1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic in S. typhimurium TA 98, , TA 100, TA 1535, TA1537 and E. coli WP2uvrA.
- CHO/HPRT Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): not mutagenic.
- HL/MNT in vitro (OECD 487, GLP, K, rel. 1): not clastogenic and not aneugenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 12 to August 29, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on August 30, 2005/ signed on November 21, 2005)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 from liver of male Sprague- Dawley rats induced with phenobarbitone/β-naphthoflavone (oral)

Test concentrations with justification for top dose:

Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix using the direct plate incorporation method.
Range finding study: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and 50, 150, 500, 1500 and 5000 µg/plate in E. coli WP2 uvr A, with and without S9-mix using the direct plate incorporation method.
Main study: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and 50, 150, 500, 1500 and 5000 µg/plate in E. coli WP2 uvr A, with and without S9-mix using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley and Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Range finding study and mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

OTHERS:
- After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

No data

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: Test material was immiscible in sterile distilled water at 50 mg/mL.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY:
The test material was non-toxic to the bacterial background lawns of the strains of bacteria used (TA 100 and WP2uvrA-). However, a substantial decrease in the frequency of revertant colonies was observed in TA 100.

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation test: The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains initially at 500 µg/plate with and without S9. No toxicity was observed in strain WP2uvrA-, with or without S9. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

Table 7.6.1/2: Preliminary toxicity study

S9 mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
- S9	TA 100	88	95	87	82	67	88	84	75	56	42	10
+ S9	TA 100	109	71	101	101	109	74	79	103	88	77	12
- S9	WP2 uvr A	19	22	13	14	24	15	12	24	21	21	21
+ S9	WP2 uvr A	31	20	27	25	26	24	29	18	30	22	25

Conclusions:

The test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assayand ranged between 5 and 5000μg/plate, depending on strain type. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels (5 and 15 μg/plate) were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains initially at 500 µg/plate with and without S9. No toxicity was observed in strain WP2uvrA-, with or without S9. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January to 12 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 17 June 2015/ signed on 24 September 2015)

Type of assay:

other: hprt assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Chinese Hamster Ovary (CHO-K1) cell line, isolated by Kao and Puck (1967) and cloned by O’Neill et al. (1977) was used.
- Source of cells: ECACC, Salisbury, Wiltshire, UK
- Cell line has a high cloning efficiency (ca. 85%) and has a rapid doubling time (11 to 14 h). CHO-K1 cells have been demonstrated to be sensitive to many chemical mutagens.
- Stock of cells is stored in liquid nitrogen

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: A sample of cells was removed before the start of the study and grown in Hams F12 medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics, (penicillin/streptomycin at 100 units/100 μg per mL) at approximately 37 °C with 5% CO2 in humidified air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; cells are cleansed of mutants by culturing in HAT medium for four days. This is Hams F12 growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT-free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2% S9; S9 was prepared from rats induced with Phenobarbital/β-Naphthoflavone

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL for the 4 h exposure without and with metabolic activation. However, due to excessive toxicity being demonstrated in the absence of S9, this exposure group was repeated using the following concentrations: 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 μg/mL

Mutagenicity Test:
- 4 h exposure without metabolic activation: 0, 1.5, 3, 6, 12, 16, 20, 24, 28 and 32 μg/mL
- 4 h exposure with metabolic activation: 0, 20, 40, 60, 80, 100, 120 and 160 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 50 mg/mL but was soluble in DMSO at 500 mg/mL in solubility checks performed in-house.
- Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethyl benzanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: Hams F12 medium

CELL CULTURE:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were grown in Hams F12 medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics, (penicillin/streptomycin at 100 units/100 μg per mL)
Cell density at seeding: Cells were seeded at 1 x 10^7/225 cm2 flask approximately 48 h before being exposed to the test or control items. This was demonstrated to provide at least 20 x 10^6 cells available for dosing in each flask, using a parallel flask counted for cell number at the time of dosing.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air

SELECTION AGENT (mutation assays): Hams F12 with 5% serum supplemented with 10 μg/mL 6-Thioguanine (6-TG),

NUMBER OF REPLICATIONS: Single culture for preliminary toxicity test; Duplicate cultures for mutagenicity test

NUMBER OF CELLS EVALUATED: 200 cells/flask were seeded for cloning efficiency and 2 x 10^5 cells were analyzed for mutant frequencies.

