thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key description in vitro

Key; M-000903-01-1; Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and E. coli WP2uvrA; 313 – 5000 µg/plate; negative with and without metabolic activation (S9 mix)

Key, M-611809-01-1; MNT (OECD 487); GLP; human primary lymphocytes; 13.2 – 2000 µg/mL; negative with and without metabolic activation (S9 mix)

Key, M-000799-01-1; HPRT (OECD 476); GLP; 9 – 1000 µg/mL; negative with and without metabolic activation (S9 mix)

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Dec 1994 - 7 Apr 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

less than 2 million cells were seeded, data was not pooled for data analysis

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells, provided by University of Ulm, Germany
- Suitability of cells: standard cell for this assay
- Normal cell cycle time (negative control): 10-14 h
For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: subculturing twice a week
- Cell cycle length, doubling time or proliferation index: 10-14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: cells were cultured in hypoxanthine-free Eagle's Minimal Essential Medium (MEM), supplemented with nonessential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHC03 , penicillin (100 units/mL), streptomycin (100 μg/mL) and heat-inactivated fetal calf serum (FCS, 10%). This medium is the culture medium. For treatment, the FCS was reduced to 2%. Cells were kept at 37 °C, 5% CO2.

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system was cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with Aroclor 1254. S9 fraction was purchased commercially.

The protein concentration of the S9 preparation was 37.4 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 40% v/v in the S9 mix. The cofactors in the final S9 mix were concentrated as follows: 8 mM MgCI2 x 6 H2O, 33 mM KCI, 5 mM Glucose-6-phosphate, 1 mM NADP in buffer.

The final concentration of S9 in the culture was 5% (S9 mix) and 1.6% (S9 preparation).

The S9 was tested for its capability to activate the positive control 7,12-dimethylbenzanthracene (DMBA) during the test.

Test concentrations with justification for top dose:

The concentrations in the main experiment were based on a pre-test. Nine concentrations were tested ranging from 9 to 1000 µg/mL for 5 h +/- S9 mix. Without S9 mix, cytotoxicity was not seen up to the highest concentration. Precipitation occurred at 500 μg/mL and above.

Therefore, the following concentrations were chosen for the main test: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
- Percentage of solvent in the final culture medium: 1 % (v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2 (- S9 mix), 3 (+ S9 mix)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4x10^6 cells per 250 mL flask (exposure), 1.5 x 10^6 cells (expression), 3x10^5 (selection)
- Test substance added in medium (2% FCS)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment: 13 - 14 days

FOR GENE MUTATION:
- Expression time: 7 days (subcultured once)
- Selection time: 6-7 days
- Fixation time: 13-14 days
- Selective agent: 6-TG, final concentration 10 μg/mL, 8 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment (relative cloning efficiency), relative population growth, the ability of cells to form colonies at the time of mutant selection (absolute cloning efficiency)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Mutation frequency = (number of mutation colonies * 100/number of evaluated dishes x 3 x 10^5 x CE)
with CE = cloning efficiency

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 476 version dated 1984, which was the standard at the time the study was conducted.

Evaluation criteria:

Acceptability
The assay is considered acceptable if it meets the following criteria
- the assay is performed twice
- at least 5 dishes per conditions are used to determine mutant frequency (better: 8)
- the numbers of mutant colonies per million cells found in the negative and/or solvent controls fall within the laboratory historical control data range
- the positive control substances produce a significant increase in mutant colonies (three times of
positive control)
- the average cloning efficiency of the negative and/or solvent controls must exceed 50%
- spontanous mutant frequency should be lower than 25x10^-6 cells
- absolute CE should be 10% or greater

An assay is considered positive if an increase in mutant frequencies is
- concentration-dependent (desirable over 3 concentrations, but at higher concentrations, reproducibiliy is sufficient to classify as mutagenic)
- significant (at least two to three times the mutant frequency of the solvent control)
- reproducible (seen in parallel cultures)

The results should be reproduced in the second experiment.

A test substance is considered equivocal if there is no concentration-dependency but one or more concentrations induced a reproducible, significant mutant frequency in all assays.

The assay is considered negative if none of the criteria described above are met (for a range of applied concentrations which extends to toxicity causing about 30% survival or less).

