clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in bacteria

WoE, M-103604-02-1, batch 1, Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 102; negative, both in absence and presence of metabolic activation (S9 mix).

WoE, M-036520-01-1, batch 2, Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and E. coli WPA; negative for TA 98, TA 100, TA 1537, and E. coli WPA in absence and presence of metabolic activation (S9 mix), but  positive for S. typhimurium TA 1535 at under the same conditions.

WoE, M-009769-02-1, batch 3 and batch 2, Ames (OECD 471); no GLP; S. typhimurium TA 1535, negative, both in absence and presence of metabolic activation (S9 mix).

WoE, M-009777-02-1, batch 4, Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 102; negative both in presence and absence of metabolic activation (S9 mix).

WoE, M-036420-01-1, batch 5, Ames (OECD 471, not mentioned but assumed); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and E. coli WPA; negative both in presence and absence of metabolic activation (S9 mix).

 

Chromosome aberration in mammalian cells

WoE M-105153-01-1, batch 4, CA (OECD 473), GLP, V79 cells; positive in the absence of S9 mix, however at precipitating concentrations and 45 - 52 % cell numbers in % of the controls.

WoE M-103614-01-1, batch 1, CA (OECD 473), GLP, V79 cells; negative, both in absence and presence of S9 mix.

WoE M-036479-02-1, batch 2, CA (OECD 473), GLP, CHL cells; positive, both in presence and absence of S9 mix.

 

Gene mutation in mammalian cells

WoE M-103610-01-1, batch 1, HPRT (OECD 476), GLP, V79 cells; negative, both in absence and presence of S9 mix.

WoE M-105147-01-1, batch 4, HPRT (OECD 476), GLP, V79 cells; negative, both in absence and presence of S9 mix.

WoE M-036462-02-1, batch 2, MLA (OECD 476), GLP, L5178Y cells; positive, both in presence and absence of S9 mix, but at concentration levels that were cytotoxic.

WoE M-009761-02-1, batch 2, HPRT (OECD 476), GLP, V79 cells; negative, both in absence and presence of S9 mix.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 May - 16 Jun 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

2-AA was used as the sole indicator of the efficacy of the S9 mix

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254 five days before sacrifice.

The protein concentration of the S9 preparation was 28.4 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. 70 mL cofactor solution are composed as follows: 162.6 mg MgCI2 x 6 H2O, 246.0 mg KCI, 179.1 Glucose-6-phosphate disodium salt, 315.0 mg NADP disodium salt in 100 mM phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 18.5% (S9 mix) and 1.8% (S9 fraction).

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.

Test concentrations with justification for top dose:

- Preliminary experiment I (plate incorporation): 16, 50, 158, 500, 1581, 5000 µg/plate

No bacteriotoxic or mutagenic effects were found up to the highest concentration. Therefore, the concentrations were used in the second experiment as well. 5000 µg/plate is recommended as the highest test concentration by the guideline.

- Experiment II (preincubation): 16, 50, 158, 500, 1581, 5000 µg/tube (all strains)
- additional test (preincubation): 16, 32, 48, 64, 80, 96, 110 µg/tube (only TA 102 with S9 mix)
The additional test was neccessary because the revertant count for TA 102 with S9 at 50 µg/tube was ca. 100 colonies higher than control colony count.

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:

sodium azide

cumene hydroperoxide

other: Nitrofurantoin (NF), TA 100, 0.2 µg/plate, -S9 4-Nitro-1,2-phenylene diamine (4-NPDA), TA 1537, 10 µg/plate, and TA 98, 0.5 µg/plate, -S9 2-Aminoanthracene (2-AA), all strains, 3 µg/plate, +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 (+1 additional test)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for pre-incubation test (experiment II and additional test)

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn, reduction in revertant colonies, reduction in titer

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

The substance was considered mutagenic when a reproducible and dose-related increase in mutant count was observed in any strain with and/or without metabolic activation. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. If results are questionable, investigation should continue.

Statistics:

Mean values and standard deviation were calculated. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

S. typhimurium TA 102

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: No precipitation of the test item was reported up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary plate incorporation experiment I, the concentration range of the test item was 16 - 5000 µg/plate. No bacterial mutagenicity could be evidenced for all tested S. typhimurium strains, both with and without S9 mix. No cytotoxicity was noticed at concentrations up to an including 5000 µg/plate

STUDY RESULTS
Bacteriotoxic effects were not observed up to and including 1581 µg/tube, titer reduction was seen occasionally at 5000 µg/tube. Since cytotoxicity was rather slight and only reported for some strains, assessment was not impaired.
No substantial increase in revertant colony numbers for any of the five strains investigated was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. In experiment II, TA 102 at 50 µg/tube with metabolic activation had about 100 revertant colonies more than the controls. This was not dose-dependent and the result could not be confirmed in the additional experiment with concentrations between 16 and 110 µg/tube, so that the finding is considered incidental. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. For summarized results of both experiment I and experiment II, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent and positive controls were within the range of the laboratory's historical control data. For details, please refer to Attachment 2 in the attached background material.

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. 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 TA 102 with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Nov - 22 Nov 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

only 1 strain was tested (TA 1535), 2 batches of the test material were used

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

yes

Remarks:

only 1 strain was tested (TA 1535), 2 batches of the test material were used

Principles of method if other than guideline:

The aim of present study was to compare the mutagenic potential of two different batches (batch 3 and batch 2) of the the test item, using Salmonella typhimurium TA 1535 als test system.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats which received single injection of 500 mg/kg bw Aroclor 1254 five days before sacrifice.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix.
The protein concentration of the S9 preparation was 28.4 mg/mL in all experiments.

70 mL cofactor solution are composed as follows: 162.2 mg MgCI2 x 6 H2O, 246 mg KCI, 179.1 mg Glucose-6-phosphate disodium salt, 315 mg NADP disodium salt in 100 mM sodium phosphate buffer (pH 7.4).

Each batch of S9 was routinely tested for its capability to activate the known mutagens (not specified) and 2-aminoanthracene in the Ames test.

The concentration of S9 mix in the final culture was 18.5% (S9 mix) and 2.7 % (S9 fraction).

Test concentrations with justification for top dose:

2 different batches of the test material were tested for purpose of comparison, referred to as batch 2 (analytical purity 96%) and batch 3 (analytical purity 98.6%).

Experiment IA (preliminary plate incorporation assay with batch 3, with and without S9 mix): 1000, 2000, 3000, 4000 and 5000 µg/plate
Experiment IB (preliminary plate incorporation assay with batch 2, with and without S9 mix): 3000, 5000 and 7000 µg/plate

Based on the results of the preliminary trial, the test concentrations were set as follows for the main trial.

Experiment IIA (main preincubation assay with batch 3, with and without S9 mix): 1000, 2000, 4000, 6000 and 8000 µg/tube
Experiment IIB (main preincubation assay with batch 2, with and without S9 mix): 1000, 2000, 4000, 6000 and 8000 µg/tube

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:

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for pre-incubation test (experiment II)

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and clearing or diminution of the background lawn as well as titer reduction

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

Assessment criteria
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants
exceeding the threshold of twice the colony count of the corresponding solvent control is observed. This should be reproducible and dose-related.


Acceptability
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the
following criteria:
- the spontaneous reversion rates in the negative and solvent control are in the range of the
historical data
- positive controls showed sufficient increase in mutant counts and
- Titer determinations had to demonstrate sufficient bacterial density in the suspension

Statistics:

Mean values and standard deviation were calculated. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at 5000 µg/plate and above (both batches) based on reduction of titer

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: Substance precipitation occurred at the highest tested concentration of 8000 µg/tube in the main trial.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary plate incorporation tests, the concentration range of the test item was 1000 - 5000 µg/plate and 3000 - 7000 µg/plate for batch 3 and 2, respectively. For both batches, no bacterial mutagenicity could be evidenced in the tester strain S. typhimurium TA 15335, both with and without S9 mix. No cytotoxicity was noticed up to 4000 µg/plate, whereas at the highest concentration levels of 5000 and 7000 µg/plate, a titer reduction was noticed.

STUDY RESULTS
With respect to the main experiments, IIA and IIB, no concentration-related and/or biologically relevant increase in revertant counts over negative controls could be evidenced of each of the tested batches. Further, there was no indication of cytotoxicity at test concentrations up to and including 4000/µg per tube. The total bacteria counts consistently were comparable to the negative controls. Higher test concentrations only were weakly cytotoxic and thus, were used for assessment purposes.
The results from the positive control substances were as expected, and thus, confirmed the suitability and sensitivity of the test system used.
For summarized results of both experiment I and experiment II, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent and positive controls were within the range of the laboratory's historical control data. For details, please refer Attachment 2 in the attached background material.

Conclusions:

The aim of present study was to compare the mutagenic potential of two different batches (different purity levels) of the test item, using Salmonella typhimurium TA 1535 als test system. The test conduct in present study followed the OECD guideline 471 but was not GLP compliant. Under the conditions of the assay, both batches of the test item were not mutagenic in S. typhimuirum strain TA 1535 with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

5 Aug - 13 Sep 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

2-AA was the sole control of efficacy for the S9 mix

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium), trp operon (E.coli)

Species / strain / cell type:

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

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats which received a single treatments of 500 mg/kg bw Aroclor 1254 (i.p.) five days before sacrifice.

The protein concentration of the S9 preparation was not reported.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 9% v/v in the S9 mix. 55 mL cofactor solution are composed as follows: 5 mL S9 preparation, 1 mL 1.65 M KCI/0.4 M MgCI2, 2.5 mL 0.1 M Glucose-6-phosphate, 2 mL 0.1 M NADP 25 mL 0.2 M sodium phosphate buffer (pH 7.4) and 14.5 mL sterile distilled water.

Concentration of S9 mix and S9 in the final culture medium were 18.5% (S9 mix) and 1.67% (S9 fraction).

Each batch of S9 was routinely tested for its sterility, and its ability to activate the known mutagen 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Pre-experiment: 50, 150, 500, 1500, 5000 µg/plate (only TA 100 and WP2uvrA)
No toxicity was observed, so that the concentrations used in the main experiments were 50, 150, 500, 1500 and 5000 µg/plate for both plate incorporation experiments I and II.

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:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene 2AA, 1 µg/plate for TA 100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA, 0.5 µg/plate for TA 98

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Pre-Experiment: test substance added in agar (plate incorporation, only for TA 100 and WP2 uvrA)
- Experiment I and II: test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

A test item is considered as a mutagen if a dose-related and statistically significant increase in the number of revertants is observed. The increase should exceed the threshold of twice the colony count of the corresponding solvent control and should occur at sub-toxic dose levels. If the two experiments give conflicting results, a third experiment should be performed.

Statistics:

Mean values and standard deviation were calculated. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

increase >2 fold of control colonies

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 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:

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:

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: No precipitation of the test item was reported up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed, so that the concentrations used in the main experiments were 50, 150, 500, 1500 and 5000 µg/plate for both plate incorporation experiments I and II.

STUDY RESULTS
No toxic effects, evident as a reduction in the number of revertants were observed in both experiments neither with nor without S9 mix. In strains TA 98, 100, 1537 and WP2uvrA, no substantial increase in revertant colony numbers was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. For TA 1535, 5000 µg/plate caused a slight but significant increase in colonies with and without metabolic activation. This increase was 1.6-fold (experiment I, without metabolic activation), 2.5-fold (experiment I, with metabolic activation), 1.9-fold (experiment II, without metabolic activation) and 2.1-fold (experiment II, with metabolic activation). However, the number of revertant colonies for TA 1535 at 5000 µg/plate remained equal or below the maximum number of revertant colonies as available from historical control data.

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

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent and positive controls were within the range of the laboratory's historical control data. For details, please refer to Attachment 3 in the attached background material under.

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA 1537 and in E.coli WP2 uvrA with and without metabolic activation. For S. typhimurium TA 1535, a reproducible and statistically significant increase in the frequency of revertant was noticed both, in the absence and presence of S9 mix. The finding however was comparatively stronger in presence of metabolic activation. The responses in the presence of S9 mix were consistent and achieved a two-fold increase over the concurrent solvent controls. Thus, based on the results obtained with S. typhimurium TA 1535, the test substance was considered mutagenic in bacteria.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Mar - 23 Apr 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

2-AA was the sole control of efficacy for the S9 mix, no historical control data was provided

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicological Studies", Japan's MAFF, 59 NohsanNo. 4200

Version / remarks:

January 28, 1985

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium), trp operon (E. coli)

Species / strain / cell type:

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

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received Phenobarbital (PB, 1 x 30 mg/kg bw and 3 x 60 mg/kg bw) and 5,6-benzoflavone (5,6-BF, 1 x 80 mg/kg bw) via intraperitoneal injection.

The protein concentration of the S9 preparation was 28.4 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. 1 mL cofactor solution are composed as follows: 16.3 mg MgCI2, 24.6 mg KCI, 17.0 Glucose-6-phosphate, 36.2 mg NADPH, 30.5 mg NADH, 119.6 mg Na2HPO4, 24.7 mg NaH2PO4, 0.1 mL S9 preparation, pH of the buffer was 7.4.

Concentration of S9 mix and S9 in the final culture medium were 18.5% (S9 mix) and 1.8% (S9 fraction).

Each batch of S9 was tested for its capability to activate the known mutagen 2-aminoanthracene and for sterility in the Ames test.

Test concentrations with justification for top dose:

- Experiment I: 100, 500, 1000, 2000, 5000 µg/plate (preliminary experiment)
No bacteriotoxicity was found in this test, so that 5000 µg/plate were used as the highest concentration in the main experiment (experiment II).
- Experiment II: 313, 625, 1250, 2500, 5000 µg/plate

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:

sodium azide

furylfuramide

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine, 2HC1(ICR-191), 1 µg/plate, TA 1537, -S9 2-Aminoanthracene (2AA), 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and 1537, 20 µg/plate for WP2uvrA, 0.5 µg/plate for TA 98, DMSO, +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/EXPOSURE
- Test substance added in agar for plate incorporation test (experiment I and II)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of mutants and clearing or diminution of the background lawn

Rationale for test conditions:

none given

Evaluation criteria:

Evaluation criteria
The test substance was considered mutagenic, when the number of revertant colonies increased more than twice that of the negative control in a dose dependent manner and the finding was reproducible.

Statistics:

The mean was calculated from the duplicates. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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: No precipitation of the test item was reported up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
The results of the preliminary experiment I showed that the number of revertant colonies was less than twice that of the solvent control, regardless of the presence and the absence of the metabolic activation system in all strains and at any dose levels of the test substance. No cytotoxicity was evident, neither with nor without S9 mix.


STUDY RESULTS
No cytotoxicty, evident as a reduction in the number of revertants was observed in the main experiment neither with nor without S9 mix. No substantial increase in revertant colony numbers of any of the five strains investigated was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. For results of both experiment I and experiment II, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
No historical control data was provided by the testing facility.

Conclusions:

The study was performed similarly to OECD guideline 471 and compliant with GLP. 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.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Sep - 17 Oct 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

2-AA was the sole control of efficacy for the S9 mix

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified from Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received triple treatments of 80 mg/kg bw phenobarbital i.p. and ß-naphthoflavone p.o. on consecutive days.

The protein concentration of the S9 preparation was 35.7 mg/mL in the both experiments. The S9 fraction was mixed with cofactors to final concentrations of: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium phosphate buffer (pH 7.4).
S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 15 % 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 were18.5% (S9 mix) and 2.77% (S9 fraction).

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate (with and without metabolic activation) - only strain TA 98 and 100. Since no toxic effects were observed in this pre-experiment, 5000 µg/plate was chosen as maximal concentration.

Experiment I and II: 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)

Experiment I was a plate incorporation test, experiment II was conducted as pre-incubation test.

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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD TA 1537 50 μg/plate and TA 98 10 μg/plate in DMSO; -S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for pre-incubation test (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 h
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and clearing or diminution of the background lawn

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Acceptability:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in colony count

Statistics:

Mean values and standard deviation were calculated. According to the OECD guideline 471, statistical analysis of the data is not mandatory.

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:

S. typhimurium TA 102

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: No precipitation of the test item occurred up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate and only in strains TA 98 and 100. Neither cytotoxicity nor genotoxicity was observed. Data can be found in Attachment 1 in the attached background material.

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. No substantial increase in revertant colony numbers of any of the five strains investigated was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation.
There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In experiment II, the negative and solvent control were slightly below the historical control data in strain TA 100 with metabolic activation. Since the alteration was small it is considered incidental. In both experiments, the positive control in TA 1535 and 1537 exceeded the historical control data without and with metabolic activation, respectively. This does not compromise the assay but is considered a sign of sensititivity of the assay.