At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
- In triplicate at 200 cells/25 cm2 flask in 5 mL of Hams F12 with 5% serum to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
- At 2 x 10^5 cells/75 cm2 flask (10 replicates per group) in Hams F12 with 5% serum supplemented with 10 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were counted manually and the number recorded for each flask.
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
- The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

Rationale for test conditions:

Maximum dose tested was the maximum recommended dose level for a UVCB substance

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in either of the experimental exposure conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is considered to be concentration-related.
- The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in both of the experimental exposure conditions examined:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- There is no concentration related increase.
- The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

There is no requirement for verification of a clearly positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful. Discussion with the study monitor would normally occur in the event of any equivocal result in order to determine whether or not any further experimentation is required and/or agree on the overall study conclusions.

Statistics:

When there is no indication of any increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated.
- Precipitation: Yes

RANGE-FINDING/SCREENING STUDIES:
- A precipitate of the test item was observed at the end of exposure period at and above 156.25 µg/mL in the 4 h exposure group in the presence of S9. In the 4 h exposure in the absence of S9, precipitate was observed at and above 39.06 µg/mL at the end of the exposure period in the initial experiment. There was no precipitate observed in the repeat 4 h exposure group in the absence of S9 at the end of the exposure period.
- The toxicity response curve in both exposure groups was extremely steep. In the absence of S9 there was modest toxicity demonstrated at 16 µg/mL with a 20% reduction in cloning efficiency and the next higher dose level (32 µg/mL) had no surviving cells at the end of the exposure period and was therefore not plated. The 4 h exposure group in the presence of S9 demonstrated a 28% reduction in cloning efficiency at 78.13 µg/mL and there were no surviving dose levels at and above 156.25 µg/mL.
- The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 32 µg/mL in the absence of S9 and 160 µg/mL in the presence of S9.

HISTORICAL CONTROL DATA
- Vehicle control mutation frequency values were all within the acceptable historical control range

MUTAGENICITY TEST:
- A precipitate of the test item was seen at the end of the exposure period in the presence of S9 at 160 µg/mL. The level of toxicity observed in the Mutagenicity Test was considered to be similar to that observed in the Preliminary Toxicity Test. In the absence of S9 the dose levels of 24, 28 and 32 µg/mL were too toxic for plating for mutant frequency and Day 7 cloning efficiency. In the presence of S9, the dose levels of 120 and 160 µg/mL were too toxic for plating for Day 7 cloning efficiency and mutant frequency. Both exposure groups demonstrated adequate clonal survival at Day 0 at the maximum dose levels plated for mutant frequency to meet the acceptability criteria of the test.
- In the absence of S9, the toxicity curve very steep with a modest reduction in cloning efficiency of 11% at 16 µg/mL and an 83% reduction in cloning efficiency at 20 µg/mL. In the presence of S9 there was a clear toxicity dose response demonstrated by the Day 0 cloning efficiencies with a reduction in cloning efficiency of 17, 36 and 86% at 60, 80 and 100 µg/mL, respectively. There was no marked reduction in the Day 7 cloning efficiencies of either exposure group indicating full recovery from the test item-induced toxicity observed at the end of exposure period.
- There were no significant or dose related increases in mutation frequency per survivor in either the absence or presence of S9.

None

Conclusions:

Under the test conditions, test item was shown to be non-mutagenic to CHO cells at the HPRT locus.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to test item for 4 h, with and without metabolic activation (2 % S9).