Statistics:

Mutation frequencies were submitted to weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. The weighted analysis of variance is followed by a Dunnett's test (nominal significance level of a = 0.05).
Mutation frequencies of 5 plates are too few to be considered for statistics. If the relative population growth was below 10%, the concentration is also discarded for evaluation.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated at 500 μg/mL and above. At 500 μg/mL (the highest soluble concentration), osmolality was not affected.

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was conducted with a series of 9 concentrations ranging from 9 μg/mL to 1000 μg/mL, with and without S9 mix. Cytotoxicity was not affected up to the highest concentration tested but precipitation occurred at 500 µg/mL and above. Based on these results, 6 concentrations were retained for the main experiments ranging from 15.6 μg/mL to 500 μg/mL both, with and without S9 mix.

CYTOTOXICITY:
No toxicity occurred up to the highest concentration tested, both with and without S9 mix.

STUDY RESULTS
Without metabolic activation:
No relevant increase in mutation frequencies was observed. At 62.5 μg/mL, an increased mutant frequency compared to the vehicle controls was observed but this was not reproducible, not concentration-related and did not exceed the range, which is typical of vehicle control variation between trials. Therefore, the finding was considered incidental.

With metabolic activation:
No relevant increase in mutation frequencies was observed. A slight increase occurred at 125, 250 and 500 μg/mL in one culture compared to the vehicle controls but this was not reproduced in the parallel culture or the other trials. The joint statistical assessment of the 3 trials resulted in no statistically significant increase of the mutation frequency over the concurrent vehicle controls at any of the tested concentrations

Solvent, negative and positive controls gave the expected outcome.

HISTORICAL CONTROL DATA
Historical control data was provided by the testing facility. The data came from 17 and 16 experiments with and without metabolic activation, respectively. The experiments were conducted from February 1994 to April 1995.

Mutation frequency of negative controls
- S9-activation 6.1 ± 3.8 (range 0.5 to 15.0)
+ S9-activation 5.7 ± 5.9 (range 0.5 to 28.8)
Mutation frequency of vehicle controls
- S9-activation 5.6 ± 4.4 (range 1.0 to 22.6)
+ S9-activation 5.0 ± 5.5 (range 0.5 to 26.7)
Mutation frequency of positive controls
- S9-activation (EMS) 887.3 ± 193.9 (range 531.1 to 1291.5)
+ S9-activation (DMBA) 93.4 + 42.2 (range 21.8 to 240.2)

Conclusions:

The present study was conducted according to the OECD guideline 476 dated 1984, under GLP conditions. Under the conditions of the assay, the test item did not induce any gene mutation at the HPRT locus in V79 cells, neither in the presence nor absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun - 21 Aug 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

S9 mix was only tested with 2-AA, no historical control data provided

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon (Salmonella strains), Trp operon (E.coli strain)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of male rats which received phenobarbital and 5-6-benzoflavone.

The S9 fraction was mixed with Cofactor-I immediately before testing operation to result in a final concentration of approx. 10 % v/v in the S9 mix. Each batch of S9 was routinely tested for its capability to activate the known mutagen 2-aminoanthracene in the Ames test.

Concentration of S9 mix and S9 in the final culture medium was 71.4% (S9 mix during pre-incubation) and 7.14% (S9 fraction during pre-incubation).

Test concentrations with justification for top dose:

Experiment I and II: 313, 625, 1250, 2500 and 5000 μg/plate (with and without metabolic activation)

Experiment I and II were pre-incubation tests.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Justification for percentage of solvent in the final culture medium: not reported

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

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

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in bacterial suspension for pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: bacterial growth inhibition

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

Mean numbers of the revertant colonies of each concentration of each test system was calculated and compared to that of the solvent control. If a concentration-related increase in revertant colonies was found that was more than two fold compared to the solvent control, it was evaluated as positive in terms of mutagenicity. No further statistical analysis was done.

Statistics:

No statistical analysis was done.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: crystallization was observed for all test strains at 5000 µg/plate

STUDY RESULTS
No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed in both experiments neither with nor without S9 mix. In both experiments (I and II), no substantial increase in number of revertant colonies was observed for any of the five tester strains used, at any concentration level applied, neither in the presence nor absence of metabolic activation.

The positive and solvent controls induced the expected results, confirming the suitability and sensitivity of the assay.

