant increases in the number of binucleated cells with micronuclei. However, these increases were within the 95% control limits of the distribution of the historical vehicle control database. Since it was not possible to test up to concentrations showing the required cytotoxicity or test item precipitation at the end of treatment, the dose-related and statistically significant increases could be biologically relevant. Therefore, the results are considered equivocal. In the second cytogenetic assay, the test item induced a dose-related and statistically significant increase in the number of binucleated cells with micronuclei. The statistically significant increase was above the 95% control limits of the distribution of the historical vehicle control database. This increase was considered biologically relevant. It is concluded that this test is valid and that the test item induces the formation of micronuclei in human lymphocytes in the absence of S9-mix after 24-hour exposure to the test item under the experimental conditions described in this report. In the presence of S9-mix, the test item is considered equivocal for its ability to induce the formation of micronuclei.  Since the test item induces the micronuclei frequency in the absence of S9-mix in binucleated cells only, it may be considered a clastogenic compound.

In vitro gene mutation in mammalian cells

An in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro micronucleus study.

Justification for classification or non-classification

According to the criteria laid down in the CLP regulation EC 1272/2008, the test substance is considered classified as mutagenic category 2, based on the positive in vitro micronucleus test and absence of in vivo genotoxicity data.