 

The dose levels used in the Main Mutagenicity Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

4 h exposure without metabolic activation: 0, 1.5, 3, 6, 12, 16, 20, 24, 28 and 32 μg/mL

4 h exposure with metabolic activation: 0, 20, 40, 60, 80, 100, 120 and 160 μg/mL

Vehicle (dimethyl sulphoxide) and positive controls were included.

 

The assay acceptance criteria were achieved, in both exposure groups, in terms of cloning efficiency, number of cells plated, etc. The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

 

The test item demonstrated no significant increases in mutant frequency at any dose level, either in the absence or presence of metabolic activation. The dose range used in both exposure groups included the appropriate range of toxicity including one dose level that induced approximately >80% but <90% growth inhibition.

 

Under the test conditions, test item was shown to be non-mutagenic to CHO cells at the HPRT locus.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 28 to June 15, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 10, 2012/ signed on November 30, 2012)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

20% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male rats induced with Phenobarbitone/β-Naphthoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 8.61, 17.23, 34.45, 68.91, 137.81, 275.63, 551.25, 1102.5 and 2205 μg/mL; 4 h exposure with and without S9-mix; 20 h continuous exposure to the test item without S9-mix followed by a 28 h incubation period in treatment-free media

Experiment 1:
4 h exposure to the test item formulations without S9-mix, followed by a 28 h incubation period in treatment-free media: 8.5, 17, 25.5, 34, 51 and 68 μg/mL
4 h exposure to the test item formulations with S9-mix (2%), followed by a 28 h incubation period in treatment-free media: 17, 34, 51, 68, 85 and 102 μg/mL

Experiment 2:
20 h continuous exposure to the test item without S9-mix, followed by a 28 h incubation period in treatment-free media: 17, 34, 51, 68, 85 and 102 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in minimal essential medium (MEM) at 22.1 mg/mL but was soluble in DMSO at 220.5 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
The molecular weight of the test item was given as 220.5, therefore, the maximum dose level was 2205 μg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level. The purity of the test item was 96% and was accounted for in the test item formulations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: demecolcine

Remarks:

mitomycin C: 0.2 μg/mL; demecolcine: 0.075 μg/mL without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5 μg/mL with S9 mix

Details on test system and experimental conditions:

TEST SYSTEM: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 h under typical experimental exposure conditions.
The details of the donors used are: Preliminary Toxicity Test: male, aged 32 years; Experiment 1 and 2: male, aged 18 years

CELL CULTURE: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine (FBS) serum, at approximately 37 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) in Experiment 1, 20 h continuous exposure (-S9) in Experiment 2
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B.

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (4.5 μg/mL) was added to the cultures.
STAIN (for cytogenetic assays): 5 % Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the cytokinesis block proliferation index (CBPI) value expressed as a percentage of the vehicle controls.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Cytotoxicity = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture

OTHER:
The criteria for identifying micronuclei was that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Bi-nucleate cell were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

The following criteria was used to determine a valid assay:
Negative Control: The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the laboratory historical control data range. The level of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered to be valid.
Positive Control Values: All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9 mix.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analysis may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