For results of both experiment I and experiment II, please refer to the attached background material (Attachment 1 and 2).

HISTORICAL CONTROL DATA
Not provided

Conclusions:

The study was performed according to OECD guideline 471 (as adopted 1983) and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic up to 5000 µg/plate in the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jul - 4 Sep 2017

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 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human blood was collected from healthy, non-smoking donors not receiving medication.
- Sex, age and number of blood donors: 2 male donors (27 and 33 years old)
- Whether whole blood or separated lymphocytes were used: whole blood was collected
- Whether blood from different donors were pooled or not: no, blood from the 27 year old donor was used for experiment I, and from the 33 year old donor for experiment II
- Mitogen used for lymphocytes: 2 % Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™ and supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL), for treatment, serum-free medium was used.
- CO2 concentration, humidity level, temperature: 37 °C with 5.5% CO2 in humidified air

Cytokinesis block (if used):

cytochalasin B (4 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared according to the SOP of the testing facility for liver S9 preparation from phenobarbital/β-naphthoflavone treated rats. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

The protein concentration of the S9 preparation was 32.2 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution. The cofactors in the final S9 mix are concentrated as follows: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 5% (S9 mix) and 0.75 mg/mL (S9 protein).

Test concentrations with justification for top dose:

A pre-experiment was conducted to determine the concentrations used in the main experiment. Cytotoxicity was assessed by a reduced cytokinesis-block proliferation index (CBPI) and expressed as cytostasis. Cytotoxicity should not exceed 55 ± 5 % cytostasis by counting 500 cells per culture. Since no cytotoxicity was seen in the pre-experiment, it was considered to serve as experiment I.
The concentrations used were therefore as follows:

Pre-experiment/experiment I:
4 h - S9, 13.2, 23.3, 40.6, 71.0, 124, 217, 380, 666, 1165, 2039 µg/mL
4 h, + S9, 13.2, 23.3, 40.6, 71.0, 124, 217, 380, 666, 1165, 2039 µg/mL

Experiment II
20 h, -S9, 16.4, 28.7, 50.2, 87.8, 132, 198, 296, 444, 667, 1000 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
- final percentage of solvent in the final culture medium: 1%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Pulse exposure 4 h (with and without S9-mix), continuous exposure 20 h (without S9 mix)
- Harvest time after the end of treatment (sampling/recovery times): for pulse exposures 16 h in test-substance free medium plus 20 h with Cytochalasin B (Cyto-B) in total ca 36 h, for continuous exposure no recovery time was scheduled, Cyto-B was added immediately after exposure with fresh serum-containing medium, so harvest time was 16 h after treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance, its concentration, and duration and period of cell exposure: Cyto-B was added to a final concentration of 4 μg/mL to the culture medium 20-22 h prior to harvest.
- Methods of slide preparation and staining technique: At the defined sampling time (40 h after beginning of treatment), cultures were centrifuged, the supernatant removed and the cells resuspended in 5 mL saline G. After an additional centrifugation, cells werere suspended in 5mL (hypotonic) 0.0375 M KCl at 37±1°C and incubated for 20 minutes. Cells were fixed by adding 1 mL of cold methanol/glacial acetic acid (19:1, v/v) into the KCl suspension. The fixative was changed by centrifugation and resuspension, which was repeated as necessary. For slide preparation, cells were again centrifuged and resuspended in a minimal amount of fresh fixative (twice for 20 minutes each). Several drops of cell suspension were spread onto clean microscope slides and stained with Giemsa.
- Number of cells spread and analyzed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture, so 2000 cells per concentration
- Criteria for scoring micronucleated cells (selection of analyzable cells and micronucleus identification): The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics to the main nuclei, and
2. Micronuclei were smaller than approximately one third the diameter of the main nuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI); 500 cells per culture were used. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Rationale for test conditions:

The tests were performed according to current regulatory test guidelines.

Evaluation criteria:

Acceptability
The assay was considered acceptable if the following criteria were met:
- The quality of the slides is sufficient for adequate cell scoring.
− The solvent control were within the laboratory historical solvent control data range.
− The positive controls produced a statistically significant increase in the micronucleus frequency and were be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control were considered to be acceptable.