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

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent, untreated and positive controls were within the range of the laboratory's historical control data. For details, please refer to the attached background material (Attachment 3) under "Attached background material"

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. 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 TA 102 with and without metabolic activation.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Aug - 23 Oct 1996 (main experiments)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

only 200 metaphases scored instead of 300as recommended by the actual guideline version

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by The Department of Health of the Government of the United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese Hamster Lung Cells, National Institute of Health Sciences - Cell Bank
- Suitability of cells: standard cells
- Normal cell cycle time (negative control): 12-16 h

For cell lines:
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: cultures were established approximately 24 h prior to treatment, 0.3 to 0.45 x 10E+6 cells were seeded per flask for the 6, 12 and 24 h time-points and 0.1 x 10E+6 cells were seeded per flask for the 48 h time-point.
- Cell cycle length, doubling time or proliferation index: 11 h
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: not reported
- Periodically ‘cleansed’ of spontaneous mutants: not reported

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's minimum essential medium, 10% fetal bovine serum and antibiotics, 37 °C, 5% CO2 in humidified air

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254 five days before sacrifice.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 50 % v/v in the S9 mix. The cofactors in the final S9 mix are concentrated as follows: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 5 mM NADP in 30 mM phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 10% S9 mix and 5% S9 preparation (1.23 mg/mL S9 protein).

Each batch of S9 was tested for its capability to activate the positive control cyclophosphamide in the assay.

Test concentrations with justification for top dose:

Experiment I: 4 h (8 h, + S9), 12 h (- S9): 156.25, 312.5, 625, 937, 1250, 1870 µg/mL
Experiment II: 4 h (8 h) and 12 h: 78.13, 156.25, 312.5, 625, 937.5, 1250 µg/mL (- S9 mix); 312.5, 625, 937.5, 1250, 1875 µg/mL (+ S9 mix)
24 h: 78.13, 156.25, 312.5, 625, 937.5, 1250 µg/mL (- S9 mix)
48 h: 39, 78.13, 156.25, 312.5, 625, 937.5 µg/mL (- S9 mix)

The test concentrations were based on a preliminary toxicity test in which cell cultures were treated for 12, 24 and 48 h with the test item without metabolic activation and for 6 h and 4 h with metabolic activation and a recovery time of 18 and 8 h, respectively. Concentrations ranged from 9.77 to 2500 µg/mL and cytotoxicity was assessed as the reduction of cell numbers. The test material precipitated at the highest concentrations.
A number of cells < 50% of the solvent control was observed after 48 h of exposure from 312.5 µg/mL on. However, also in the other conditions without S9 mix, cell numbers were decreased in a concentration-dependent manner (up to 62% after 4 h treatment, 8 h recovery, 70% after 6 h treatment, 18 h recovery, 62% 24 h, all at 1250 µg/mL). Cell count was not evaluated in the highest concentration due to precipitation. No metaphases were seen at 2500 µg/mL iunder any condition and only few metaphases were seen at 1250 µg/mL after 24 and 48 h exposure.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen based on its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.45 x 10^6 cells/flask for 12 h exposure -S9 and 4 h exposure +S9, 0.3 x 10^6 cells/flask for 6 (+/-S9) and 24 h (-S9) exposure, 0.1 x 10^6 cells/flask for 48 h treatment
Test substance added in medium: Eagle's Minimal Essential medium with Earle's Salts (MEM), supplemented with 10% fetal bovine serum and antibiotics

TREATMENT AND HARVEST SCHEDULE:
Experiment I: a) 12 h exposure (- S9), 0 h recovery b) 4 h exposure (+ S9 mix), 8 h recovery
Experiment II: a) 12 h exposure, 0 h recovery, 24 h exposure, 0 h recovery, 48 h, 0 h recovery, 6 h exposure, 18 h recovery (all -S9 mix), b) 4 h exposure (+ S9 mix), 8 h recovery and 6 h exposure (+ S9 mix), 18 h recovery

SPINDLE INHIBITOR
0.1 μg/mL colcemid was added 1 h prior to harvest

SLIDE PREPARATION AND METAPHASE ANALYSIS
- Methods of slide preparation and staining technique used including the stain used: At harvest, cells were treated with a hypotonic solution (0.075 M KCl) for 20 min at 37 °C. After that they were fixed in a mixture of methanol and glacial acid (3:1, changed several times). For preparation of metaphase spreads, cells were prepared onto microscope slides and and stained with 2% Gurrs Giemsa R66 (5 min).
- Number of cells spread and analysed per concentration: 100 cells in metaphase were analysed per culture (200 per test condition)
- Analysis of chromosome aberration: Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: mitotic index (MI, % cell in mitosis, calculated fromm 1000 cells), reduced cell numbers

Rationale for test conditions:

These were complying with the recommendations of the OECD test guideline 473 version dated 1983, which was referred to in the study report.

Evaluation criteria:

not reported

Statistics:

not reported

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity described as reduced cell number and reduced mitotic index

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: precipitation of the test item occurred at
- Influence on other factors: no influence on pH (change of 0.12 units) or osmolality was observed (within 50 mOsm limiting rate)

RANGE-FINDING/SCREENING STUDIES
In the preliminary cytotoxicity test a dose-related
increase in cell toxicity in most of the exposure groups could be evidenced, with precipitation noticed at the highest concentration level of 2500 µg/mL. Furthermore, assessment of the slides prepared from the preliminary cytotoxicity test revealed metaphases present at up to 1250 µg/mL in the 6 (18), 4 (8) and 12 h exposure groups and 625 µg/mL in the 24 and 48 h exposure cases.

CYTOTOXICITY
In experiment I and II, cytotoxicity was seen at similar concentrations, comparable to those of the preliminary toxicity experiment described above. Higher cytotoxicity was seen at higher exposure times. For example, in the 48 h treatment group, cell number was reduced to 50% at 315.5 µg/mL, in shorter exposure times, cytotoxicity was observed in higher concentrations of for example 1250 µg/mL (6 h treatment, 18 h recovery, - S9 mix). In general, mitotic indices were more sensitive to indicate cytotoxicity.

Summarized data can be found in Attachment 1 in the attached background material.

STUDY RESULTS
Experiment I
No increase in the frequency of chromosome aberrations or polyploid cells was seen in any treatment group.

Experiment II
High statistically significant increased frequencies of chromosomal aberrations were found in the highest concentration groups under the following test conditions: 6 h exposure, 18 h recovery + S9 mix (1875 µg/mL), 48 h exposure - S9 (625 µg/mL).
In contrast, rather small statistically significant increases were reported under the following conditions: 6 h exposure, 18 h recovery - S9 mix (1250 µg/mL), 12 h exposure (+/- S9 mix, 1562.5 and 937.5 µg/mL, respectively) and at 625 µg/mL in the 24 h treatment group. In the latter, the highest dose did not result in higher increases in chromosomal aberrations.
No increased polyploidy was found in any treatment group.

The positive and vehicle controls were acceptable. All positive controls resulted in increased chromosomal aberrations with the exception of the 12 h harvest time point (experiment I and II without S9 mix), which was considered typical for the 12 h time point and a result of toxicity-induced cell-cycle delay. The negative controls gave frequencies of aberrations within the range expected for the CHL cell line.

Summarized data can be found in Attachment 2 in the attached background material.

HISTORICAL CONTROL DATA
Historical control data can be found in Attachment 3 in the attached background material.

Conclusions:

The conduct of present study was compliant with the OECD 473 test guideline dated 1983 and followed GLP. Under the conditions of the assay, the test item induced clastogenic effects in CHL cells in vitro.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Sep - 12 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

only 200 metaphases scored instead of 300 as recommended by the actual guideline version.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

the top concentration in the vehicle (DMSO) was a test item suspension showing precipitation.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

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, Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, Germany)
- Suitability of cells: standard cell line

For cell lines:
- Absence of Mycoplasma contamination: yes, checked
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: subcultured twice weekly
- Cell cycle length, doubling time or proliferation index: 12 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: not reported

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Minimal Essential Medium with 10% fetal calf serum, 37 °C, n a humidified atmosphere with 1.5% carbon dioxide (98.5% air).

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received 80 mg/kg bw. Phenobarbital i.p. and beta-Naphthoflavone p.o. on three consecutive days.

The protein concentration of the S9 preparation was 34.6 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution. Cofactor solution was composed as follows: 8 mM MgCI2, 33 mM KCI,5 mM Glucose-6-phosphate disodium salt, 4 mM NADP in 100 mM sodium-ortho-phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 50 μl/mL (S9 mix) and 0.75 mg protein/mL (S9 fraction).

Each batch of S9 was tested for its capability to activate the known mutagen cyclophosphamide in the chromosome aberration test.

Test concentrations with justification for top dose:

Experiment I: 250, 500, 750, 1000, 1500, 2000 µg/mL (4 h exposure, +/- S9-mix)
Experiment II: 100, 200, 400, 600, 800, 1000 µg/mL (18 h exposure, - S9-mix) and
250, 500, 750, 1000, 1500, 2000 µg/mL (4 h exposure, + S9-mix) and
400, 600, 800, 1000 µg/mL (28 h exposure, - S9-mix)

The highest concentration was chosen according to the OECD guideline 473 and with regard to a pre-test . In the pre-test concentrations from 19.5 to 2500 μg/mL were tested for 4 (+/- S9 mix) and 24 h (- S9 mix) . Precipitation of the test item occurred from 1250 μg/mL (+/- S9 mix). Cytotoxicity was defined as reduced number of cells (< 50%). Without S9-mix, cytotoxicity was observed at 1250 µg/mL and above (24 h). With metabolic activation, no cytotoxicity was observed up to the highest concentration. Based on this results, 2000 μg/mL was chosen as the top dose in experiment I (4 h exposure). For 24 h exposure, 1000 μg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix and 2000 µg/mL in the presence of S9 mix.

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: 0.5 % (v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 with and without S9 mix, and an additional experiment conducted only without S9 mix

METHOD OF TREATMENT/ EXPOSURE
- Cell density at seeding: 10000 - 60000 cells per chamber
- Test substance added in medium: MEM with (-S9 mix) or without (+S9 mix) 10% FCS (v/v)

TREATMENT AND HARVEST SCHEDULE
- Exposure duration: 4, 18, and 28 h without S9 mix, 4 h with S9-mix
- Recovery time: 14 h (experiment I, +/- S9-mix), 0 h (experiment II, - S9 mix, both time points), 24 h (experiment II, + S9 mix)
- Preparation interval: 18 h (experiment I and II), 28 h (experiment II)


SPINDLE INHIBITOR
0.2 μg/mL colcemid was added approx. 2.5 h prior to harvest

SLIDE PREPARATION AND METAPHASE ANALYSIS
- Methods of slide preparation and staining technique used including the stain used: At harvest, cells were treated with a hypotonic solution (0.4% KCl) for 20 min at 37 °C. After that they were fixed in a mixture of methanol and glacial acid (3:1) and stained with Giemsa.
- Number of cells spread and analysed per concentration: 100 cells in metaphase were analysed per slide (in total 200 metaphases per concentration)
- Analysis of chromosome aberration: Breaks, fragments, deletions, exchanges, and chromosome
disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI, % cell in mitosis), reduced cell numbers (therefore, two additional cultures per test item were prepared but not treated with colcemid. Cell number of treatment groups is given in percent to the solvent control)

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 473 version dated 1997, which was the standard at the time the study was conducted.

Evaluation criteria:

Acceptability:
An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the negative control rate was in the expected range according to the laboratory's experience.

Assessment criteria:
A test item was considered clastogenic if a concentration-related and/or significant increase in the chromosome aberration rate occurred that was out of the historical control data (0.0-4.0% aberrant cells, excluding gaps).
A test item was considered non-clastogenic if there was no such increase at any concentration tested.

Moreover, a test item is considered aneugenic if the number of induced numerical aberrations is not in the range of the test laboratorys historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

The Fisher’s exact test (p < 0.05) was used for statistical evaluation and statistical significance was discussed regarding biological significance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was seen after 18 and 28 h of treatment without metabolic activation and after 4 h treatment at concentration within precipitation range

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: precipitation of the test item occurred at 1000, 1500 and 2000 μg/mL (+/-S9 mix, experiment I) and 750, 1000, 1500 and 2000 μg/mL (+ S9 mix, experiment II)
- Influence on other factors: no influence on pH or osmolality was observed (exp. I: solvent control
407 mOsm, pH 7.3 versus 380 mOsm and pH 7.2 at 2000 μg/mL)
- Evaluated experimental points: 750, 1000, 1500 μg/mL (+/-S9 mix, experiment I), 200, 400, 600 µg/mL (- S9 mix, experiment II), 500, 750, 1000 μg/mL (+ S9 mix, experiment II), 600 µg/mL (- S9 mix, experiment III, higher concentrations could not be tested because of strong toxic effects, seen as reduced cell numbers and low metaphase numbers, also see below)

RANGE-FINDING/SCREENING STUDIES
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 h of treatment with 1250 μg/mL and above in the absence of S9 mix. In the presence of S9 mix no clear cytotoxicity was observed up to the highest test item concentration. Considering inhomogeneity of the stock solution, the toxicity data and the precipitation observed in the pre-test, 2000 μg/mL was set as top test concentration for the first main experiment (Experiment I) with and without S9 mix. With respect to the second main experiment (Experiment II), the pre test also showed clearly reduced cell numbers after 24 h of treatment with 625 and 1250 μg/mL. Therefore, 1000 μg/mL was retained as top test concentration for continuous exposure in the absence of S9 mix.

CYTOTOXICITY
Cytotoxicity was defined as reduced cell numbers below 50% of the solvent control or reduced mitotic indeces. This occurred only at 18 and 28 h continous treatment without metabolic activation. At 18 h and 400 µg/mL treatment, cell numbers were reduced to 41% of the control, and to 51% after 28 h of 600 µg/mL treatment. Cytotoxicity in the 4 h exposure scenario (experiment I) was only observed in concentrations where the test item preticipated (2000 µg/mL).

STUDY RESULTS
Experiment I
No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed with and without metabolic activation. Only one statistically significant increase was seen at 1500 μg/mL in the presence of S9 mix (3.0%, p < 0.05) but this was in the range of the historical control data (0.0-4.0%) and similar to the response of the negative control in experiment I (3.0 %) and therefore not biologically relevant.

Experiment II
No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed with and without metabolic activation. Only one statistically significant increase was seen at 1000 μg/mL in the presence of S9 mix (3.0%, p < 0.05) but this was in the range of the historical control data (0.0-4.0%) and similar to the response of the negative control in experiment I (3.0%) and therefore not biologically relevant.

No biologically relevant increase occurred in the rate of polyploid metaphases after treatment with the test item (1.3 - 4.2%) as compared to the rates of the solvent controls (1.6 - 2.5%).

- Concurrent vehicle negative and positive control data:
The positive controls mitomycin C and cyclophosphamide resulted in a clear and statistically significant rise in aberration rates.

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Historical control data can be found in Attachment 2 in the attached background material.

Conclusions:

The present study was conducted according to the OECD guideline 473 dated 1997, and under GLP conditions. Under the conditions of the assay, the test item did not induce any clastogenic effects in V79 cells in vitro, both in the absence and presence of S 9 mix.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Sep - 18 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

only 200 metaphases scored instead of 300

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

the top concentration in the vehicle (DMSO) was a test item suspension showing precipitation.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79, Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, Germany
- Suitability of cells: standard cell line
- Normal cell cycle time (negative control): 12 h

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: not reported
- Cell cycle length, doubling time or proliferation index : 12 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: not reported

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: 37° C, in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).MEM (Minimal Essential Medium); supplemented with 10 % fetal calf serum (FCS)

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received 80 mg/kg bw Phenobarbital i.p. and beta-Naphthoflavone p.o. on three consecutive days.

The protein concentration of the S9 preparation was 34.6 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution. Cofactor solution was composed as follows: 8 mM MgCI2, 33 mM KCI,5 mM Glucose-6-phosphate disodium salt, 4 mM NADP in 100 mM sodium-ortho-phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 50 µl/mL (S9 mix) and 0.75 mg protein/mL (S9 fraction).

Each batch of S9 was tested for its capability to activate the known mutagen cyclophosphamide in the chromosome aberration test.

Test concentrations with justification for top dose:

Without S9 mix: 250, 500, 750, 1000, 1500, 2000 µg/mL *
With S9 mix: 250, 500, 750, 1000, 1500, 2000 µg/mL

The highest concentration was chosen according to the OECD guideline 473 and with regard to a pre-test (not detailed reported here). In the pre-test concentrations from 19.5 to 2500 µg/mL were tested for 4 (+/- S9 mix) and 24 h (- S9 mix) . Precipitation of the test item occurred from 1250 µg/mL (+/- S9 mix). Cytotoxicity was defined as reduced number of cells (< 50%). Without S9-mix, cytotoxicity was observed at 625 and 2500 µg/mL (4 h) and at 1250 and 200 µg/mL (24 h). With metabolic activation, no cytotoxicity was observed up to 2500 µg/mL. Based on this results, 2000 µg/mL was chosen as the top dose in this experiment. Since the test item was considered to be clastogenic after 4 h treatment a second experiment was not performed.