PRELIMINARY TOXICITY TEST:
- A greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 275.63 μg/mL, in both the 4 h exposure groups. Cloudy precipitate was also seen at the end of the exposure period at 275.63 to 1102.5 μg/mL in the 4 h exposure group in the absence of S9 and at 137.81 μg/mL in the 4 h exposure group in the presence of S9. Cloudy precipitate and greasy/oily precipitate was observed in the parallel blood-free cultures at the end of the exposure, at and above 551.25 μg/mL in the 20 h continuous exposure group.
- Haemolysis was observed following exposure to the test item at and above 34.45 μg/mL in the 4 h exposure group in the absence of S9. In the 20 h continuous exposure group and the 4 h exposure group, in the presence of S9, haemolysis was observed at and above 68.91 μg/ml at the end of the exposure period. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 34.45 μg/mL in the 4 h exposure group in the absence of metabolic activation (S9). The maximum dose with binucleate cells present in the 4 h exposure group in the presence of S9 and in the 20 h continuous exposure group was 68.91 μg/mL. The test item induced marked evidence of toxicity in all three exposure groups.
- The selection of the maximum dose level was based on toxicity and was 68 μg/mL and 102 μg/mL for the 4 h exposure groups in the absence and presence of S9, respectively in Experiment 1. For the 20 h exposure group used in Experiment 2, 102 μg/mL was the maximum dose selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of micronuclei found in the solvent control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the 34 μg/mL in the absence of S9. In the presence of S9 the maximum dose level of the test item with binucleate cells suitable for scoring was 85 μg/mL. Precipitate was not seen at the end of exposure in either exposure group. Haemolysis was observed at the end of exposure at and above 25.5 μg/mL and 34 μg/mL in the absence and presence of S9 respectively.
- CBPI data confirm the qualitative observations in that a dose-related inhibition of CBPI was observed, and that 15% inhibition of cell proliferation was achieved at 34 μg/mL in the absence of S9. The higher dose level of 51 μg/mL in the absence of S9 did not have any binucleate cells for scoring due to the steepness of the toxicity curve. In the presence of S9 a dose-related inhibition of CBPI was observed and 47% inhibition of cell proliferation was achieved at 85 μg/mL with the dose level of 102 μg/mL having no binucleate cells for scoring due to toxicity. The maximum dose level selected for analysis of binucleate cells was 34 μg/mL and 85 μg/mL in the absence and presence of S9 respectively.

Experiment 2:
- The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the 68 μg/mL in the absence of S9. Precipitate was not seen at the end of the exposure period. Haemolysis was observed at the end of exposure at and above 68 μg/mL.
- CBPI data confirm the qualitative observations in that a dose-related inhibition of CBPI was observed, and that 52% inhibition of cell proliferation was achieved at 68 μg/ml. Therefore, the maximum dose level selected for binucleate cell analysis was 68 μg/mL where optimum toxicity was achieved.

Table 7.6.1/1: Cytokinesis block proliferation index (CBPI) and Micronucleus Data

Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Control CBPI	Micronuclei (MN) per 1000 Binucleate cells			%Cells with MN	Mean % Cells with MN
		Mono	Bi	Multi			1 MN	2 MN	>2 MN
Experiment 1 – 4 h Exposure Without Metabolic Activation (S9)
0	A	187	201	72	1.77	100	5	1	0	0.60	0.70
	B	180	240	80	1.80		8	0	0	0.80
17	A	191	242	67	1.75	97	3	0	0	0.30	0.40
	B	181	253	66	1.77		5	0	0	0.50
25.5	A	170	270	60	1.78	98	6	0	0	0.60	0.40
	B	183	252	65	1.76		2	0	0	0.20
34	A	217	250	33	1.63	85	7	0	0	0.70	0.60
	B	178	292	30	1.70		5	0	0	0.50
MMC 0.2	A	209	271	20	1.62	74	67	3	1	7.10	7.90***
	B	250	230	20	1.54		76	8	3	8.70
Experiment 1 - 4 h Exposure With Metabolic Activation (S9)
0	A	286	174	40	1.51	100	7	0	0	0.70	0.50
	B	265	188	47	1.56		3	0	0	0.30
51	A	247	187	66	1.64	137	7	0	0	0.70	0.55
	B	159	265	76	1.83		4	0	0	0.40
68	A	254	216	30	1.55	111	6	0	0	0.60	0.45
	B	222	235	43	1.64		3	0	0	0.30
85	A	397	94	9	1.22	53	8	0	0	0.80	0.75
	B	336	156	8	1.34		5	0	2	0.70
CP 5	A	379	119	2	1.25	51	81	6	4	9.10	8.20***
	B	357	138	5	1.30		65	6	2	7.30
Experiment 2 – 20 h Exposure Without Metabolic Activation (S9)
0	A	91	366	43	1.90	100	9	0	0	0.90	1.25
	B	94	366	40	1.89		16	0	0	1.60
34	A	137	335	28	1.78	94	9	0	0	0.90	1.05
	B	81	386	33	1.90		11	1	0	1.20
51	A	167	325	8	1.68	78	0	1	0	0.10	0.40
	B	142	358	0	1.72		6	1	0	0.70
68	A	290	208	2	1.42	48	5	0	0	0.50	0.35
	B	285	214	1	1.43		2	0	0	0.20
DC 0.075	A	189	282	29	1.68	115	36	5	6	4.70	4.20***
	B	40	229	231	2.38		31	6	0	3.70