Evaluation criteria
The test article was considered to give a negative result under the following conditions:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
− There is no concentration-related increase.
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data.

The test article was considered to give a positive result under the following conditions:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
− The increase is concentration-related in at least one experimental condition.
− The results are outside the range of the laboratory historical solvent control data.

If results are ambiguous, they should be reevaluated and if needed, additional cells can be scored.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05). A statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: cultured human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to clear at the highest concentration without and with metabolic activation, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no changes at maximum concentration
- Data on osmolality: osmolality at the highest concentration was 96.5% of the solvent control
- Precipitation and time of the determination: at the end of treatment, precipitation was observed in experiment I and II at 666/667 µg/mL and above (+/- S9 mix). Therefore, evaluation was performed up to and including 666/667 µg/mL.

PRE EXPERIMENT
No cytotoxicity was seen in the pre-experiment, it was considered to serve as experiment I.

STUDY RESULTS
In the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The values in fact, were within the following ranges:
4 h -S9 mix: 0.55 - 0.80%
4 h +S9-mix: 0.35 - 0.50%
20 h -S9-mix: 0.30 - 0.40%

and thus, they were within the historical control data ranges:
4 h -S9 mix: 0.08 - 1.12%
4 h +S9-mix: 0.16 - 1.08%
20 h -S9-mix: 0.12 - 1.03%


Concurrent vehicle negative and positive control data:
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition, which did not lead to statistically significant increase of micronuclei. All positive control compounds (CPA, MMC, Demecolcine) induced statistically significant increases in the proportion of cells with micronuclei. The study is therefore considered valid.

Summarized data can be found in Attachment 1.

CYTOTOXICTY
Moderate cytotoxicity was seen at the highest evaluated concentration without metabolic activation (cytostasis: exp. I 32.3%, exp. II 43.9%), with metabolic activation, clear cytotoxicity (cytostasis 61.6%) was observed at the highest evaluated concentration.

Conclusions:

The study was performed according to OECD guideline 487 and compliant with GLP. The test item was found not to induce micronuclei in cultured human peripheral blood lymphocytes neither in the presence nor absence of a metabolic activation system up to the concentration of 666/667 µg/mL, where precipitation occurred. The test item is therefore not considered to induce mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key description in vivo

Key, M-000775-01-1; MNT (OECD 474); GLP; mouse bone marrow cells; 50, 75 and 100 mg/kg bw; negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb - 21 Mar 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

intraperitoneal injection not recommended as exposure route.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 27 - 43 g
- Assigned to test groups randomly: yes
- Housing: singly in type I cages with bedding of soft wood granules, type S 8/15
- Diet: Altromin 1324 Standard Diet (Altromin GmbH, lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

0.5 % aqueous Cremophor emulsion

Details on exposure:

Preparation of dosing solutions:
The test substance was suspended in 0.5 % aqueous Cremophor emulsion, using sonication for 30 minutes. The substance was injected intraperitoneally. The administered volume was 10 mL/kg bw. The same conditions were used for the positive control substance cyclophosphamide that was dissolved in deionized water.

Duration of treatment / exposure:

single injection

Frequency of treatment:

once

Post exposure period:

Test substance: 16, 24, 48 h
Control animals: 24 h
Positive control: 24 h

Dose / conc.:

60 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): proven cytostatic agent and known clastogen
- Route of administration: injected intraperitoneally
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses were based on a pre-test in which groups of five animals received the test substance intraperitoneally at 10, 50, 75 and 100 mg/kg bw. For up to one hour, apathy, reduced motility, spasm, twitching, shivering and difficulty in breathing was observed in animals receiving 50 mg/kg bw or higher. In addition, 1 of 5 and 5 of 5 animals died in the 75 and 100 mg/kg bw groups, respectively. Therefore, the test substance was administered at a dose level of 60 mg/kg bw in the main test.

TREATMENT AND SAMPLING TIMES:
After the designated time, animals were sacrificed. At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). The bone marrow was flushed out with fetal calf serum. The serum-bone marrow suspension was centrifuged and most of the supernatant discarded. The pellet was resuspended in the remaining liquid to produce a homogeneous suspension.