* the experiment had to be repeated (same concentrations) because in the first trial the negative, solvent and positive controls were not evaluable

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: DMSO was chosen based on its solubility properties and its relative nontoxicity to the cell cultures.
- Percentage of solvent in the final culture medium: 0.5% (v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates (cells were seeded into at least 2 chambers of Quadriperm dishes with microscopic slides)
- Number of independent experiments: 1 (only one experiment as recommended by the test guideline, since the test item was considered to be mutagenic after 4 hrs treatment.)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10000 - 60000 cells per chamber
- Test substance added in medium: MEM with (-S9 mix) or without (+S9 mix) 10% FCS (v/v)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h, after that the cells were washed twice and cultured in complete medium
- Recovery time: 14 h
- Harvest time: 18 h after beginning of treatment

SPINDLE INHIBITOR
0.2 μg/mL Colcemid was added approx. 2.5 h prior to harvest

SLIDE PREPARATION AND METAPHASE ANALYSIS
- Methods of slide preparation and staining technique used including the stain used: At harvest, cells were treated with a hypotonic solution (0.4% KCl) for 20 min at 37 °C. After that they were fixed in a mixture of methanol and glacial acid (3:1) and stained with Giemsa.
- Number of cells spread and analysed per concentration: 100 cells in metaphase were analysed (for 500 µg/mL + S9-mix: 200 metaphases analysed)
- Analysis of chromosome aberration: Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: mitotic index (MI, % cell in mitosis), reduced cell numbers (therefore, two additional cultures per test item were prepared but not treated with colcemid. Cell number of treatment groups is given in percent to the solvent control)

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 473 version dated 1997, which was the standard at the time the study was conducted.

Evaluation criteria:

Acceptability:
An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the negative control rate was in the expected range according to the laboratory's experience.

Assessment criteria:
A test item was considered clastogenic if a concentration-related and/or significant increase in the chromosome aberration rate occurred that was out of the historical control data (0.0-4.0% aberrant cells, excluding gaps).
A test item was considered non-clastogenic if there was no such increase at any concentration tested.

Moreover, a test item is considered aneugenic if the number of induced numerical aberrations is not in the range of the test laboratorys historical control data (0.0 - 8.5% polyploid cells).

Statistics:

The Fisher’s exact test (p < 0.05) was used for statistical evaluation and statistical significance was discussed regarding biological significance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

clastogenic at concentrations of 1500 and 2000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic effects were observed at 2000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at concentrations of 1500 µg/mL and above

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: precipitation of the test item occurred at 1500 and 2000 µg/mL (-S9 mix) and 750, 1000, 1500 and 2000 µg/mL (+S9 mix)
- Influence on other factors: no influence on pH or osmolality was observed (main
experiment: solvent control 391 mOsm, pH 7.2 versus 392 mOsm and pH 7.2 at 2000 µg/mL).
- Evaluated experimental points: 750, 1000, 1500, 2000 µg/mL (-S9 mix), 500, 750, 1000 µg/mL (+S9 mix, higher concentrations could not be tested because of strong toxic effects, seen as reduced cell numbers and low metaphase numbers, also see below)

RANGE-FINDING/SCREENING STUDIES
Clear toxic effects were observed after 4 h treatment with 625 and 2500 µg/mL in the absence of S9 mix. In the presence of S9 mix no clear toxic effects were observed up to the highest test item concentration. After 24 h continuous treatment with 1250 µg/mL and above in the absence of S9 mix clear toxic effects were observed. Taking into account the inhomogeneity of the stock solution in DMSO in the pre-experiment, the highest applicable concentration for the cytogenetic experiment was set at 2000 µg/mL.

CYTOTOXICITY
Cytotoxicity was defined as reduced cell numbers below 50% of the solvent control. This occurred at 4 h treatment with 2000 µg/mL (-S9 mix). With metabolic activation, reduced mitotic index was observed in the precipitating concentration range only (1500 and 2000 µg/mL). These data points were excluded from the evaluation.

STUDY RESULTS
Without S9 mix
A dose-related increase of chromosome aberrations occurred from 1000 µg/mL on compared to solvent controls. At 1500 and 2000 µg/mL, this was statistically and biologically significant (8.0% and 24.0%, respectively) compared to solvent controls. Also, exchanges were increased in a dose-related manner from 750 (0.0%) to 1000 (1.0%), 1500 (5.5%) and 2000 µg/mL (10.5%), all compared to solvent controls.

With S9 mix
No biologically relevant increase in chromosome aberrations was observed compared to solvent controls. A statistically significant increase was observed at 500 µg/mL (3.8%, p< 0.05). However, this was not considered biologically significant since it was within the historical control data (0.0-4.0%). Also, more metaphases had to be scored for this data point (200 per slide compared to 100 per slide) since results were inhomogeneous at 100 metaphases per slide. All treatment data was therefore close to the solvent control (1.5 - 3.8% aberrant cells, exclusive gaps compared to 0.5% aberrant cells, exclusive gaps in the solvent control).

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.0 - 3.9 %) as compared to the solvent controls (1.3 %, each).

- Concurrent vehicle negative and positive control data:
The positive controls mitomycin C and cyclophosphamide resulted in a clear and statistically significant rise in aberration rates.

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Historical control data can be found in Attachment 2 in the attached background material.

Conclusions:

The present study was conducted according to the OECD guideline 473 dated 1997 and under GLP conditions. Under the conditions of the assay, the test item induced clastogenic effects in V79 cells in vitro in the absence of S9 mix. However, these effects occured at concentrations, at which the test item precipitated and cell numbers were 45 - 52% of the solvent control.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Sep - 11 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 cultured during the expression period and plated for mutant selection, mutant frequency not corrected for cloning efficiency

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

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, Laboratory for Mutagenicity Testing; Technical University, Darmstadt, Germany)
- Suitability of cells: standard cell line for the assay
- Normal cell cycle time (negative control): 12-16 h

For cell lines:
- Absence of Mycoplasma contamination: each batch was screened for mycoplasma contamination
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: subcultured twice weekly
- 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: MEM
(minimal essential medium), supplemented with 10% fetal calf serum (FCS), 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received 80 mg/kg bw Phenobarbital i.p. and beta-Naphtoflavone p.o. on three consecutive days.

The protein concentration in the S9 preparation was 26.2 mg/mL in the pre-experiment and 34.6 mg/mL in experiment I.

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, 5 mM NADP in 100 mM sodium-orhto-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:

The concentrations in the main experiment were based on a pretest.
No cytotoxicity was observed up to the highest concentration but precipitation of the test item occurred at 625 µg/mL and above in the presence and absence of metabolic activation. pH and oasmolarity remained stable. Based on these results the concentrations for the main experiment were chosen as follows:

Experiment I:
Without S9 mix: 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL
With S9 mix: 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL

Experiment II
Without S9 mix: 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL

78.1 µg/mL in experiment I and II and 156.3 µg/mL in experiment I were not evaluated after day 4 since four concentrations are required by the guideline.

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: 0.5 % (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: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5x10^6 (single culture, for mutation rate) and 5x10^2 cells (in duplicate, for toxicity)
- Test substance added in medium (without FCS for 4 h exposure, for 24 h with FCS for experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment I: 4 h, Experiment II: 24 h
- Harvest time after the end of treatment (sampling/recovery times): approximately 18 days

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days
- Fixation time: approximately 18 days
- Selective agent: 6-TG, final concentration 11 µg/mL, 8 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment (cloning efficiency I), relative population growth and cloning efficiency (CE II, viability, cloning efficiency determined after the expression period to measure viability of the cells without selective agent, absolute and relative)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- number of mutant colonies per per 10^6 cells , induction factor (mutant colonies per 10^6 cells / mutant colonies per 10^6 cells of the corresponding solvent control)

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 476 version dated 1997, 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 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 significanincrease in mutant colonies
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50%

Evaluation
An assay is considered positive if
A a concentration-dependent and reproducible increase in mutant frequency is observed. The mutagenic response should be at least three times that of the negative controls in at least one concentration.

An assay is considered negative if there is no such concentration-dependent or reproducible increase.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an
adequate statistical method is not available.

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 1250 and 2500 µg/mL under all experimental conditions.

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment increasing precipitation occurred at 625 μg/mL and above in the presence and absence of S9 mix at both treatment intervals of 4 and 24 hours. No cytotoxicity was observed. The concentration range of the main experiments was based upon the results generated in the pre-experiment.

CYTOTOXICITY:
No toxicity occurred up to the highest concentration tested , both, in presence and absence of S9 mix.

STUDY RESULTS (MAIN TESTS)
Experiment I:
No relevant increase in mutation frequencies was observed. At 1250 µg/mL without metabolic activation, the mutant frequency was increased in one of the duplicate cultures (above the historical control data). However, the induction factor did not reach the threshold of three times the corresponding solvent control and the effect could not be reproduced in the second experiment. Therefore it was considered incidental.

Experiment II:
No relevant increase in mutation frequencies was observed.

Solvent, negative and positive controls gave the expected outcome.

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Histroical control data was provided by the testing facility in a separate statement (M-581971-01-1) and can be found Attachment 2 in the attached background material.

Conclusions:

The present study was conducted according to the OECD guideline 476 dated 1997, 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.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 Sep - 18 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 cultured during the expression period and plated for mutant selection, mutant frequency not corrected for cloning efficiency

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

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, Laboratory for Mutagenicity Testing; Technical University, Darmstadt, Germany)
- Suitability of cells: standard cell line for the assay
- Normal cell cycle time (negative control): 12-16 h

For cell lines:
- Absence of Mycoplasma contamination: each batch was screened for mycoplasma contamination
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: subcultured twice weekly
- 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: MEM (minimal essential medium), supplemented with 10% fetal calf serum (FCS), 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received 80 mg/kg bw Phenobarbital i.p. and beta-Naphtoflavone p.o. on three consecutive days.

The protein concentration in the S9 preparation was 26.2 mg/mL in the pre-experiment and 34.6 mg/
mL in experiment I.

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, 5 mM NADP in 100 mM sodium-orhto-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:

The concentrations in the main experiment were based on a pretest in which V79 cells were treated with the test item up to 2500 µg/mL. Incubation lasted 4 h (+/- S9 mix) and 24 h (- S9 mix). No cytotoxicity was observed up the highest concentration at the 4 h treatment with and without metabolic activation but cell growth was completely suppressed at 1250 µg/mL and 2500 µg/mL after 24 h treatment (- S9). Up to and including 625 µg/mL, no cytotoxic effects were observed. Precipitation of the test item occurred at 1250 µg/mL and above in the presence and absence of metabolic activation in both treatments. pH and oasmolarity remained stable. Based on these results the concentrations for the main experiment were chosen as follows:

Experiment I:
Without S9 mix: 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL
With S9 mix: 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL

Experiment II
Without S9 mix: 62.5, 125, 250, 500, 750, 1000, 1500 µg/mL

78.1 µg/mL in experiment I and 62.5 µg/mL in experiment II were not evaluated after day 4 since four concentrations are required by the guideline.

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: 0.5 % (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: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5x10^6 (single culture, for mutation rate) and 5x10^2 cells (in duplicate, for toxicity)
- Test substance added in medium (without FCS for 4 h exposure, for 24 h with FCS for experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment I: 4 h, Experiment II: 24 h
- Harvest time after the end of treatment: approximately 18 days

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days
- Fixation time: approximately 18 days
- Selective agent: 6-TG, final concentration 11 µg/mL, 8 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment (cloning efficiency I), relative population growth and cloning efficiency (CE II, viability, cloning efficiency determined after the expression period to measure viability of the cells without selective agent, absolute and relative)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- number of mutant colonies per 10^6 cells, induction factor (mutant colonies per 10^6 cells / mutant colonies per 10^6 cells of the corresponding solvent control),

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 476 version dated 1997, 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 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 significanincrease in mutant colonies
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50%

Evaluation
An assay is considered positive if a concentration-dependent and reproducible increase in mutant frequency is observed. The mutagenic response should be at least three times that of the negative controls in at least one concentration.

An assay is considered negative if there is no such concentration-dependent or reproducible increase.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1250 and 2500 µg/mL (- S9) and at 500 µg/mL (+S9) and above.

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: The test item precipitated at 1250 and 2500 µg/mL in experiment I (- S9) and at 2500 µg/mL (+ S9). In experiment II, precipitation occurred at 1500 µg/mL only.

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment increasing precipitation occurred at 1250 μg/mL and above in the presence and absence of S9 mix at both treatment intervals. Cytotoxicity was noticed for the 24 h treatment, at 625 µg/mL; due to the precipitation, the concentrations above 625 µg/mL could not be assessed in terms of toxicity (no S9 mix). The concentration range of the main experiments was based upon the results generated in the pre-experiment.

CYTOTOXICITY:
In experiment I, cytotoxicity occurred at 1250 and 2500 µg/mL (- S9) as evidenced by the reduced cloning efficiency I (survival). In the experiment II, cytotoxicity was observed at 500 µg/mL and above.

It was noticed that the relative cloning efficiency (CE I) was below the 10% limit at 2500 µg/mL (in one culture, culture II)) in the first experiment without metabolic activation and at 750 µg/mL and above in the second experiment (all cultures). However, the data were still judged as acceptable since the cell density at the first subcultivation remained well above this limit.

STUDY RESULTS (MAIN TEST)
Experiment I
No relevant increase in mutant frequencies was observed. At 1250 and 2500 µg/mL without metabolic activation, the mutant frequency was increased more than three times that of the solvent control in one of the duplicate cultures. However, this was not found in the second culture and was considered related to the low mutant colonies/10^6 cells of the solvent controls. Therefore, this finding was considered incidental.

Experiment II
No relevant increase in mutant frequencies was observed. At 1000 µg/mL without metabolic activation, the mutant frequency was increased more than three times that of the solvent control in one of the duplicate cultures. However, this was not found in the second culture and no concentration-dependency could be established. Therefore this finding was considered incidental.

Solvent, negative and positive controls gave the expected outcome.

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Histroical control data was provided by the testing facility in a separate statement (M-589714-01-1) and can be found Attachment 2 in the attached background material.

Conclusions:

The present study was conducted according to the OECD guideline 476 dated 1997, 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.

Reference 11

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 Aug - 2 Oct 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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:

Please refer to "principles of method if other than guideline"

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 1997

Deviations:

no

Principles of method if other than guideline:

The assay was based on Cole and Arlett (1984). Small (representing large genetic changes involving chromosome 11b and suggesting clastogenic activity) and large (representing base-pair substitutions or deletions) colonies were counted.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by the Department of Health of the Government of the United Kingdom

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y +/- 3.7.2c mouse lymphoma cells, obtained from the MRC Cell Mutation Unit, University of Sussex, Birghton, UK
- Normal cell cycle time (negative control): 12 h

For cell lines:
- Absence of Mycoplasma contamination: not reported
- Methods for maintenance in cell culture: not reported
- Cell cycle length, doubling time or proliferation index : 12 h
- Modal number of chromosomes: not reported
- Periodically checked for karyotype stability: not reported
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium, supplemented with 10% donor horse serum and 20 mM Hepes buffer (R10) at 37 °C with 5% CO2 in air. For the study, RPMI with 20% donor horse serum (R20) and without serum (R0) was used

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague
Dawley rats which received 500 mg/kg bw Aroclor (single injection) five days before sacrifice.

S9 fraction was thawed and mixed with S9 cofactor solution (10% S9 preparation in S9 mix). The cofactors in the final S9 mix are concentrated as follows: 25 mM Glucose-6-phosphate, 25 mM NADP in R0.

The final concentration of S9 in the culture was 10% (S9 mix) and 1% (S9 preparation).

Efficiency of the S9 mix was only assessed by the positive control during the assay.

Test concentrations with justification for top dose:

A pre-test was performed to find appropriate concentrations for the main study.
Concentrations used: 39, 78.1, 156.25, 312.5, 625, 625, 1250, 2500 µg/mL
Based on the results of the pre-test, the test concentrations for the main test were set as follows:

Experiment I
312.5, 625, 1250, 1667 and 2500 µg/mL
Experiment II
300, 600, 1200, 1600, 2000 and 2400 µg/mL

The top dose of 2500 µg/mL corresponds to approximatly 10 mM.

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate (quadruplicate for vehicle controls)
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10 x10^6 cells per flask
- Test substance added in medium (R0)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h
- Harvest time after the end of treatment: approximately 16 days

FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: 10-14 days
- Fixation time: approximately 16 days
- Selective agent: 5-trifluorothymidine (TFT), final concentration 4 μg/mL, 10-14 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: plating efficiency using the zero term of the Poisson distribution [P(0)] method (P(0)= number of negative wells/total wells plated, PE % = [(-In P(0)x 100]/[number of cells per well])

METHODS FOR MEASUREMENT OF GENOTOXICITY
-Method: Mutation frequency (M.F. per survivor = [(-In P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium)

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
An assay was considered acceptable if
- the mutant frequency per survivor in the vehicle control was in the normal range (10-125 x 10^-6), values greater than 150 x 10^-6 mutants per survivor in the vehicle control will make the experiment unvalid.
- positive controls give a marked increase in mutant frequency per survivor (at least 4 - 5 fold, ideally 10 fold or greater)

Evaluation
A test item was considered positive if at leat two of the following criteria were met:
- A statistically significant increase in mutant frequency.
- A greater than three-fold increase in the mutant frequency per survivor compared to the vehicle control.
- A dose-related increase in the mutant frequency per survivor.
- An increase in the absolute number of mutants.

If only one of these requirements were met, a test material was considered equivocal.

Statistics:

Data from both main experiments was statistically analysed by means of an computer program following acknowledged statistical guidelines.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

statistically significant increases in the mutant frequency were only observed at concentration levels that were cytotoxic

Cytotoxicity / choice of top concentrations:

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: not reported

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was conducted with concentrations ranging from 39 µg/mL to 2500 µg/mL, both, with and without S9 mix. At the highest concentration with and without metabolic activation cytotoxicity occurred. The reduction in plating efficiency after 10 - 14 days was especially marked in absence of S9 mix.