 

MMC = Mitomycin C; CP = Cyclophosphamide; DC = Demecolcine; *** = P<0.001

Conclusions:

Under the test conditions, test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (2% S9 mix). In Experiment 1, test material was tested at the concentrations of 8.5, 17, 25.5, 34, 51 and 68 μg/mL (without S9-mix) and 17, 34, 51, 68, 85 and 102 μg/mL (with S9-mix) for a 4 h exposure time. In Experiment 2, test material was tested at the concentrations of 17, 34, 51, 68, 85 and 102 μg/mL (without S9-mix) for 20 h continuous exposure. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei. Preliminary toxicity test was performed before the main test.

 

All vehicle (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

 

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments using a dose range which included a dose level which achieved approximately 50% reduction in cytokinesis block proliferation index (CBPI) in the 4 h exposure group in the presence of S9 and in the 20 h exposure group. The toxicity curve in the 4 h exposure group in the absence of S9 was very steep so that there was only a modest reduction in CBPI at the maximum dose scored although the test item was tested to toxic limits.

 

Under the test conditions, test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6.1/1: Summary of Genetic Toxicity Tests:

 

Test n°	Test guideline / reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 (Safepharm, 2008)	Ames Test (OECD 471) K, rel.1	Gene mutation	TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA	-S9 +S9	Up to limit concentrations	-S9 : non mutagenic +S9 : non mutagenic
2 (Harlan, 2013)	CHO/hprt test (OECD 476) K, rel.1	Gene mutation	Chinese Hamster Ovary cells	-S9 +S9	Up to cytotoxic concentrations	-S9 : non mutagenic +S9 : non mutagenic
3 (Envigo, 2016	HL/MNT vitro (OECD 487) K, rel.1	Chromosomal aberration	Human lymphocytes	-S9 +S9	Up to cytotoxic concentrations	Not clastogenic Not aneugenic

Gene mutation Assays (Tests n° 1-2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammalian cells using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO/hprt test) (Test n°2). None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The substance does not induce forward mutations at the hprt locus in CHO cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The substance is therefore considered as negative for inducing forward mutations at the hprt locus in CHO cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

Chromosomal aberration (test n°3)

The clastogenic and aneugenic potential of the substance was determined using an in vitro human lymphocytes micronucleus assay (Test n°4), which identifies substances that cause micronuclei. These micronuclei may originate from acentric fragments or whole chromosomes, and the test thus has the potential to identify both clastogenic and aneugenic chemicals. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments using a dose range which included a dose level which achieved approximately 50% reduction in cytokinesis block proliferation index (CBPI) in the 4 h exposure group in the presence of S9 and in the 20 h exposure group. The toxicity curve in the 4 h exposure group in the absence of S9 was very steep so that there was only a modest reduction in CBPI at the maximum dose scored although the test item was tested to toxic limits. The substance is therefore considered as negative for inducing micronuclei in cultured human lymphocytes under activation and non-activation conditions used in this assay. The test substance is therefore considered as non-clastogenic and non-aneugenic in vitro.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).