DETAILS OF SLIDE PREPARATION:
One drop of the viscous suspension was placed on a well-cleaned slide and spread to allow proper evaluation of the smear. The slides were dried overnight and stained automatically with an Ames Hema- Tek Slide Stainer (Miles Company). The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

METHOD OF ANALYSIS:
Micronuclei were analyzed with a light microscope at a magnification of about 1000. 1000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. Additionally, the number of normochromatic erythrocytes showing micronuclei was assessed.

Evaluation criteria:

A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control at any of the time intervals investigated.

A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.

A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.

Acceptability
A test was considered acceptable if the solvent and positive control fall withing the expected range, in accordance with the laboratory's experience and/or the available literature data

Statistics:

When the negative control mean was superceded by any group, the test group with the highest mean and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. Significance was set at p below 5%.

The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided Chi 2-test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality and clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The doses were based on a pre-test in which groups of five animals received the test substance intraperitoneally at 10, 50, 75 and 100 mg/kg bw. For up to one hour, apathy, reduced motility, spasm, twitching, shivering and difficulty in breathing was observed in animals receiving 50 mg/kg bw or higher. In addition, 1 of 5 and 5 of 5 animals died in the 75 and 100 mg/kg bw groups, respectively.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: apathy, roughened fur, spasm and difficulty in breathing for up to 16 h after administration
- Mortality: 3/40 treated animals died during the test period
- Induction of micronuclei: no difference between control and treatment groups
- Ratio of PCE/NCE: no difference between control and treatment groups
- Positive control: the samples from the positive control had statistically significant increases in
the number of polychromatic erythrocytes compared with the concurrent control group which demonstrated that the test system was capable of detecting a known clastogen.

Summarized results can be found in Attachment 1.

- Historical control data: the negative and positive control are within the historical controls (refer to the attached background material 2).

Conclusions:

The study was performed according to OECD guideline 474 dated 1983 and compliant with GLP. In the present bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following an intraperitoneal injection of 60 mg/kg bw of the test item, in both male and female mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the endpoint summary. 

Additional information

 To characterize the mutagenic/genotoxic potential of the present test substance, several in vitro and in vivo studies have been conducted, almost according to currently acknowledged test guidelines. 

 

In-vitro testing

Bacterial mutagenicity

With respect to bacterial mutagenicity, the key study refers to an Ames test conducted according to OECD Guideline 471 (1983) and in compliance with GLP (M-000903-01-1). A standard battery of tester strains including 4 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537) and one Escherichia coli strain (WP2uvrA) was used. The test substance was completely soluble in Dimethyl sulfoxide (DMSO) and used at six concentrations (313, 625, 1250, 2500 and 5000 μg/plate) Two independent experiments were performed as pre-incubation experiments (both with and without metabolic activation by S9 mix). Precipitation was observed in the incubation medium at the highest concentration only. The test substance did not induce relevant increases in revertant colony numbers for any tester strain neither in the absence nor presence of metabolic activation. Further, no cytotoxicity was observed. Untreated, solvent and positive controls were considered as valid, but no historical control data was provided. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in E.coli WP2uvrA with and without metabolic activation.

 

An additional study served as a supporting study (M-000694-01-1). This study was an Ames test conducted according to OECD Guideline 471 (1983) and in compliance with GLP but with only four Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537). Two experiments were performed, the first being a plate-incorporation assay and the second a preincubation test. The plate incorporation assay served as a range finding experiment for the second experiment. In both experiments, six concentrations of the test substance were used (16, 50, 158, 500, 1581, 5000 µg/plate) and the assay was conducted with and without metabolic activation by S9 mix. No precipitation of the test item occurred. Bacteriotoxicty was only observed at the highest concentration tested as a weak, strain-specific effect. The plates could still be assessed for mutagenicity. The test substance did not induce relevant increases in revertant colony numbers for any tester strain neither in the absence nor presence of metabolic activation. Untreated, solvent and positive controls were considered as valid, since they were within the range of the historical control data. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation.