CYTOTOXICITY
Experiment I: At 2500 µg/mL, the test material induced cytotoxicity (seen as reduced colonies at Day 0 and Day 2 of microtitre plate counts), supported by reduced relative total growth (RGT) with and without metabolic activation. The 2500 µg/mL without S9 mix was excluded from the evaluation
Experiment II: Relative survival was decreased at high concentrations. Without metabolic activation, 2000 µg/mL of the test item were too toxic to provide evaluable data. With S9 mix, all data points could be evaluated but dose-dependend toxicity occurred (up to ca. 81% relative toxicity at 2400 µg/mL).

STUDY RESULTS
Experiment I
No statistically significant or dose related (linear-trend) increases in the mutant frequency x10^-6 per viable cell were seen, neither in the presence nor absence of S9 mix. However, a non-statistically significant increase occurred at 1667 µg/mL (above the upper level of 125 x 10^-6 per viable cells for control cultures) with and without metabolic activation. Dose-dependency could not be established because the higher value of 2500 µg/mL was excluded from evaluation due to toxicity.

Experiment II
Mutant frequency increased significantly and in a dose-dependent manner both in the absence and presence of S9 mix. This increase was mostly due to small colonies indicating clastogenic activity of the test item.

Solvent and positive controls produced the expected results so that the assay can be considered valid.

Summarized data can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Historical control data was provided in the study report and can be found in Attachment 2 in the attached background material.

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 was found to be mutagenic to L51 78Y cells; however, statistically significant increases in the mutant frequency were only observed at concentration levels that were cytotoxic.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Jan - 3 Apr 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

for expression, 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 non-essential 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 Sprague Dawley rats which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254 five days before sacrifice.

The protein concentration of the S9 preparation in the concentration selection experiment was 40.8 mg/mL and in the mutagenicity experiment 39.0 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 are 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 DMBA during the test.

Test concentrations with justification for top dose:

The concentrations in the main experiment were based on a pre-test. The doses tested were 62.5, 125, 250, 500, 1000, 1250, 2000, 2500 µg/mL for 5 h +/- S9 mix. Without S9 mix, cytotoxicity was seen at 2500 µg/mL but in no other condition. Precipitation occurred at 2000 µg/mL and above. Therefore the following concentrations were chosen for the main test:

Experiment I and II: 156, 313, 625, 1250, 2500, 5000 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: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4x10^6 cells in 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 (absolut 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 are 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 is 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 2000 μg/mL and above. At 1250 µg/mL (the highest soluble concentration), osmolality was not affected.

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was conducted with a series of 8 concentrations ranging from 62.5 µg/mL to 2500 µg/mL, both, with and without S9 mix. Precipitation of the test substance occurred at and above 2000 µg/mL. Cytotoxicity was evident at a concentration of 2500 µg/mL in absence of S9 mix, whereas no cytotoxic effects could be seen with S9 mix. Based on these results, 6 concentrations were retained for the main experiments ranging from 156 µg/mL to 5000 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 (MAIN TEST)
Without metabolic activation:
No relevant increase in mutation frequencies was observed. At 156 µg/mL, one culture showed increased mutant frequency (10 instead of 0.4-6.0 seen in solvent and negative controls) but this was not reproduced in the parallel culture or the second trial and in the range of historical control data for the controls. Therefore the finding was considered incidental.

With metabolic activation:
No relevant increase in mutation frequencies was observed. A slight increase occurred at 2500 µg/mL in one culture (9.1 instead of 0.1-1.6 seen in solvent and negative controls) but this was not reproduced in the parallel culture or the second trial and in the range of historical control data for the controls. Therefore the finding was considered incidental.

Solvent, negative and positive controls gave the expected outcome.

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
Histroical control data was provided by the testing facility and can be found Attachment 2 in the attached background material. The data came from 20 experiments with and without metabolic activation conducted from April 1995 to August 1996.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus tests, rat and mouse

WoE, M-618171-01-1, batch 6, MNT in vivo (OECD 474); GLP; rat bone marrow; oral (gavage), negative

WoE, M-105161-01-1, batch 4, MNT in vivo (OECD 474); GLP; mouse bone marrow; injection (i.p.), negative

WoE, M-103617-01-1, batch 1, MNT in vivo (OECD 474); GLP; mouse bone marrow; injection (i.p.), negative

WoE, M-036435-02-1, batch 2, MNT in vivo (OECD 474), GLP, mouse bone marrow, oral (gavage), negative

 

Comet assays, rat

WoE, 589564-01-1, batch 6, Comet assay in vivo (OECD 489); GLP; rat liver, kidney and stomach; oral (gavage), negative

 

Unscheduled DNA synthesis assay, rat

WoE, M-103622-01-1, batch 1, UDS in vivo (OECD 486), GLP, rat primary hepatocytes, 1000 and 2000 mg/kg bw, negative

WoE, M-105184-01-1, batch 4, UDS in vivo (OECD 486), GLP, rat primary hepatocytes, oral (gavage), negative

WoE, M-009751-03-1, batch 2, UDS in vivo (OECD 486), GLP, rat primary hepatocytes, oral (gavage), negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Jul - 30 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

Animals did not receive enriched environments, only males were used (justification was provided)

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Food safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

other: Crl:CD(SD) (SPF)

Details on species / strain selection:

commonly used for micronucleus tests, historical data is available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc, Shiga, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 302.7-338.4 g
- Housing: plastic cages (W290xD340xH170 mm, Crea Japan Inc., Tokyo, Japan) with shavings (Whiteflake, Charles River Laboratories Japan, Inc.), in groups of maximum three animals
- Diet: Laboratory animal chows (CRF-1, Oriental Yeat Co., Ltd., Tokyo, Japan), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.0 - 24.1
- Humidity (%): 49.6 - 61.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

0.5% aqueous cremophor (Kolliphor EL) was used as a vehicle due its relative non-mutagenicicty and non-toxicity

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance was weighed to the approriate amount, ground with pestle and mortar and mixed with the cremophor solution. Constant stirring was achieved using a magnetic stirrer at room temperature. Stability of the test substance in the vehicle was proven for 2 h at room temperature for concentrations of 1 and 250 mg/mL. The dose volume was 10 mL/kg bw. The individual dose volumes were calculated based on body weights taken just before administration.

The positive control cyclophosphamide (CP) was dissolved in purified water (12 mg/mL) and administered once at 5 mL/kg bw (orally via gavage).

Duration of treatment / exposure:

2 days

Frequency of treatment:

Rats were dosed twice, once per day (24 h in between)
rats that received the positive control were only dosed once, together with the second dosing in animals receiving the test item and solvent control

Post exposure period:

24 h

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (for 0, 500 and 1000 mg/kg bw/day, and for the positive control), 7 (for 2000 mg/kg bw group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: known mutagen
- Route of administration: orally via gavage
- Doses / concentrations: as described above, CP was solubilized at 12 mg/mL and administered at 5 mL/kg bw

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
No range finding study was conducted but doses were based on an alkaline comet assay in rats. The rats were of the same strain, age and were treated in the way described for this study (including dosing volume, route, frequency). No toxicity differences between the sexes were found and the rats tolerated all doses, up to the highest of 2000 mg/kg bw/day. As this is the limit dose recommended by the guideline, the doses in this experiment were chosen accordingly.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Groups of 5 or 7 male rats were dosed twice, approximately 24 h apart, with the vehicle alone (negative control) or the test item (500, 1000 or 2000 mg/kg bw/day). At the end of the observation period (approximately 24 h after the second test item administration), rats were euthanized by carbon dioxide inhalation.

OBSERVATIONS
Clinical signs and mortality were observed immediately, 3 h and 1 day after dosing. Body weights were recorded immediately before each dosing and 1 day after second dosing.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were washed out from the femurs with 1.0 mL of fetal bovine serum with 25 mM EDTA. After that, the cell suspension was centrifuged and the pellet was resuspended in residual serum and diluted with fetal bovine serum (including 25 mM EDTA). Small portions of this solution were smeared on a glass slide and the slides were fixed with methanol for 5 min within one day after preparation.

METHOD OF ANALYSIS:
The cells were analysed using acridine orange (40 µg/mL) using a fluorescent microscope (B excitation) using a 40-fold objective lens and 15-fold eye lens in a dry system. Erythrocytes with yellowish green fluorescence emitting particles (nuclei) and sharp outlines were identified as micronucleated erythrocytes. Due to the staining, polychromatic erythrocytes (PCEs) appeared bluish-grey and normochromatic erythrocytes (NCEs) appeared red. The incidences of micronucleated cells were scored in 4000 PCEs (per animal) and the incidence of PCEs in 1000 erythrocytes (including PCEs and NCEs) was examined.

Evaluation criteria:

Acceptability
The test was considered acceptable if
- incidence of PCEs (micronucleated) in the vehicle control group was within the acceptable negative control range from the historical control data
- the positive control induced a significant increase in micronucleated PCEs when compared with the concurrent vehicle control group.

Evaluation
An assay was considered positive if
- there was a statistically significant increase in the incidence of micronucleated PCEs when compared to the solvent or vehicle control
- the increase was dose-related
- the incidence of micronucleated PCEs in the dose group showing the increase exceeds the historical control range.

Statistics:

To statistically evaluate the incidences of PCEs, Kastenbaum-Bowman tables were used. Levels of significance were p=0.05 and 0.01. In case of an increased incidence, the dose-response relationship was tested with the Cochran-Armitage trend test (one-tailed, levels of significance were p=0.025 and 0.005).

The ratio of PCEs to total erythrocytes and animal body weight changes were evaluated using the t-test. The Student's t-test was used if p >= 0.05 and the F-test and the Welch's t-test was used if p<0.05 in the F-test (all two-tailed, levels of significance p=0.05 and 0.01).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Clinical signs: no clinical signs were observed in any of the treatment or control groups
- Body weight: Body weight and body weight gain was reduced in treatment groups before second dosing (500 and 1000 mg/kg bw/day) and one day after second dosing (all treatment groups)
- ratio of PCEs: the ratio of PCEs to total erythrocytes was decreased significantly (at all dose levels), a sign for bone marrow toxicity
- Induction of micronuclei: incidences of micronucleated PCEs were similar to vehicle controls for all dose levels and within the range of historical control data. Occassional incidences of slightly elevated increases were considered incidental and not of biological importance.
- Positive control: the positive control induced a statistically significant increase in the number of PCEs with micronuclei.

Historical control data
The historical control data was provided by the testing facility and can be found in Attachment 2 in the attached background material

The study is considered acceptable since 

1) the vehicle controls fell within the historical control range,
2) a sufficient number of animals were evaluable and
3) the positive control induced the expected result, confirming the sensitivity of the assay. 

ADME data had already been generated and decrease in % PCEs confirmed target organ exposure. 

Conclusions:

The study was performed according to OECD guideline 474 and in compliance with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the limit dose of 2000 mg/kg bw/day in male rats.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Dec 2016 - 16 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other:

Remarks:

Crl:CD(SD) [SPF]

Details on species / strain selection:

commonly used, recommended by the guideline, historical data is available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc, Shiga, Japan
- Age at study initiation: 8 weeks (dose-finding study), 7.5 weeks (main experiment)
- Weight at study initiation:
Dose-finding study: 309 - 328 g (males), 196 - 203 g (females)
Main experiment: 306 - 336 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: metallic mesh cages, in groups of maximum two animals
- Diet: Laboratory animal chows (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8-23.2
- Humidity (%): 44-58
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

0.5% aqueous cremophor (Kolliphor EL) was used as a vehicle due its relative non-mutagenicity and non-toxicity

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance was weighed to the approriate amount, ground with pestle and mortar and mixed with the cremophor solution just before administration. Stability of the test substance in the vehicle was proven for 2 h at room temperature for concentrations between 1 and 250 mg/mL. The dose volume was 10 mL/kg bw.
The positive control cyclophosphamide (CP) was dissolved in purified water (20 mg/mL, dose received 200 mg/kg bw).

Duration of treatment / exposure:

2 days

Frequency of treatment:

Rats were dosed once per day on two consecutive days (21 h in between)

Post exposure period:

3 h

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Males, main study

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Males, main study

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Males, main study and males and females in the dose-finding study

No. of animals per sex per dose:

Dose finding study: 3 males/females
Main study: 6 males (vehicle control + treatment), 4 males (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate, known mutagen
- Route of administration: injection
- Doses / concentrations: 200 mg/kg bw

Tissues and cell types examined:

Liver (metabolising organ and target organ for toxicity), kidney (target organ for toxicity) and stomach (site of first contact, target organ for toxicity)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted with 3 males and females receiving either the vehicle or 2000 mg/kg bw of the test item. Based on the results of this dose-finding study (for details please refer to "Additional information on results"), three dose levels including 2000 mg/kg bw/day as highest dose as recommended by OECD 489 were selected. As no substantial differences between sexes had been observed, the main study was performed in male rats only.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Groups of 6 or 4 male rats were dosed twice, approximately 21 h apart, with the vehicle alone (negative control), with the positive control (200 mg/kg bw/day EMS) or the test item (500, 1000 or 2000 mg/kg bw/day). At the end of the observation period (approximately 3 h after the second test item administration), rats were euthanized by carbon dioxide inhalation.

SLIDE PREPARATION
Portions of liver (about 5-mm cube from the left lateral lobe), kidney (cranial half of the left kidney) and stomach were collected. Cell suspensions were prepared as follows: Liver and kidney were homogenized, the glandular stomach was rinsed and afterwards incubated in homogenizing buffer to remove the surface epithelium. The surface epithelium was then discarded. The epithelium of the stomach was scraped gently into fresh homogenizing buffer with a blade to release the cells into the buffer. This formed the cell suspenison from the stomach.
For slide preparation, a superfrosted glass slide was pre-coated with 1.0% agarose gel and 0.5% low-melting agarose gel was added to the cell suspenisons (90 µL agarose gel + 10 µL cell suspension). 90 µL of this mixture was added to the glas slide and covered with a non-coated superfrosted glass slide. Another layer of 0.5% low-melting agarose (90 μL) was added in the same manner.
The slides were placed in lysing solution (at refrigerated conditions in the dark) overnight (or longer).
Per organ, three slides were prepared of which two were evaluated.

Electrophoresis of the slides was carried out using submarine-type electrophoresis chamber (BE-540, BIO CRAFT). After 20 min of unwinding in the chamber, electrophoresis was carried out (0.7 V/cm (25V) (initial current: 300 mA) for 20 minutes).

EXAMINATION
After neutralisation and dehydration in ethanol (>= 99.5%),, slides were stained with SYBR® Gold nucleic acid gel stain, diluted 5000 fold with TE buffer (pH 8.0) for analysis. Images of DNA migration of cells were examined using a fluorescence microscope with IB excitation and auxiliary absorbing filters.
Per animal, 150 cells (clear defined non-overlapping and without unusual staining artefacts) were analyzed, meaning 75 cells per slide and 750 cells per dose group. Additionaly, 150 cells per animal (75 cells per slide) were analyzed using the fluorescence microscope (×200) for the number of hedgehogs.

ANALYSIS
A percentage of DNA in the tail to the total (% tail DNA: Tail % intensity) was used as an indicator for DNA damage. For each slide, the median %tail DNA was determined and for each animal, the mean slide values were calculated. Each animal value of % tail DNA was logarithmically transformed prior to statistical analysis.

Evaluation criteria:

Acceptability
The study was considered valid if the negative control data was within the range of the historical control data of the laboratory and treatment with the positive control substance gives a statistically significant increase in %tail DNA compared to the negative control group. In addition, the biological relevance of the results was taken into consideration for final judgement.

Evaluation of results
A test item was considered to give a positive outcome if there was a statistically significant increase of %tail DNA in at least one of the treatment groups. This increase should also be dose-dependent and outside of the range of historical control data.

Statistics:

For %tail DNA (mean of animal values): Dunnett’s multiple comparison test (one-sided, significant level of 0.025) was used. To compare negative and positive control, Aspin-Welch’s t test (one-sided, significant level of 0.025) was used.

Body weight
Homogenity of variances was analyzed using Bartlett’s test (two-sided, significance level of 0.05). If data was homogenious, Dunnett’s multiple comparison test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group. If data was not homogenious, Steel’s test (two-sided, significance level of 0.05) was used.
Body weight gain was first analyzed by the F-test for homogeneity of variances (two-sided, significance level of 0.05). If data was homogenious, Student’s t test (two-sided, significance level of 0.05) was performed. If homogeneity was not detected, Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

seen by reduced body weight and clinical signs (tremor) at 1000 and 2000 mg/kg bw/day. In addition, ptosis was observed at 2000 mg/kg bw/day.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: the test item was soluble and stable in the vehicle. No precipitation occurred.
- Clinical signs of toxicity in test animals: At 2000 mg/kg bw/day neither mortality nor moribundity were observed in either sex. Only locomotor activity was decreased in one male at 2000 mg/kg bw. 2000 mg/kg bw is the limit dose recommended by the guideline, so that the doses in this experiment were chosen accordingly.
- Evidence of cytotoxicity in tissue analysed: None

RESULTS OF DEFINITIVE STUDY
% tail DNA
No statistically significant increase was noted in any of the treatment groups compared with the negative control group in any organ (liver, kidney and stomach). Also, the incidences of hedgehogs were not increased. The positive control induced a significant increase and both positive and solvent control were within the historical control data of the laboratory.