 

Cytogenetic assay in mammalian cells

In a key study (M-611809-01-1), the test substance was assessed for its potential to induce micronuclei in human lymphocyte in vitro (clastogenic or aneugenic activity) by a GLP-compliant study according to OECD Guideline 487 (2017). Human lymphocytes obtained from 2 donors were exposed to the test substance in the presence and absence of metabolic activation in two independent experiments. During the first experiment, cells were treated with 13.2 – 2000 µg/mL of the test substance for 4 h in the presence and absence of metabolic activation (S9 mix). After that, they were incubated in fresh medium for 16 h. In the second experiment, the cells were treated with the test item in the same concentration range for 20 h in the absence of a metabolic activation system. After treatment (and recovery), Cytochalasin B was added in experiment I and II for ca 20 h, followed by harvest of the cells. 1000 binucleate cells from each culture (2000 per concentration) were analyzed for micronuclei. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range. The positive control substances, mitomycin C, cyclophosphamide, and demecolcin led to the expected increase in the number of cells containing micronuclei. At the end of treatment, precipitation was observed in experiment I and II at 666/667 µg/mL and above (+/- S9-mix). Therefore, evaluation was performed up to and including 666/667 µg/mL. Without metabolic activation, moderate cytotoxicity was seen at 666/667 µg/mL, the highest evaluated concentration (cytostasis: 32.3% after 4 h + 16 h + 20 h, 43.9% after 20 h + 20 h). With metabolic activation, clear cytotoxicity (cytostasis 61.6%) was observed at 666 µg/mL, the highest evaluated concentration after 4 h + 16 h + 20 h. The test substance did not induced micronuclei in human peripheral blood lymphocytes neither in the presence nor in the absence of metabolic activation in experiment I or II. The test item is therefore not considered to induce mutagenic effects.

 A further GLP-compliant study was performed according to the OECD guideline 473 (1983) to determine clastogenic properties of the test substance by chromosomal aberration assay (M-000772-01-1). Since the number of metaphases was lower than recommended by the guideline, the study serves as a supporting study.

V79 cells were exposed to the test substance in the presence and absence of metabolic activation in concentrations of 75, 300 and 750 µg/mL for 4 hours. After treatment, cells were incubated for further 14 hours (all concentrations) or 26 hours (only high concentration and solvent control) in complete cell culture medium without the test substance for recovery. The concentrations were chosen based on a pre-experiment in which concentrations from 1 to 750µg/mL were tested for 4 h (+/- S9 mix) followed by a recovery period of 20 h. No precipitation of the test item occurred. At harvest time of 18 h, test substance treatment with metabolic activation resulted in reduced cell viability (73%). The mitotic index was not reduced after 18 h with metabolic activation but was reduced without metabolic activation. When cells were harvested after 30 h, cell viability without metabolic activation was reduced to 62.3% but no effect on the mitotic index occurred. No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 18 (with and without metabolic activation) or 30 hours (with and without metabolic activation) culture time. The positive controls were considered valid, as mitomycin C and cyclophosphamide had a clear clastogenic effect. In total, the test item is considered to not induce clastogenic effects in V79 cells in vitro. The study was considered valid to serve as a supporting study.

 

Two additional GLP compliant studies were performed in vitro. For these studies, no robust study summaries were included in the IUCLID data set since they are not recommended anymore by OECD guidelines. However, they will be described and discussed here for purpose of data completeness. One was a UDS study with rat hepatocytes (M-000790-01-1) and the other a Rec-assay in bacteria (M-009213-01-1). In the UDS assay, the cells were exposed to the test substance in concentrations from 75 µg/mL to 500 µg/mL in the presence of 10 µCi/mL3H-thymidine (13.6 Ci/mmole) for 16-24 h before slide preparation. 1.0 µg/mL 2-Acetylaminofluorene (2-AAF) served as the positive control. 150 cells per testing condition were assessed. Cytotoxicty was determined with trypan blue in additional cultures. No precipitation of the test item occurred. Cytotoxicty was observed at 450 and 500 µg/mL, all other concentrations and the positive control were non-toxic The number of net grains per nucleus, cells in repair and heavily labelled nuclei remained unaltered in all concentrations. Positive and solvent controls were acceptable. The study was conducted according to OECD guideline 482 which was deleted on 2ndof April 2014 . Under the experimental conditions reported, the test item did not induce any unscheduled DNA-synthesis.