Summarized data can be found in Attachment 1 in the attached background material.

Gross necropsy
No treatment-related findings occurred. Incidental findings were a hepatodiaphragmatic nodule in one animal of the control group and a cyst in the left kidney in one animal that received 1000 mg/kg bw/day.

Body weight and clinical signs
- 1000 mg/kg bw: Tremor and decreased body weight
- 2000 mg/kg bw: Tremor, decreased body weight, ptosis

Summarized results can be found in Attachment 2 and 3.

HISTORICAL CONTROL DATA
Historical control data was provided by the testing facility and can be found in Attachment 4 in the attached background material.

Conclusions:

The study was conducted under GLP and according to the OECD guideline 489. Under the conditions of the assay, the test item did not induce DNA damage in rats, when administered by gavage at doses up to and including the maximum recommended dose of 2000 mg/kg bw/day.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Sep - 03 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

no bodyweight was recorded at termination, only 2000 PCEs were scored per animal (instead of 4000), no justification for route of administration (i.p.), animals were housed individually without enrichment, humidity was higher than recommended

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Füllinsdorf, Switzerland
- Age at study initiation: 5 - 7 weeks (males), 7 - 9 weeks (females)
- Weight at study initiation: males mean value 39.4 g (SD +/- 2.8 g), females mean value 33.5 g (SD +/- 1.8 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: individually in Makrolon Type I, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 80
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

0.5% aqueous cremophor was used as a vehicle due to its relative non-mutagenicity and non-toxicity. The volume administered was 10 mL/kg bw.

Details on exposure:

The test item was formulated at the desired concentration in the vehicle on the day of the experiment. Animals were weighed before dosing so that the administered dose could be assigned to the respective bodyweight (10 mL/kg bw).

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single dose

Post exposure period:

24 h (male/female of all groups), 48 h (male/female of highest treatment group)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals/sex/dose for all dose groups and 6 additional animals/sex/dose for the 48 hrs sampling in the high dose group (200 mg/kg bw)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: known mutagen
- Route of administration: i.p.
- Doses / concentrations: single dose, 40 mg/kg bw, 10 mL/kg bw, solubilized in deionised water

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. Two animals per sex and group received a single dose of the test item i.p. in 0.5% cremophor at 10 mL/kg bw. They were examined for acute toxic symptoms 1, 2-4, 6, 24, 30 and 48 h after administration. The dose levels used were 100, 200, 300 and 1000 mg/kg bw. Males and females showed symptoms at 100 mg/kg bw, namely reduction of spontaneous activtiy, abdominal position, eyelid closure, ruffled fur and apathy. At 300 mg/kg bw, one female died, at 1000 mg/kg bw, both females and one male died. On the basis of these results, 200 mg/kg bw was chosen as the highest dose (maximum tolerated dose) for the test.

TREATMENT AND SAMPLING TIMES:
After treatment, the animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1, 2-4, 6 and 24 h after administration of the test item. Sampling of bone marrow was performed in all groups at 24 hours post-treatment, and in the animals of the highest dose group additionally at 48 hours post-treatment. Therefore, the animals were sacrificed with CO2.


DETAILS OF SLIDE PREPARATION:
Out of the six animals per sex and dose used, 5 animals per sex and dose were used for slide preparation.
For collection of bone marrow cells, the femora was removed, the epiphyses were cut off and the bone marrow was flushed out with fetal calf serum (using a syringe). The cell suspension was centrifuged (1500 rpm, 10 min) and the pellet was resuspended. Small portions of this solution were smeared on a glas slide, air dried and stained with May-Grünwald (Merck, Darmstadt, Germany) / Giemsa (BDH Limited Poole, Great Britain). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
The prepared slides were analyzed microscopically. At least 2000 polychromatic erythrocytes (PCE) were analyzed per animal for micronuclei. Moreover, the ratio of PCEs to erythrocytes was recorded (in 2000 erythrocytes) as a measurement of cytotoxicity.

Evaluation criteria:

Acceptability
The test was considered acceptable if
- incidence of PCEs (micronucleated) in the vehicle control group was within the acceptable negative control range of the historical control data
- the positive control induced a significant increase in micronucleated PCEs and were in the range of the historical control data
- at least 80% of animals were evaluable
- PCE to erythrocyte ratio was at least 20% of the negative control

Evaluation
An assay is considered positive if
- there was a statistically significant increase in the incidence of micronucleated PCEs when compared to the solvent or vehicle control
- there was a dose-related increase in the incidence of micronucleated PCEs.

Statistics:

For statistical evaluation, the nonparametric Mann-Whitney test was used.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs occurred at 50 mg/kg bw, treatment-related deaths occurred at 200 mg/kg bw.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY
The findings of the range finding study are discussed above. 200 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
50 mg/kg bw: reduction of spontanous activity, abdominal position, eyelid closure, ruffled fur
100 mg/kg bw: reduction of spontanous activity, abdominal position, eyelid closure, ruffled fur
200 mg/kg bw: Animals showed reduction of spontanous activity, abdominal posture, eyelid closure, ruffled fur and tremor. Two males died 6 h after treatment.

Summarized Results can be found in Attachment 1 in the attached background material.

Induction of micronuclei:
Not affected

Ratio of PCE/NCE (for Micronucleus assay):
There was no change in the ratio of PCEs per 2000 erythrocytes under any of the testing conditions.

Summarized results can be found in Attachment 2 in the attached background material.

-Statistical evaluation:
Only the positive control induced a statistically significant induction of PCEs with micronuclei (p < 0.0001). 50 and 100 mg/kg bw were not tested, as well as 200 mg/kg bw after 48 h. After 24 h and treatment with 200 mg/kg bw, significance was p = 0.3079 (not significant).

The positive and vehicle control were in the range of historical control data.

HISTORICAL CONTROL DATA
Historical control data was provided by the laboratory as follows:
Time frame: 1999 - 2001
Negative controls:
Percent micronucleated polychromatic erythrocytes
Range: 0.010 to 0.150, Mean: 0.066* +/- 0.032

Positive controls:
Cyclophosphamide; 40 mg/kg bw [CPA]
Percent micronucleated polychromatic erythrocytes
Range: 0.910 to 2.975, Mean: 1.644* +/- 0.446
*= mean of 95 experiments

Conclusions:

Under the conditions of the assay, there were no indications of a clastogenic effect after single i.p. treatment of male and female mice with the test substance up to a dose level of 200 mg/kg bw. The test was conducted according to OECD guideline 474 (1997) and under GLP conditions.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 Sep - 24 Nov 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

no bodyweight was recorded at termination, only 2000 PCEs were scored per animal (instead of 4000), no justification for route of administration (i.p.), animals were housed individually without enrichment, humidity was higher than recommended

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Füllinsdorf, Switzerland
- Age at study initiation: 5-7 weeks (males), 7-9 weeks (females)
- Weight at study initiation: males mean value 39.2 g (SD +/- 2.3 g), females mean value 33.5 g (SD +/- 1.8 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: individually in Makrolon Type I, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

0.5% aqueous cremophor was used as a vehicle due to its relative non-mutagenicity and non-toxicity. The volume administered was 10 mL/kg bw.

Details on exposure:

The test item was formulated at the desired concentration in the vehicle on the day of the experiment. Animals were weighed before dosing so that the administered dose could be assigned to the respective bodyweight (10 mL/kg bw).

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single exposure

Post exposure period:

24 h (male/female of all groups), 48 h (male/female of highest treatment group)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals/sex/dose for all dose groups and 6 additional animals/sex/dose for the 48 h sampling in the high dose group (300 mg/kg bw)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: known mutagen
- Route of administration: i.p.
- Doses / concentrations: single dose, 40 mg/kg bw, 10 mL/kg bw, solubilized in deionised water

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. Two animals per sex and group received a single dose of the test item i.p. in 0.5% cremophor at 10 mL/kg bw. They were examined for acute toxic symptoms 1, 2-4, 6, 24, 30 and 48 h after administration. The dose levels used were 100, 300, 400, 500 and 1000 mg/kg bw. Males and females showed symptoms at 100 mg/kg bw, namely reduction of spontaneous activtiy, abdominal position, eyelid closure, ruffled fur and apathy, even though some of the symptoms were reversible. Deaths occurred at 400 mg/kg bw (1 female), 500 mg/kg bw (1 male, 2 females) and 1000 mg/kg bw (2 males/2 females). On the basis of these results, 300 mg/kg bw was chosen as the highest dose (maximum tolerated dose) for the test.

TREATMENT AND SAMPLING TIMES:
After treatment, the animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1, 2-4, 6 and 24 h after administration of the test item. Sampling of bone marrow was performed in all groups at 24 hours post-treatment, and in the animals of the highest dose group additionally at 48 hours post-treatment. Therefore, the animals were sacrificed with CO2.

DETAILS OF SLIDE PREPARATION:
Out of the six animals per sex and dose used, 5 animals per sex and dose were used for slide preparation.
For collection of bone marrow cells, the femora was removed, the epiphyses were cut off and the bone marrow was flushed ot with fetal calf serum (using a syringe). The cell suspension was centrifuged (1500 rpm, 10 min) and the pellet was resuspended. Small portions of this solution were smeared on a glas slide, air dried and stained with May-Grünwald (Merck, Darmstadt, Germany) / Giemsa (BDH Limited Poole, Great Britain). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
The prepared slides were analyzed microscopically. At least 2000 polychromatic erythrocytes (PCE) were analyzed per animal for micronuclei. Moreover, the ratio of PCEs to erythrocytes was recorded (in 2000 erythrocytes) as a measurement of cytotoxicity.

Evaluation criteria:

Acceptability
The test was considered acceptable if
- incidence of PCEs (micronucleated) in the vehicle control group was within the acceptable negative control range of the historical control data
- the positive control induced a significant increase in micronucleated PCEs and were in the range of the historical control data
- at least 80% of animals were evaluable
- PCE to erythrocyte ratio was at least 20% of the negative control

Evaluation
An assay was considered positive if
- there was a statistically significant increase in the incidence of micronucleated PCEs when compared to the solvent or vehicle control
- there is a dose-related increase in the incidence of micronucleated PCEs.

Statistics:

For statistical evaluation, the nonparametric Mann-Whitney test was used.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs were observed from 75 mg/kg bw onwards. At 300 mg/kg bw, mortality occurred.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY
The findings of the range finding study are discussed above. 300 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
75 mg/kg bw: reduction of spontanous activity, abdominal position, eyelid closure, ruffled fur
150 mg/kg bw: reduction of spontanous activity, abdominal position, eyelid closure, ruffled fur, tremor
300 mg/kg bw: Animals showed reduction of spontanous activity, abdominal posture, eyelid closure, ruffled fur and tremor. Two males and one female died 6 h after treatment.

Summarized Results can be found in Attachment 1 in the attached background material.

Induction of micronuclei:
Not affected

Ratio of PCE/NCE (for Micronucleus assay):
There was no change in the ratio of PCEs per 2000 erythrocytes under any of the testing conditions.

Summarized results can be found in Attachment 2 in the attached background material.

-Statistical evaluation:
Only the positive control induced a statistically significant induction of PCEs with micronuclei (p < 0.0001). For treatment groups, significance levels ranged from p = 0.0560 (300 mg/kg bw, 24 h) to p=0.3264 (150 mg/kg bw, 24 h), none of these were significant.

The positive and vehicle control were in the range of historical control data.

HISTORICAL CONTROL DATA
Historical control data was provided by the laboratory as follows:
Time frame: 1999 - 2001
Negative controls:
Percent micronucleated polychromatic erythrocytes
Range: 0.010 to 0.150, Mean: 0.066* +/- 0.032

Positive controls:
Cyclophosphamide; 40 mg/kg bw [CPA]
Percent micronucleated polychromatic erythrocytes
Range: 0.910 to 2.975, Mean: 1.644* +/- 0.446
*= mean of 95 experiments

Conclusions:

Under the conditions of the assay, there were no indications of a clastogenic effect after single oral treatment of male and female mice by gavage with the test substance up to a dose level of 300 mg/kg bw. The test was conducted according to OECD guideline 474 (1997) with the deviations stated above and under GLP conditions.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 Aug - 25 Sep 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

no bodyweight was recorded at termination, only 1000 PCEs were scored per animal (instead of 4000)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by The Department of Health of the Government of the United Kingdom

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK), Limited, Margate, Kent, UK
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 21-26 g (male), 20-25 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: standard diet (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 53-57
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil BP

Details on exposure:

The test item was formulated at the desired concentration in the vehicle on the day of the experiment. Animals were weighed before dosing so that the administered dose could be assigned to the respective bodyweight (10 mL/kg bw).

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

24 h, 48 h, 72 h

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex, per dose and per sampling time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: known to induce micronuclei
- Route of administration: orally via gavage
- Doses / concentrations: 50 mg/kg bw, 10 mL/kg bw in distilled water

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. The test substance was administered orally (by gavage) to 2 males and females in doses of 100, 200, 300 and 400 mg/kg bw (in the 200 mg/kg bw group, 4 males and females were used). Animals were observed one hour after dosing and subsequently once daily for up to three days. Moreover, two additional groups of 2 females and males were administered with 100 and 200 mg/kg bw to obtain data about the PCE/NCE ratio after 24 h.
Deaths were observed at 200 mg/kg bw and above, clinical signs occurred at all dose levels and included hunched posture, decreased respiratory rate, ptosis, lethargy, laboured respiration, ataxia, splayed gait, pilo-erection and occasional body tremors. No cytotoxic response was found in the bone marrow at 100 and 200 mg/kg bw. Based on these results, the highest dose in the main study was set to 100 mg/kg bw (maximum tolerated dose).

TREATMENT AND SAMPLING TIMES:
After treatment, all animals were examined for toxic symptoms one hour after administration and then daily for the observation period of three days. One group of mice (5 males and females) from each dose level was killed by cervical dislocation 24-hours following treatment, a second group from each dose level at 48-hours and a third group at each dose level at 72-hours. At each time point, a vehicle control group was killed as well. The animals of the positive control group were sacrificed 24-hours post-treatment.

DETAILS OF SLIDE PREPARATION:
For collection of bone marrow cells, the bone marrow was flushed out of the femur with fetal calf serum (using a syringe). The cell suspension was centrifuged and the pellet was resuspended. Small portions of this solution were smeared on a glas slide, air dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa.

METHOD OF ANALYSIS:
The prepared slides were analyzed microscopically. At least 1000 polychromatic erythrocytes (PCE) were analyzed per animal for micronuclei. Moreover, normochromatic erythrocytes (NCEs) associated with 1000 erythrocytes were counted. The ratio of PCEs to NC erythrocytes was recorded as a measurement of cytotoxicity.

Evaluation criteria:

Evaluation
An assay is considered positive for mutagenicity if
- there is a statistically significant increase in the incidence of micronucleated PCEs at any dose level and/or any time point when compared to the solvent or vehicle control

A test item was considered positive in bone marrow toxicity if
- group mean PCE/NCE ratio was statisticaly significent lower than the solvent control

Statistics:

The data was analysed following (x+1)^(1/2) transformation using Student's t-test (two tailed) and
any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs were observed in all treatment groups, mortality occurred at 100 mg/kg bw in three females

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY
The findings of the range finding study are discussed above. 100 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
Hunched posture, decreased respiratory rate, laboured respiration, ptosis and lethargy were found in animals of all treatment groups at all time points (24, 48 and 72 h). In the 48 h group that received 100 mg/kg bw of the test item, three females died prematurely.

Induction of micronuclei:
Not affected

Ratio of PCE/NCE (for Micronucleus assay):
There was no change in the ratio of PCE/NCE under any of the testing conditions, except for the positive control.

Summarized results can be found in Attachment 1 in the attached background material.

The positive and vehicle control were in the range of historical control data.

HISTORICAL CONTROL DATA
Histrorical control data was provided by the laboratory and can be found in Attachment 2 in the attached background material.

Conclusions:

The study was performed according to OECD guideline 474 (1983) with the restrictions stated above and compliant with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the MTD of 100 mg/kg bw in mice.

Reference 6

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Sep - 13 Nov 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

Hanlbm: WIST (SPF)

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: Mean value 172.0 g (SD ± 12.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 0vernight (2 hours treatment) or approximately 6 hours (16 hours treatment)
- Housing: individual housing in Makrolon Type II, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet (Altromin, Lage/Lippe, Germany, ad libitum), ad libitum with the exception of the fasting period
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: 0.5% Cremophor
- Justification: non-toxicity for the animals
Volume used: 10 mL/kg bw
Application: single dose, orally

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% Cremophor.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single dose

Post exposure period:

sacrifice after 2 and 16 h

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

2 hour sacrifice interval:
Substance: DMH; N,N´-dimethylhydrazinedihydrochloride
Dose used: 40 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: 0.9% NaCl solution
Application: single dose, orally (gavage)

16 hour sacrifice interval:
Substance: 2-AAF; 2-acetylaminofluorene
Dose used: 100 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: dimethylsulfoxide/polyethylene glycol 400 (1 + 9)
Application: single dose, orally (gavage)

Tissues and cell types examined:

primary hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. Two fasted animals per sex and group received a single dose of the test item (2000 mg/kg bw) orally in 0.5% cremophor at 10 mL/kg bw. They were examined for symptoms of acute toxicity 1, 2 - 4, 6 and 24 h after administration. Observed signs were reduction of spontaneous activity, eyelid closure and ruffled fur for up to 6 hours. No mortality was observed, so that 2000 mg/kg bw was set as the maximum dose. Since there were no sex specific toxicity symptoms observed the main experiment was carried out using only male rats.

TREATMENT AND SAMPLING TIMES:
At sampling, animals were anaesthetised with Na-thiopental. To obtain hepatocytes, the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented (HBSS) with collagenase (0.05 % (w/v)) adjusted to pH 7.4 and maintained at 37 °C. The isolated hepatocytes were washed twice with HBSS and the cell suspension was filtered through a stainless steel mesh to obtain a single cell suspension. After centrifugation, cells were resuspended into Williams medium E (WME), containing hepes (2.38 mg/mL), penicillin (100 units/mL), streptomycin (0.10 mg/mL), L-glutamine (0.29 mg/mL), insulin (0.5 µg/mL) and fetal calf serum (FCS, 10%). Three cultures were established per animal.

DETAILS OF SLIDE PREPARATION:
Aliquots of the cell suspension (2.0 x 10E5 viable cells/mL) was seeded into 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine. Then, cells attached after 1.5 h (95% air/5% CO2, 37 °C). After that the culture medium was discarded and cells were rinsed with PBS. 3HTdR (5 μCi/mL, specific activity 20 Ci/mmol) was added in 2 mL culture medium (supplemented with 1% FCS only) and labelling was performed for 4 h. Cells were washed with culture medium (1% FCS) and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. On the next day, the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate (10 min to swell the nuclei) and cells were fixed with methanol:acetic acid (3+1 v/v, 3 x 20 minutes each), rinsed with 96 % (v/v) ethanol, and air-dried. After that, cover slips were mounted on glass slides and coated with KODAK NTB2 photographic emulsion in the dark. For drying, they were then placed in light-proof boxes in the presence of a drying agent for 14 days at 4 °C and the photographic emulsion was developed with Ilford Phenisol at room temperature, fixed in Rapid fixer and stained with hematoxylin/eosin.

METHOD OF ANALYSIS:
The number of silver grains above the nucleus were counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). The number of grains of one nuclear-sized cytoplasm area adjacent to the nucleus was counted as well. Three animals per group were evaluated per animal, two slides were investigated with at least 100 cells (50 per slide).

Evaluation criteria:

A test item is considered to give a positive response if the mean number of net grains is higher than five per nucleus at any concentration or time point. Ideally, this increase is dose dependent (nuclear and net grains and/or an increase of the percentage distribution of the nuclear grain counts). A group average between 0 and 5 net grains is considered as a marginal response.

Statistics:

Statistical analysis was done with the non-parametric Mann-Whitney test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs as described.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The findings of the range finding study are discussed above. 2000 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
2 h: reduction of spontaneous activity and ruffled fur for both treatment groups (1000 and 2000 mg/kg bw)
16 h: reduction of spontaneous activity and ruffled fur for up to 16 hours, eyelid closure for 2 hours were observed in both treatment groups (1000 and 2000 mg/kg bw)

Viability and number of hepatocytes
No effect in any treatment group and time point. The viability of the isolated hepatocytes was over 70% for all tested animals.

Mean nucleus, cytoplasmic area, and net grains
2 h: No effect observed
16 h: No effect observed

The positive controls were in the range of historical control data. For the solvent control, the mean net grain values after 2 h were slightly below the lower value of the historical control range (-15.87 vs -13.51). This could be due to the fact that these data were obtained using different instruments for counting. However, this did not have an impact on the outcome of the results.

Summarized results can be found in Attachment 1 (2 h ) and Attachment 2 (16 h) in the attached background material.

- Statistical evaluation: A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

HISTORICAL CONTROL DATA
Historical control Data was provided by the laboratory and can be found in Attachment 3 in the attached background material.

Conclusions:

Under the experimental conditions reported, the test item did not induce unscheduled DNA-synthesis. The study was performed under GLP and according to the OECD guideline 486.

Reference 7

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 Sep - 20 Nov 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

Hanlbm: WIST (SPF)

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: Mean value 173.0 g (SD ± 12.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 0vernight (2 hours treatment) or approximately 6 hours (16 hours treatment)
- Housing: individual housing in Makrolon Type II cages, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet (Altromin, Lage/Lippe, Germany, ad libitum), ad libitum with the exception of the fasting period
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: 0.5% Cremophor
- Justification: non-toxicity for the animals
Volume used: 10 mL/kg bw
Application: single dose, orally

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% Cremophor.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single dose

Post exposure period:

sacrifice after 2 h and 16 h

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

2 hour sacrifice interval:
Substance: DMH; N,N´-dimethylhydrazinedihydrochloride
Dose: 40 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: 0.9% NaCl solution
Application: single dose, orally (gavage)

16 hour sacrifice interval:
Substance: 2-AAF; 2-acetylaminofluorene
Dose: 100 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: dimethylsulfoxide/polyethylene glycol 400 (1 + 9)
Application: single dose, orally (gavage)

Tissues and cell types examined:

primary hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. Two fasted animals per sex and group received a single dose of the test item (2000 mg/kg bw) orally in 0.5% cremophor at 10 mL/kg bw. They were examined for symptoms of acute toxicity 1, 2 - 4, 6 and 24 h after administration. Observed signs were reduction of spontaneous activity, eyelid closure and ruffled fur for up to 24 hours. No mortality was observed, so that 2000 mg/kg bw was set as the maximum dose. Since there were no sex specific toxicity symptoms obeserved the main experiment was carried out using only male rats.

TREATMENT AND SAMPLING TIMES:
At sampling, animals were anaethetised with Na-thiopental. To obtain hepatocytes, the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented (HBSS) with collagenase (0.05 % (w/v)) adjusted to pH 7.4 and maintained at 37 °C. The isolated hepatocytes were washed twice with HBSS and the cell suspension was filtered through a stainless steel mesh to obtain a single cell suspension. After centrifugation, cells were resuspended into Williams medium E (WME), containing hepes (2.38 mg/mL), penicillin (100 units/mL), streptomycin (0.10 mg/mL), L-glutamine (0.29 mg/mL), insulin (0.5 µg/mL) and fetal calf serum (FCS, 10%). Three cultures were established per animal.

DETAILS OF SLIDE PREPARATION:
Aliquots of the cell suspension (2.0 x 10E5 viable cells/mL) were seeded into 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine. Then, cells attached after 1.5 h (95% air/5% CO2, 37 °C). After that the culture medium was discarded and cells were rinsed with PBS. 3HTdR (5 μCi/mL, specific activity 20 Ci/mmol) was added in 2 mL culture medium (supplemented with 1% FCS only) and labelling was performed for 4 h. Cells were washed with culture medium (1% FCS) and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. On the next day, the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate (10 min to swell the nuclei) and cells were fixed with methanol:acetic acid (3+1 v/v, 3 x 20 minutes each), rinsed with 96 % (v/v) ethanol, and air-dried. After that, cover slips were mounted on glass slides and coated with KODAK NTB2 photographic emulsion in the dark. For drying, they were then placed in light-proof boxes in the presence of a drying agent for 14 days at 4 °C and the photographic emulsion was developed with Ilford Phenisol at room temperature, fixed in Rapid fixer and stained with hematoxylin/eosin.

METHOD OF ANALYSIS:
The number of silver grains above the nucleus were counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). The number of grains of one nuclear-sized cytoplasm area adjacent to the nucleus was counted as well. Three animals per group were evaluated. Per animal, two slides were investigated with at least 100 cells (50 per slide).

Evaluation criteria:

A test item is considered to give a positive response if the mean number of net grains is higher than five per nucleus at any concentration or time point. Ideally, this increase is dose dependent (nuclear and net grains and/or an increase of the percentage distribution of the nuclear grain counts). A group average between 0 and 5 net grains is considered as a marginal response.

Statistics:

Statistical analysis was done with the non-parametric Mann-Whitney test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

only minor clinical signs were observed

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The findings of the range finding study are discussed above. 200 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
2 h: reduction of spontaneous activity, eyelid closure and ruffled fur for both treatment groups (1000 and 2000 mg/kg bw)
16 h: reduction of spontaneous activity and ruffled fur for up to 16 hours, eyelid closure for 1 hour for both treatment groups (1000 and 2000 mg/kg bw)

Viability and number of hepatocytes
No effect in any treatment group and time point. The viability of the isolated hepatocytes was over 70% for all tested animals.

Mean nucleus, cytoplasmic area, and net grains
2 h: No effect observed
16 h: No effect observed

The positive controls were in the range of historical control data. For the solvent control, the mean net grain values after 16 h were slightly below the lower value of the historical control range ( -16.25 vs -13.51). This could be due to the fact that these data were obatined using different instruments for counting. However, this did not have an impact on the outcome of the results.

Summarized results can be found in Attachment 1 (2 h) and Attachment 2 (16 h) in the attached background material.

- Statistical evaluation: A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

HISTORICAL CONTROL DATA
Historical Control Data was provided by the laboratory and can be found in Attachment 3 in the attached background material.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce unscheduled DNA-synthesis. The study was performed under GLP and according to the OECD guideline 486.

Reference 8

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 Feb - 17 Jul 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

adopted 1997

Deviations:

no

Remarks:

The study was conducted before the guideline was published, so it was based on a draft. However, the requirements of the guideline are met.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

Crl:(Wi)BR (SPF)

Details on species / strain selection:

Standard animal for the test, historical data is available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 120-140 g
- Assigned to test groups randomly: yes
- Fasting period before study: overnight (4 hours treatment) or approximately 6 hours (16 hours treatment)
- Housing: individual housing in Makrolon Type II cages, with granulated soft wood bedding
- Diet: pelleted standard diet (Altromin 1324, Lage/Lippe, Germany), ad libitum with the
exception of the fasting period
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 41-69
- Air changes (per hr): ca 10 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: 0.5% Cremophor
- Justification: non-toxicity for the animals
Volume used: 20 mL/kg bw
Application: single dose, orally, stomach tube

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% Cremophor.

Duration of treatment / exposure:

single treatment

Frequency of treatment:

single dose

Post exposure period:

4 h, 16 h

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

nominal dose

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 (6 for vehicle control, 16 h)

Control animals:

yes, concurrent vehicle

Positive control(s):

4 hour sacrifice interval:
Substance: DMH; N,N´-dimethylhydrazinedihydrochloride
Concentration used: 40 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: DMSO
Application: single dose, orally, stomach tube

16 hour sacrifice interval:
Substance: 2-AAF; 2-acetylaminofluorene
Concentration used: 100 mg/kg bw
Volume used: 10 mL/kg bw
Solvent: corn oil
Application: single dose, orally by stomach tube

Tissues and cell types examined:

primary hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was conducted on acute toxicity to find appropriate doses. Three fasted male rats per group received a single dose of the test item (4000 and 5000 mg/kg bw) orally. They were examined for symptoms of acute toxicity for at least 72 h after administration.
Observed signs were roughened fur, sunken flanks, languor and slitted eyes. No mortality was observed, so that 5000 mg/kg bw was set as the maximum dose.

TREATMENT AND SAMPLING TIMES:
At sampling, animals were anaesthetized with Nembutal Sodium Solution. To obtain hepatocytes, the liver was perfused through the vena portae with EGTA solution (HBSS containing EGTA, Hepes, and gentamycin sulfate) followed by collagenase solution (Williams medium E (WME), containing Hepes, gentamycin sulfate, NaOH and collagenase Type IV) adjusted to pH 7.4 and maintained at 37 °C. The isolated hepatocytes were filtered and washed with cold WME twice. After that the cell suspension was centrifuged several times and the pellet was resuspended in WME. For culturing of cells, the cells were resuspended at the last step in WME supplemented with L-glutamine (2 mM) , gentamycin sulfate (50 µg/mL) , dexamethasone (2.4 µM) and Biochrom heat-inactivated fetal calf serum (FCS, 10%). Two cultures were established per animal. An aliquot of the cell suspension was used for the determination of cell viability and cell concentration by the method of trypan blue exclsion.

DETAILS OF SLIDE PREPARATION:
The cell suspension was seeded into 60 mm Petri-dishes for cell viability measurements (7.5E5 viable cells per dish) and into 6-well plates (3.75E5 vialble cells per well) for genotoxicity assessment. The six-well plates contained one 25 mm round plastic coverslip per well coated with collagen. Then, cells attached for 0.5 h (5% CO2, 37 °C). After that the culture medium was discarded and cells were rinsed with PBS. 3HTdR (10 μCi/mL, specific activity 15.3-15.6 Ci/mmol) was added in culture medium (supplemented with 1% FCS only, without dexamethasone) and labelling was performed for 4 h. Cells were washed with culture medium (1% FCS) and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. On the next day, the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate (5-10 min to swell the nuclei) and cells were fixed with ethanol:acetic acid (3+1 v/v, 3 times, 30 min total), rinsed with deionised water and air-dried. After that, cover slips were mounted on glass slides and coated with KODAK NTB-2 photographic emulsion in the dark. For drying, they were then placed in light-tight boxes in the presence of a drying agent for 11-14 days at -20 °C and the photographic emulsion was developed for 2-4 min in Kodak D-19 developer below 15 °C, fixed in Kodak fixer, air dried and stained with hematoxylin/eosin.

METHOD OF ANALYSIS:
Per animal, three slides were investigated with at least 150 cells (50 per slide).
Nuclear net grain (NNG) count was calculated as follows: counting nuclear grains and subtracting
the average number of grains in 3 cytoplasmic areas of the same size as the corresponding nucleus. The number of cells in repair (nuclei with 5 or more net grains) was also determined.

Evaluation criteria:

Acceptance
An assay was considered acceptable if
- the viability of the hepatocytes in the vehicle control is > 70%
- viability of the monolayer cell cultures of animals treated with 0.5% aqueous Cremophor is > 65%
- sufficent cells are evaluable for data recording
- cytoplasmic background counts in hepatocytes of vehicle control animals are below 30 grains per nuclear-sized area
- the average NNG value in hepatocytes of vehicle control animals is between -8 and 0. No more than 5% of the cells should be in repair
- the positive controls yield values of 2-15 NNG per dose group with 5%-80% of the cells with
greater than or equal to 5 NNG

Evaluation Criteria
A test item is considered to give a positive response in the assay if NNG is 2 or higher (dose group average) and 11% or more of the cells respond.

A dose group average equal to or below 0 NNG is considered to be a negative response. Results between 0 NNG and 2 NNG have to be assessed on a case by case basis.

Statistics:

Not reported

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clincal signs as described.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE FINDING STUDY
The findings of the range finding study are discussed above. 5000 mg/kg bw was set as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
At 5000 mg/kg bw, animals showed roughened fur, palmospasm, shivering, rapid breathing, slitted eyes and twitching.

Viability and number of hepatocytes
No effect in any treatment group and time point

Mean nucleus, cytoplasmic area, and net grains
No effect in any treatment group and time point

The positive and solvent controls were in the range of historical control data.

HISTORICAL CONTROL DATA
Historical control data was provided by the laboratory and can be found in Attachment 3 in the attached background material

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce unscheduled DNA-synthesis. The study was performed under GLP and according
to a preliminary draft of the OECD guideline 486.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective endpoint summary. 

Additional information

To characterize the genotoxic potential of the test substance are available: Since different results were obtained, based on the testing of different batches of the same test item, the assessment of the mutagenic/genotoxic potential of the present compound was based on a weight of evidence approach. Regarding the genetic toxicity in vitro, data are available, covering bacterial mutagenicity (5 Ames tests, all RL 2) cytogenicity in mammalian cells (3 chromosome aberration tests, all RL 2) as well as gene mutation in mammalian cells (3 HPRT test and a mouse lymphoma assay, all RL2). Regarding in vivo studies, four micronucleus test, one comet assay and three unscheduled DNA synthesis assays were evaluated. Thereby, different batches of the test material were investigated. As mentioned above, the studies were conducted with several batches of the same test item differing in terms of purity. Thus, it cannot be excluded that the different results obtained might relate to the degree of purity. In the following, all studies will be described and evaluated. To protect the data confidentiality, no original batch numbers were reported in this endpoint summary, but batches were given numbers in ascending order.  

Gene mutation in bacteria

The gene mutation test in bacteria (Ames assay) was conducted according to OECD Guideline 471 and in compliance with GLP with a standard battery of tester strains including 5 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537, TA 102; M-103603-02-1). In an additional statement (M-582751-01-1), it was shown that the bacterial strains were in exponential growth during the test phase. The test substance (batch 1) was completely soluble in Dimethyl sulfoxide (DMSO) and no precipitation was observed at any concentration tested. In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate or tube. The pre-experiment was only conducted in strain TA 98 and 100. Since no toxic effects were observed in the pre-experiment, in experiment I (plate-incubation) and experiment II (pre-incubation), six concentrations (33, 100, 333, 1000, 2500 and 5000 µg/plate) were tested. Both experiments were performed with and without metabolic activation system (S9 mix). The test substance did not induce relevant increases in revertant colony numbers of 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 as the mean numbers of revertant colonies of the solvent controls fell within the range of historical data or were lower, and positive control treatments increased the number of revertants significantly. 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 TA 102 with and without metabolic activation.