In the Rec-Assay, the test substance was investigated for its DNA damaging effect in spores using Bacillus subtilis strains HI7 (Rec+) and M45 (Rec-) for DNA damaging effects. Concentrations ranging from 416 to 6660 µg/plate were tested in both strains with and without metabolic activation. Mitomycin C and 2-aminoanthracene were used as positive controls without and with metabolic activation, respectively. Spore suspensions were mixed with molten nutrient agar medium and poured into sterilized plates. Paper disks with the respective substance were put on the plate and incubated for 24 h. After the incubation, the diameter of inhibition zones appearing around the disk were measured. Growth inhibition was not observed for any test substance concentration. Positive and solvent controls were acceptable. Under the experimental conditions reported, the test item did not induce any DNA damaging effects.

 

Gene mutation in mammalian cells

The potential of the test item to induce genotoxicity in mammalian cells was determined in a study performed according to OECD Guideline 476 (1984) and GLP (M-000799-01-1).

A preliminary cytotoxicity test was conducted with a series of 9 concentrations ranging from 9 μg/mL to 1000 μg/mL, with and without S9 mix. No cytotoxicity was noted up to the highest concentration tested but precipitation occurred at 500 µg/mL and above. Therefore, the following concentrations were chosen for the main test: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL

In the main experiment, three and two tests were performed with and without metabolic activation, respectively. Therefore, V79 cells were treated with the test substance for 5 h. For all testing conditions, duplicate cultures were used to assess mutagenicity. The test substance induced no cytotoxicity under any test condition and did not produce any biologically relevant change in mutant frequency. Without metabolic activation, the only increase in mutant frequency was observed at 62.5 μg/mL, with metabolic activation, a slight increase occurred at 125, 250 and 500 μg/mL in one culture. However, none of these were considered treatment related and/or biologically significant because they were not reproducible, not concentration-dependent (without metabolic activation), did not exceed the range, which is typical of vehicle control variation between trials and/or did not result in statistical significance when all trials were jointly evaluated. The positive controls ethylmethanesulphonate and dimethylbenzanthracene revealed a clear mutagenic effect in all experiments. The vehicle and positive control mutant frequencies were all in the normal range of the historical control data. In total, the test item is considered to be non-mutagenic in the HPRT test using V79 cells with and without metabolic activation.

 

Cytogenetic assay in vivo

A key study (M-000775-01-1) is available to test the potential of the test substance to induce micronuclei in bone marrow cells of rats by a GLP-compliant study according to OECD Guideline 474 (1983). Groups of 5 male and female mice received a single intraperitoneal injection of the vehicle alone (negative controls) or 60 mg/kg bw of the test substance in 0.5% aqueous Cremophor emulsion. The dose was based on a pre-test in which groups of five animals received the test substance intraperitoneally at 10, 50, 75 and 100 mg/kg bw. For up to one hour, apathy, reduced motility, spasm, twitching, shivering and difficulty in breathing was observed in animals receiving 50 mg/kg bw or higher. In addition, 1 of 5 and 5 of 5 animals died in the 75 and 100 mg/kg bw groups, respectively. Therefore, the dose in this test was set at 60 mg/kg bw. An injection of 20 mg/kg bw cyclophosphamide in sterilized water served as the positive control.The animals were sacrificed 16, 24 and 48 h injection. For the vehicle and positive control, the time of sacrifice was 24 h after injection. Bone marrow cells were prepared for evaluation.

Apathy, roughened fur, spasm and difficulty in breathing were observed in treated animals for up to 16 h after administration. 3/40 treated animals died during the test period. Since no increase in micronuclei occurred and the ratio of polychromatic erythrocytes (PCEs) to normochromic erythrocytes (NCE) stayed stable, no cytogenicity could be evidenced.. Mice dosed with cyclophosphamide revealed statistically significant increases in the number of micronucleated polychromatic erythrocytes (PCEs) compared with the vehicle group. Both the vehicle and positive controls gave frequencies of micronucleated cells within the historical control data range. In total, there was no evidence of clastogenicity or aneugenicity following a single intraperitoneal injection of the test item at 60 mg/kg bw in male and female mice. Thus, the test substance was neither clastogenic nor aneugenic in the mouse bone marrow micronucleus assay.

 

Conclusion on genetic toxicity of the test substance

Several reliable in vitro and in vito studies are available. Neither mutagenicity nor genotoxicity could be evidenced with respect to the present test substance.  

 

Justification for classification or non-classification

The available data on mutagenicity and genotoxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and
are therefore conclusive but not sufficient for classification.