In a further study conducted with another batch of the test item (M-36520-01-1, batch 2), 4 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537) and the Escherichia coli strain WP2uvrA were tested according to OECD guideline 471 and under GLP. The test substance was solubilized in DMSO and tested at concentrations of 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation using S9 mix. The selected concentration-range was based on a pre-experiment were these concentrations were tested on S. typhimurium TA 100 and E. coli WP2uvrA (without metabolic activation); no cytotoxicity was observed. Two independent experiments were performed, both as plate-incorporation tests with triplicates and an exposure duration of 48 h. No cytotoxic effects were observed up to the highest concentration tested. All solvent controls and positive control data fell in the range of the historical control data. In S. typhimurium TA 98, 100, 1537 and E. coli WP2uvrA, no substantial increase in revertant colony numbers was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. An increase was considered biologically relevant when it was 2-fold or higher compared to the control colonies. For S. typhimurium TA 1535, 5000 µg/plate caused a slight but significant increase in revertant colonies, above 2-fold with metabolic activation in both experiments (2.5- and 2.1-fold in experiment I and II, respectively). Without metabolic activation, the increase was 1.6- (experiment I) and 1.9-fold (experiment II). The positive control caused colony increases of 9.5- to 20.2-fold for this strain. However, it was noticed that the number of revertant colonies for S. typhimurium TA 1535 at 5000 µg/plate was equal or below the maximum number of revertant colonies within the historical control data. Therefore, the test material is considered mutagenic in bacteria, but the results regarding  S. typhimurium TA 1535 remain ambiguous. Accordingly, an additional comparative study with S. typhimurium TA 1535 was undertaken, using two different batches of the test item (M-009769-02-1). The study was conducted similarly to OECD guideline 471, but did not follow GLP; the batches used were batch 3 and batch 2. In the first experiment (plate incorporation), batch 3 was used from 1000-5000 µg/plate and batch 2 from 3000-7000 µg/plate. In the second experiment, both batches were used up to concentrations of 8000 µg/plate (pre-incubation of 20 min). Precipitation occurred at 8000 µg/plate. Weak bacteriotoxic effects were observed at 5000 µg/plate and above for both batches but no mutagenic effects were noticed up to the highest test concentration, both in the absence and presence of metabolic activation.

In a further study (M-009777-02-1), batch 4 was tested using the standard battery of tester strains including the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. The study was performed according to OECD guideline 471 and under GLP. During the experiment, six concentrations of the test substance (16, 50, 158, 500, 1581 and 5000 µg/plate) were tested in the presence and absence of a metabolic activation system (S9 mix). These concentrations were used both for the plate-incorporation as well as for the pre-incubation assay. Since for S. typhimurium TA 102, the colonies count at 50 µg/plate in presence of S9 mix was found to be about 100 colonies higher that the control count, the pre-incubation test was repeated for this strain only, with concentrations ranging between 16 and 110 µg/plate. The test substance did not induce relevant increases in revertant colony numbers for any tester strain in the absence or presence of metabolic activation. The additional test with S. typhimurium TA 102 did not confirm the finding of the second experiment. No cytotoxicity was observed up to the 1581 µg/plate, at 5000 µg/plate weak bacteriotoxic effects were observed (strain-specific). The controls were considered valid, as a marked increase in the revertant colonies was observed for the positive controls whereas the spontaneous revertant colonies of the solvent control was within the variance of the laboratories background data. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA102, TA 1535 and TA 1537 with and without metabolic activation.

This is in line with a fifth gene mutation test in bacteria (M-036420-01-1), using a further batch of the test material (batch 5). In this study, no guideline is mentioned but the conduct was similar to the OECD guideline 471 and the assay was conducted under GLP. However, no historical data were provided. The mutagenicity of the test item towards five bacterial strains was tested (Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA) at five concentrations (100, 500, 1000, 2000, 5000 µg/plate) in a first experiment that did not show any bacteriotoxic or mutagenic effects. In the second (main experiment) the concentrations used were 313, 625, 1250, 2500, 5000 µg/plate. Also in this experiment, no bacteriotoxic effects or mutagenic effects were observed. Both experiments were conducted as plate incorporation tests. Solvent and positive controls were considered as valid. So under the conditions of the test, the test item was not considered mutagenic in bacteria.

In conclusion, five reliable bacterial mutagenicity studies, almost conducted according the OECD guideline 471 under GPL conditions, are available. Testing within these studies was basically conducted with the same test item, however different batches were used, with purities ranging between 95 and 99.8%. Almost all tests used the standard battery of tester strains consisting of the Salmonella typhimurium strains TA98, TA 100, TA 1535, TA 1537, TA 102 and/or Escherichia coli WP2uvrA. Testing was done in presence and in absence of metabolic activation (S9 mix).

A positive result was reported in one study only, with batch 2, only for the S. typhimurium strain 1535 (study M-36520-01-1). The evaluation criteria in this study were set as follows: a test item is considered as a mutagen if a dose-related and statistically significant increase in the number of revertant colonies is observed. The increase should exceed the threshold of twice the colony count of the corresponding solvent control and should occur at sub-toxic dose levels. In present case, the highest tested concentration of  5000 μg/plate caused a slight but significant increase in number of revertant colonies with and without metabolic activation, and in the absence of any obvious cytotoxicity. The increase, depending on the experiment (two experiments done), ranged between 1.6-fold and 1,9-fold in the absence of S9 mix, and between 2.1 and  2.5-fold in the presence of S9 mix. Thus, especially in presence of metabolic activation, the criteria for a positive response were fulfilled for S. typhimurium TA 1535 treated with one of five batches (batch 2).  Nevertheless, it was noticed that the number of revertant colonies for TA 1535 at 5000 μg/plate was equal or below the maximum number of revertant colonies within historical control data. Thus, the positive result in this study remains ambiguous.

The comparison of this result with those of the remaining studies, which were negative for all tested bacteria strains,  lead to the assumption  that the findings rather might be related to the impurity fraction in the tested batches, as the batches differed from each other with respect to their purities.

For purpose of verification and confirmation, an additional comparative study with S. typhimurium TA 1535 was undertaken, using two different batches (batch 2 and 3) of the test item (M-009769-02-1). The study was conducted similarly to OECD guideline 471, but did not follow GLP; and the highest test concentration was set at 8000 µg/plate. For both batches, no increase in number of revertants colonies was noticed in this study, in presence as well as in absence of S9 mix. Thus, it remains unclear whether the positive result reported above rather was incidental or related to the impurity fraction of the test item batch used.

Thus, all data taken together do not provide sufficient evidence that would support the assumption of a bacterial mutagenic potential for the present compound.

 

Cytogenicity in mammalian cells

A GLP-compliant study was performed similar to the OECD guideline 473 to determine clastogenic properties of the test substance (batch 4) by chromosomal aberration assay (M-105153-01-1). V79 cells were exposed to the test substance in the presence and absence of metabolic activation at concentrations of 250, 500, 750, 1000 and 2000 µg/mL for 4 hours. After treatment, cells were incubated for further 14 hours in complete cell culture medium without the test substance for recovery. The concentrations were chosen based on a pre-experiment in which concentrations from 19.5 to 2500 µg/mL were tested for 4 (+/- S9 mix) and 24 h (- S9 mix). Precipitation of the test item occurred from 1250 µg/mL (+/- S9 mix).  Cytotoxicity was defined as reduced number of cells (< 50%). Without S9 mix, cytotoxicity was observed at 625 and 2500 µg/mL (4 h) and at 1250 and 200 µg/mL (24 h). With metabolic activation, no cytotoxicity was observed up to 2500 µg/mL. Based on this results, 2000 µg/mL was chosen as the top concentration in this experiment. Since the test item was considered to be clastogenic after 4 h treatment a second experiment was not performed.

The main experiment without metabolic activation by S9 mix had to be repeated because the first trial did not result in evaluable solvent, negative and positive controls. For each concentration, 200 metaphases were evaluated with the exception of 500 µg/mL with S9 mix. In this case, 400 metaphases were assessed due to inhomogeneity of the first 200 evaluated cells. Hereby it should be noticed that since the study was conducted according to a previous version of the OECD guideline (version 1997), 200 metaphases were examined rather than the 300 recommended by the newest version of this guideline (version 2016). Nevertheless, this does not affect the reliability of the study data. From the six concentrations tested, 750-2000 µg/mL without S9 mix and 500-1000 µg/mL with S9 mix were evaluated. The higher concentrations in presence of S9 mix produced marked cytotoxicity in the cells so that evaluation was not possible. The cytotoxicity was evident by reduced cell numbers and reduced mitotic indices.  Additionally, in the absence of S9 mix, reduced cell numbers were also observed at 1000, 1500 and 2000 µg/mL (51, 52 and 45%, respectively).

With metabolic activation, no significant structural chromosomal aberration was noted. The only statistical significant increase was seen at 500 µg/mL (3.8%). However, this was not dose-dependent and within the historical control data (0.0 - 4.0%) and was therefore considered incidental. In the absence of S9 mix, chromosome aberration was increased at 1500 and 2000 µg/mL (8.0 and 24.0%, respectively) and the number of cells with exchanges was dose-related increased (from 0.0 to 10.5%). The positive controls were considered valid, as mitomycin C and cyclophosphamide had a clear clastogenic effect. In total, the test item showed a clastogenic effect without S9 mix at cytotoxic concentrations of 1500 and 2000 µg/mL.

As noticed above, the study needed to be repeated (M-103614-01-1) with V79 cells according to OECD guideline 473 (1997) and GLP. Here, two experiments were performed using another batch of the test item (batch 1). In the first, V79 cells were exposed to 250 – 2000 µg/mL test substance for 4 h in the absence and presence of S9 mix, followed by 14 h of recovery time. In the second experiment, three different approaches were used: a) exposure to 100-1000 µg/mL test substance for 18 h without S9 mix and without recovery, b) exposure to 400-1000 µg/mL test substance for 28 h without S9 mix and without recovery, c) exposure to 250-2000 µg/mL test substance for 4 h with S9 mix and 24 h recovery. The concentrations were based on the pre-experiment described for M-105153-01-1. Precipitation was observed from 1000 µg/mL in experiment I and from 750 µg/mL in experiment II c). Cytotoxicity occurred at 2000 µg/mL with and without S9 mix, so that this concentration was not used for evaluation. Moreover, mitotic index was reduced from 400 µg/mL in experiment II a) and from 600 µg/mL in experiment II b).

No biologically relevant increase in the number of chromosome aberrations was observed with and without metabolic activation in both experiments. Only two statistically significant increases were seen at 1500 (experiment I) and 1000 μg/mL (experiment II) in the presence of S9 mix (each 3.0%, p < 0.05). Since the finding was in the range of the historical control data (0.0 - 4.0%) and also similar to the response obtained from the negative control in experiment I (3.0%), it is not to be considered biologically relevant. The positive controls were valid, as mitomycin C and cyclophosphamide had a clear clastogenic effect. In total, the test item showed no clastogenic effect with and without S9 mix.

A third, OECD guideline 473 (version 1983) study performed according to GLP (M-036479-02-1), was conducted with CHL cells, using test item batch 2. Two experiments were done: in experiment I, harvest time was 12 h, either after 12 h of continuous exposure without metabolic activation or 4 h exposure with metabolic activation followed by 8 h recovery. In experiment II, in addition to the treatments for experiment I, 6 h treatment with 18 h recovery was carried out in the absence and presence of S9 mix and exposure without metabolic activation was extended to 24 and 48 h (no recovery period). The concentrations of the test material ranged from 39 – 1875 µg/mL. These concentrations were based on a pre-experiment which showed that the test item caused dose-dependent cytotoxicity after different treatment times and precipitated at a concentration of 2500 µg/mL. Cytotoxicity was similar in experiment I and II, increasing with concentration and time of exposure. For example, in the 48 h treatment group, cell number was reduced to 50% at 315.5 µg/mL, in shorter exposure times, cytotoxicity was observed in higher concentrations of for example 1250 µg/mL (6 h treatment, 18 h recovery, - S9 mix). In general, mitotic indices were more sensitive to indicate cytotoxicity.

In experiment I, no influence on chromosomal aberration frequency was found. However, in experiment II, high statistically significant increased frequencies of chromosomal aberrations were found in the highest concentration groups under the following test conditions: 6 h exposure with metabolic activation and 18 h recovery at 1875 µg/mL (highest concentration tested) and for 48 h exposure without metabolic activation at 625 µg/mL (highest concentration tested). In the following conditions, small statistically significant increases were found: 6 h exposure without metabolic activation, followed by 18 h recovery (1250 µg/mL, highest concentration tested) as well as 12 h exposure (with and without S9 mix, 1562.5 and 937.5 µg/mL, respectively) and at 625 µg/mL in the 24 h treatment group (middle concentration). In the latter, the highest dose did not result in higher increases of chromosomal aberrations. No increased polyploidy was found in any treatment group neither in experiment I nor II.

The controls were considered valid, even though in the 12 h harvest time point (experiment I and II without S9 mix), the increase was only weak. This was considered typical for the 12 h time point and a result of toxicity-induced cell-cycle delay.

Under the conditions of the assay, the test material induced clastogenic effects in CHL cells in vitro.

 

In conclusion, three reliable chromosome aberration studies are available. All these studies were conducted under GLP according to OECD guideline  473 versions dated prior to 2014; accordingly, only 200 metaphases were scored instead of 300 as recommended by the actual version of the guideline. Nevertheless, these data are considered reliable. Testing within these studies was basically conducted with the same test item, however different batches were used, . From the three batches tested it is apparent that two batches induced clastogenic effects in V79 and CHL cells, while the third batch did not. Concentrations and incubation times were similar, even though the last study increased incubation time further to 48 h. It has to be noted that in the last study, higher chromosomal aberration rates occurred at cytotoxic levels. The results are therefore questionable. However, batch 2 already induced mutagenic effects in the Ames test as described above while batch 4 did not. Thus, it remains unclear whether the positive results might be related to the impurity fraction in the tested batches. All data taken together only provide an ambiguous evidence for clastogenicity to be expected from the present compound.

 

Gene mutations 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 (1997) and GLP (M-103610-01-1). It has to be noted that the number of cells seeded was lower than required by current version of the guideline (2016) and the calculation of mutagenic frequency differed.

In experiment I, V79 cells were treated with 78.1 - 2500 µg/mL of the test substance (batch 1,) in the absence and presence of metabolic activation for 4 h. The concentration ranged from 78.1 to 2500 µg/mL and was based on a pre-test were the test item did not show any cytotoxicity up to 2500 µg/mL. In experiment II, the same concentrations and cells were used, but the exposure period was prolonged to 24 h and no S9 mix was added. 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 change in mutant frequency. Only in experiment I at 1250 µg/mL without metabolic activation, the mutant frequency was increased in one of the duplicate cultures (above the historical control data). However, this was seen in only one culture, the induction factor did not reach the threshold of three times the corresponding solvent control and the effect could not be reproduced in the second experiment. Therefore, the finding was considered incidental. 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, provided in an additional statement (M-581971-01-1). In total, the test item is considered to be non-mutagenic in the HPRT test using V79 cells with and without metabolic activation.

In a similar study (M-105147-01-1), another batch was tested (batch 4) in an HPRT assay using V79 cells. This study was also performed according to the previous version of the OECD guideline 476 (1997), under GLP conditions. A pre-test was conducted to assess suitable concentrations for the test. The test item was found to be non-toxic after 4 h of treatment with and without metabolic activation but induced severe cytotoxicity after 24 h of treatment (- S9 mix) at 1250 and 2500 µg/mL. Therefore, the tested concentrations ranged from 78.1 to 2500 µg/mL in experiment I (4 h exposure, +/- S9 mix) and 62.5-1500 µg/mL in experiment II (24 h exposure, -S9 mix). Under all testing conditions, duplicate cultures were used to assess mutagenicity. Precipitation was observed at 1250 (-S9) and 2500 µg/mL (+/- S9) in experiment I and at 1500 µg/mL in experiment II. pH and osmolality remained unaltered. In experiment I, cytotoxicity occurred at 1250 and 2500 µg/mL (- S9) based on reduced cloning efficiency I (survival). Even though CEI was under 10% in experiment I for both cultures of 1250 µg/mL and one culture of 2500 µg/mL (- S9) and in experiment II for all concentrations from 750 µg/mL on, well above this limit. These discrepancies are often found between low density (used for calculating CEI) and mass cultures (used for mutagenicity) and more emphasis was put on the mass cultures. In experiment II, cytotoxicity was observed at 500 µg/mL and above. With regard to mutagenicity, no relevant increases in mutant frequencies were found. An increased induction factor (above the threshold of three times induction) occurred at 1250 and 2500 µg/mL (- S9, experiment I) and 1000 µg/mL (- S9) in experiment II in one of the duplicate cultures. However, this was not reproduced in the second culture and for experiment I, the solvent mutant frequency was considered rather low. Therefore, all the described findings were considered incidental. Solvent, negative and positive controls gave the expected outcome and were within the historical control data range. The historical control data was provided in an additional statement (M-589714-01-1). In total, the test item is considered to be non-mutagenic in the HPRT test using V79 cells with and without metabolic activation.

A further study (M-036462-02-1) assessed the mutagenicity of the test item (batch 2) to the TK locus of the mouse lymphoma cell line L5178Y. Two experiments were conducted with and without metabolic activation by S9 mix and an incubation time of 3 h each. In the first experiment, cells were incubated with 312.5, 625, 1250, 1667 and 2500 µg/mL, while in experiment II, 300, 600, 1200, 1600, 2000 and 2400 µg/mL were used. Mutation frequency served as an indicator for mutagenicity and relative survival for assessment of cytotoxicity of the test item. The study was conducted under GLP and according to OECD guideline 476 (1997). In contrast to the requirements of the current  version of the guideline (2016), the calculations were based on Cole and Arlett (1984). Small (representing large genetic changes involving chromosome 11b and suggesting clastogenic activity) and large (representing base-pair substitutions or deletions) colonies were counted. It was found that the test item caused toxicity especially in the absence of the S9 mix. Without metabolic activation, the concentrations of 2500 µg/mL (experiment I) as well as 2000 and 2400 µg/mL (experiment II) needed to be excluded from the evaluation due to severe toxicity.

All controls gave expectable results and historical control data were provided. In experiment I, no statistical significant increase of mutant frequencies occurred. However, the highest evaluable dose (1667 µg/mL) led to increased mutant frequencies both in the presence and absence of S9 mix. They were not statistically significant but higher than historical control values for the solvent controls. In experiment II, mutant frequency increased significantly and in a concentration-dependent manner both in the absence and presence of S9 mix. Significant higher frequencies occurred at 1600 µg/mL (- S9) and at 2000 and 2400 µg/mL (+S9). These increases were mostly due to small colonies indicating clastogenic activity of the test item. However, at these concentrations, relative survival was already below 50%, so that the concentrations were considered cytotoxic. Nevertheless, under the conditions of the test, the test item was considered mutagenic.

To re-test the test item batch used in study M-036462-02-1, a HPRT test was conducted with V79 cells according to OECD guideline 476 (1997) and under GLP. Two experiments were conducted with and without metabolic activation by S9 mix and an incubation time of 5 h each. The concentrations were 156, 313, 625, 1250, 2500 and 5000 µg/mL and based on a pretest in which cytotoxicity was seen only at 2500 µg/mL without metabolic activation. In the main experiment, no cytotoxicity was seen, but the test item precipitated at 2500 µg/mL and above. Also, the test item did not cause any significant increase in mutant frequency, neither in the absence nor presence of metabolic activation. Occasional increases in mutant frequency (namely in one culture at 156 µg/mL without metabolic activation and in one culture at 2500 µg/mL with metabolic activation) were considered incidental since they were not reproducible and within the range of the historical control data. All controls gave expectable results and historical control data were provided. Therefore, the test item is considered non-mutagenic under the conditions of the assay.

 

In conclusion, four reliable in-vitro mammalian cell gene mutation studies are available, comprising 3 HPRT tests and a mouse lymphoma assay (MLA). All these studies were conducted under GLP according to the OECD guideline  476 version dated 1997. Accordingly, some requirements of the current version of the guideline (2016) are not fulfilled. However, this does not affect to quality and reliability of the results obtained from these studies.

Testing within these studies was basically conducted with the same test item, however different batches were used. In the HPRT test, none of the test material batches caused gene mutation. In the MLT assay assessing the TK locus, the tested batch (batch 2) caused mutagenic effects at concentrations similar to those used in the other assays. However, these were close to or already at the cytotoxic level. The results are therefore questionable and it remains unclear whether the positive results might be related to the impurity fraction in the tested batches. Thus, there is no clear evidence for a gene mutation potential to be expected from the present compound in mammalian cells.

 

Conclusion on genetic toxicity in vitro:

Several in vitro studies are available, covering bacterial mutagenicity, as well as clastogenicity and gene mutation in mammalian cells. The studies were conducted with batches of the same test item, however, differing with respect to their purities. In the Ames test, one of five tests gave a positive result at the highest concentration, using batch 2. In fact, a slight positive result was obtained for the S. typhimurium strain 1535. However, this finding could not be reproduced in an additional comparative study testing two batches (batch 2 and 3) on S. typhimurium TA 1535. In the chromosome aberration assays, two tests were positive for clastogenic effects of the test substance (with batch 4 and batch 2) In a further study using batch 1, an increase in chromosome aberrations was noticed, which was statistically significant but within the range of historical control data. Thus, in this case a weak clastogenic effect was noticed, which however remains questionable. Gene mutation HPRT assays in mammalian cells resulted in no effect V 79 cells treated with different batches. In contrast, positive results were found in the mouse lymphoma assay using batch 2. Thus, most of the positive in vitro genotoxicity results were reported at test concentrations in the level of cytotoxicity. It also has to be noted that batch 2 and 4 had generally lower test substance purities (below 97%) than the other batches tested; this would support the assumption that positive results, in fact, were related to the impurity fraction present in each batch, rather than to the test item as such.  

 

MNT assay in vivo: rat

The test substance was assessed for its potential to induce micronuclei in bone marrow cells of rats by a GLP-compliant study according to OECD Guideline 474 (M-618171-01-1). Male Crl:CD(SD) (SPF) rats were orally treated with vehicle alone (negative controls) or 500, 1000 or 2000 mg/kg bw/day of the test substance on 2 successive days, approximately 24 hours apart. The batch used was one that was not tested in the in vitro studies (batch 6). The animals were sacrificed 24 h after the last dosing and bone marrow cells were prepared for evaluation. No animals died prematurely or showed clinical signs of toxicity. However, body weight and body weight gain were reduced in all treatment groups.

Rats dosed with cyclophosphamide served as positive control and revealed statistically significant increases in the number of micronucleated polychromatic erythrocytes (PCEs) compared with the concurrent vehicle control group. Both the vehicle and positive controls gave frequencies of micronucleated cells within the historical control data range. In addition, there were no statistically significant increases in incidents of micronucleated PCEs in male rats at any treatment level, but the ratio of PCEs in total erythrocyte counts was decreased in all treatment groups. These findings suggested that test substance treated animals showed evidence of bone marrow toxicity, demonstrating systemic exposure of the bone marrow to the test substance. In total, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the maximum dose level of 2000 mg/kg bw/day recommended by OECD guideline 474 in male rats. The test substance is considered to be neither clastogenic nor aneugenic in the rat bone marrow micronucleus assay.

 

Comet assay in vivo: rat

As the test substance was considered to be mutagenic in one of the Ames assays and in the Mouse Lymphoma Assay, an in vivo genotoxicity assay in somatic mammalian cells according to OECD 489 and GLP was conducted (M-589564-01-1, batch 6). According to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a (ECHA; version 6.0, 2017), the in vivo Comet Assay is an appropriate test for investigating potentially reactive substances that may express their mutagenic potential at the initial site of contact in the body and in potential target organs. The Comet Assay is one of the study protocols referred to as a reliable in vivo study capable of detecting gene mutations. It is therefore a suitable in vivo test method to further investigate the mutagenic potential of the test item in vivo as follow-up to the positive Mouse Lymphoma Assay.

The same batch of the test substance as in the MNT in vivo (M-618171-01-1) was used in this alkaline comet assay. In a range finding experiment, 3 male and female rats either received the test substance (2000 mg/kg bw/day) or the vehicle control (0.5% cremophor EL) orally via gavage. Neither severe toxicity nor substantial differences in toxicity between sexes were found, so that 2000 mg/kg bw/day was used as the highest dose level in the main experiment as recommended by OECD 489. Two additional doses of 500 and 1000 mg/kg bw/day were included in the study. For the main experiments, only male rats were used in groups of 6 per treatment (4 for the positive control). They received the test substance orally via gavage on two consecutive days with an interval of 21 h between administrations. 3 h after the last administration, they were sacrificed and kidney, liver and stomach cells were used for comet analysis (150 cells per animal and organ).

At 1000 and 2000 mg/kg bw/day, body weight was reduced and tremors occurred. At 2000 mg/kg bw/ day, one animal showed ptosis. Apart from that, no signs of toxicity were observed, neither in the gross necropsy nor in DNA damage. %tail DNA was similar to the control in all treatment groups, and incidences of hedgehogs (ghost cells) were unaltered. The positive control ethylmethanesulphonate, led to the expected result. The solvent control and the positive control were within the range of the historical control data provided by the laboratory. Under the conditions of the test, the test item did not cause DNA damage in kidney, liver and stomach in rats.

 

Cytogenetic assay in vivo: mouse

The test substance (batch 4) was assessed in male and female NMRI mice for a possible genotoxic effect on bone marrow cells by a GLP-compliant study according to OECD Guideline 474 (M-105161-01-1). Mice received a single intraperitoneal administration of 50, 100 or 200 mg/kg bw of the test substance in 0.5% cremophor aqueous solution. The doses were based on a range-finding study in which two animals of each sex received 100, 200, 300 and 500 mg/kg bw of the test substance in a single i.p. treatment. In the range-finding study, deaths were observed at 300 mg/kg bw and above, so that 200 mg/kg bw was set as the highest dose in the main experiment.

The groups consisted of six animals of which 5 were used for bone marrow preparation. All animals were checked for clinical signs 1, 2 - 4, 6, and 24 h after administration. The bone marrow was prepared 24 h after administration. For the highest dose group, an additional sampling time point was set at 48 h after treatment. Cyclophosphamide was used as a positive control and was also administered i.p. (40 mg/kg bw). All solutions were injected at a volume of 10 mL/kg bw.

Animals of all treatment groups showed clinical signs after treatment, namely reduction of spontaneous activity, abdominal position, eyelid closure and ruffled fur. In the highest dose group, one male showed tremor and two males died 6 h after administration. The positive control was considered valid, as a clear genotoxic effect was evident by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The test substance did not lead to a relevant increase in the number of micronucleated polychromatic erythrocytes in any of the groups. Based on the in vivo micronucleus test, there were no indications of clastogenicity after single i.p. treatment of the mouse with the test substance.

The same test set-up was used for an additional study (M-103617-01-1) in the same mouse strain with batch 1. The only difference in test design were the dose levels applied to the animals, i.e. 75, 150 and 300 mg/kg bw. These doses were also based on a range finding experiment in which 300 mg/kg bw was defined as the maximum tolerated dose. In the main study, animals of all treatment groups showed clinical signs after treatment which were comparable to those seen in the previous study (M-105161-01-1, reduction of spontaneous activity, abdominal position, eyelid closure and ruffled fur, tremor). In the highest dose group two males and one female died 6 h after administration. Both vehicle and positive control were considered valid since they showed results in the range of the historical control data.  The test substance did not lead to a relevant increase of the number of micronucleated polychromatic erythrocytes in any of the groups. Based on the in vivo micronucleus test, there were no indications of a clastogenic effect after single i.p. treatment of the mouse with the test substance.

Negative results were also obtained in mice with batch 2 (M-036435-02-1, ). In this study, the test substance was administered orally in a single gavage dose of 25, 50 and 100 mg/kg bw in arachis oil BP and animals were killed for collection of bone marrow cells 24, 48 and 72 h after administration. For each time point and dose group, 5 males and 5 females were used. The doses were based on a range finding experiment in which 100 mg/kg bw was determined as the maximum tolerated dose. Hunched posture, decreased respiratory rate, labored respiration, ptosis and lethargy were found in animals of all treatment groups at all time points (24, 48 and 72 h). In the test group that received 100 mg/kg bw of the test item and was sacrificed 48 h after administration, three females died prematurely. No relevant increase in the number of micronucleated polychromatic erythrocytes was observed in any of the groups and the ratio of PCEs to NCs remained unaffected. According to this test, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the MTD of 100 mg/kg bw in mice.

 

As all three studies in mice were conducted according to an older version of the OECD guideline 474 (1983 or 1997), there were deviations from the current version of OECD guideline 474, especially in the number of evaluated cells: only 2000 or 1000 (M-036435-02-1) PCEs were scored per animal instead of 4000 as specified in the current version.

 

Conclusion on clastogenicity in vivo:

The positive findings regarding clastogenic effects seen in vitro in several chromosomal aberration tests were not confirmed in four in vivo micronucleus tests investigating the potential of the test substance to induce clastogenic or aneugenic effects in the bone marrow of rats and mice. In these studies the same batches were investigated as in the in vitro chromosomal aberration assays in which the test materials had increased the frequency of chromosomal aberrations. No evidence of genetic toxicity was found in any of the micronucleus tests in vivo. In addition, a comet assay in vivo according to OECD 489 was conducted in rats investigating the potential for DNA damage in the target organs liver and kidney as well as in the stomach (site of first contact). In this test no DNA damage indicative of chromosomal aberrations or gene mutations was found.

However, as most of the gene mutation assays in vitro (HPRT, MLA) had given positive results, appropriate in vivo studies had to be conducted in order to confirm or disprove these results. Besides the Comet Assay in vivo, the liver UDS assay in vivo is another adequate and reliable in vivo test which can be used for detecting gene mutations in somatic cells, under the presumption that the liver is a target organ for the substance (Guidance on Information Requirements and Chemical Safety Assessment, R.7a, 2017). Therefore, three liver UDS assays were performed.

 

UDS (Unscheduled DNA Synthesis) assays

Three UDS assays were conducted with the three batches that yielded positive responses in the in vitro gene mutation studies.

In the first assay the test substance (M-103622-01-1, batch 1) was administered orally to fasted male Wistar rats in a single gavage dose of 1000 and 2000 mg/kg bw. 0.5% cremophor aqueous solution served as the vehicle. Two time points for cell preparation were chosen: 2 h and 16 h after administration. N,N´ dimethylhydrazinedihydrochloride (DMH) and 2-acetylaminofluorene (2-AAF) were used as positive controls for the 2 and 16 h time point, respectively. Groups consisted of 4 animals per treatment and dose and the dose levels were based on a range finding experiment where no mortality and only minor clinical signs were observed in male and female rats after receiving 2000 mg/kg bw of the test substance. Since no gender difference was found, only male rats were used in the main experiment.

For cell preparation, rats were anaesthetized and the liver was perfused. Primary hepatocytes were then cultured and incubated with ³H-TdR for 4 h, followed by overnight incubation with culture medium and unlabeled thymidine. After that, cells were rinsed, fixed and dried and exposure to a radiographic emulsion took place in the dark for 14 days.   

No animals died during the study and only minor clinical signs were observed in both groups receiving the test substance, namely reduction of spontaneous activity and ruffled fur and – for the 16 h preparation group – eyelid closure. Viability and number of hepatocytes, mean net nuclear grain counts remained unaltered. Positive and solvent controls were acceptable.

The study was conducted according to OECD guideline 486 and under GLP. Under the experimental conditions reported, the test item did not induce any unscheduled DNA synthesis.

A study with the same set up was conducted for batch 4 (M-105184-01-1). There were no deviations from OECD guideline 486 and the study was conducted under GLP. The same doses, fasting periods, dosing frequency and preparation intervals were used and the number of cells evaluated was equal to the previous study (M-103622-01-1). In this study, similar results were obtained. No mortality occurred, clinical signs were restricted to reduction of spontaneous activity, ruffled fur and eyelid closure. Viability and number of hepatocytes, mean net nuclear grain counts were not changed by treatment with the test substance at 1000 and 2000 mg/kg bw. Positive and solvent controls were acceptable. Under the conditions of the test, the test item did not induce DNA repair in liver cells of treated rats.

A third study also yielded negative results (M-009751-03-1). Here, batch 2 was administered orally to male Wistar rats (at least 4 per group) in a single gavage dose of 2500 and 5000 mg/kg bw. These dose levels were based on a pilot study in which rats received 4000 and 5000 mg/kg bw of the test substance and only minor clinical signs were observed up to 72 h after treatment. In the main study, rats were killed 4 or 16 h after administration and cells were isolated similarly to the procedure described above. Differences were the choice of antibiotics in the culture medium (gentamycin) and developing and fixing solution. 150 cells per animal were analyzed. At 5000 mg/kg bw, rats showed roughened fur, palmospasm, shivering, rapid breathing, slitted eyes and twitching. Apart from that, no detrimental symptoms were observed. Viability and number of hepatocytes, mean net nuclear grain counts were not changed by treatment with the test substance at all dose levels and time points. Positive and solvent controls were acceptable.

The study was conducted before the OECD guideline 486 was published but was performed according to a draft of this guideline. It can therefore be considered comparable to a guideline study. GLP practices were followed. Under the conditions of the test, the test did not induce DNA repair in liver cells.

 

Conclusion on genetic toxicity in vivo:

The positive findings regarding clastogenicity and gene mutations in vitro could not be confirmed by in vivo testing. All available in vivo tests, i.e. micronucleus, UDS and Comet assay, gave no indication for clastogenic or aneugenic effects or a potential to cause gene mutations of the test material (all batches tested). The fact that all in vivo experiments yielded negative results justifies the conclusion that the test article has no genotoxic or mutagenic potential and no further testing is required.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive and do not warrant classification.