Thiamine hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Under the conditions of the study provided, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

MLA: Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

In vitro Micronucleus: Under the conditions of this study, the test material was not clastogenic or aneugenic in human lymphocytes.

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:

28 July 2017 to 16 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 492, 878, 1568, 2800 and 5000 μg/plate (absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: A solubility test was performed in Test Facility Study No. 519034. The test material was dissolved in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
- Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment.

MUTATION ASSAY
- At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain.The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test material was tested both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Initially
tester strain TA98 in the absence of S9-mix was rejected since some of the acceptability criteria were not met. This part of the study was repeated. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
- Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
- A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium, other: TA 100, TA 1535 and 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 applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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:

DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

SECOND MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
- All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two experiments.
- The negative control values were within the laboratory historical control data ranges.
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535 in the second experiment in the presence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 10 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Table 1: Dose Range-finder and Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC Milli-Q Water 52 164 512 1600 5000	597 90 92 86 98 78 86 n NP	840 10 8 7 7 9 5 n NP	1222 24 28 29 36 28 28 n NP	1176 10 13 9 9 13 5 n NP	1317 5 4 4 5 5 4 n NP
+	PC Milli-Q Water 52 164 512 1600 5000	497 109 85 82 86 76 88 n NP	219 11 12 9 10 9 8 n NP	401 26 43 26 36 34 29 n NP	1172 13 13 16 19 14 13 n NP	252 3 5 5 4 3 4 n NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

n = Normal bacterial background lawn

 

Table 2: Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC  Milli-Q Water 492 878 1568 2800 5000 n NP	1008 114 104 98 124 108 125 n NP	790 10 5 10 8 11 13 n NP	1316 18 25 24 20 23 25 n NP	1451 14 14 9 12 9 13 n NP	1079 4 3 5 4 4 3 n NP
+	PC  Milli-Q Water 492 878 1568 2800 5000 n NP	634 84 90 83 109 83 99 n NP	60 6 8 7 5 4 6 n NP	269 32 25 37 36 31 34 n NP	380 18 11 17 16 16 19 n NP	190 5 5 3 3 3 6 n NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

n = Normal bacterial background lawn

Conclusions:

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The potential of the test material to cause genetic toxicity to bacteria was determined in accordance with the standardised guidelines OECD 471 and EU Method B13/14, under GLP conditions.

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2017 to 25 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test

Target gene:

Induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC, Manassas, USA) 2001

MEDIA USED
- Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
- Growth medium: Basic medium, supplemented with 10 % (v/v) heat-inactivated horse serum (= R10 medium). Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.
- Exposure medium: For 3 hour exposure cells were exposed in basic medium supplemented with 5 % (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure cells were exposed in basic medium supplemented with 10 % (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20) and 5 μg/mL trifluorothymidine (TFT).
- Non-selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20).
- Properly maintained: Yes. Stock cultures of the cells were stored in liquid nitrogen (-196 °C). Cell density was kept below 1 x 10^6 cells/mL.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) with a number of test material concentrations increasing by approximately half log steps.
- Dose range finding test: 125, 250, 500, 1000 and 2000 µg/mL
- Mutagenicity test: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL, selected sased on the results of the dose-range finding test.

Vehicle / solvent:

- The test material was dissolved in RPMI 1640 (exposure medium (R5)). Amber-coloured glassware or tubes wrapped in tin-foil were used when preparing the test solutions.
- The test material concentrations were used within 2 hours of preparation.
- The pH and the osmolarity of the culture medium containing the highest, non-precipitating concentration were recorded.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

MUTAGENICITY TEST
- All incubations were carried out in a humid atmosphere (80 - 100 %, actual range 29 - 98 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 27.6 - 47.7 °C).
- Eight doses of the test item were tested in the mutation assay.
- The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.

TREATMENT OF THE CELLS
- Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm^2 culture flasks at 37.0 ± 1.0 °C. Solvent and positive controls were included and the solvent control was tested in duplicate.
- In the first experiment, cell cultures were exposed for 3 hours to the test material in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test material in exposure medium for 24 hours in the absence of S9-mix.
- For the 3 hour treatment, cell cultures were exposed for to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 mL growth medium (R10).
- For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were re-suspended in 20 mL growth medium (R10). The cells in the final suspension were counted with the coulter particle counter.

EXPRESSION PERIOD
- For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were sub-cultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test material the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

DETERMINATION OF THE MUTATION FREQUENCY
- For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtitre plates/concentration) in non-selective medium.
- For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtitre plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtitre plates, each well containing 1000 cells in selective medium (TFT-selection). The microtitre plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to each well. The plates for the CEday2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF MUTANT COLONIES
- The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
- The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

CALCULATION OF THE SURVIVAL OR VIABILITY
The suspension growth (SG) for the 3 hour treatment = [Day 1 cell count/1.6 x 10^5] x [Day 2 cell count/1.25 x 10^5]

The suspension growth (SG) for the 24 hour treatment = [Day 0 cell count/1.25 x 10^5] x [Day 1 cell count/1.25 x 10^5] x [Day 2 cell count/1.25 x 10^5]

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100

The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well

The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100

The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100

CALCULATION OF THE MUTATION FREQUENCY
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CEday2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CEday2 x 10^6

Small and large colony mutation frequencies were calculated in an identical manner.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met these criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120 % in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40 % of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY OF THE TEST MATERIAL
- The test material did not precipitate in the exposure medium up to and including the concentration of 2000 μg/mL. Therefore, this concentration was used as the highest test material concentration.
- The pH and osmolarity at a concentration of 2000 μg/ml were 6.87 and 0.299 Osm/kg respectively (compared to 7.66 and 0.286 Osm/kg in the solvent control).

DOSE-RANGE FINDING TEST
- After 3 hours of treatment with various concentrations of the test material and after 24 and 48 hours of subculture, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 2000 μg/mL compared to the suspension growth of the solvent control.
- After 24 hours of treatment with various concentrations of the test material and after 24 hours of subculture, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 2000 μg/mL compared to the suspension growth of the solvent control.

FIRST MUTAGENICITY TEST
- No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

SECOND MUTAGENICITY TEST
- No significant toxicity was observed and all dose levels were evaluated.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

ACCEPTABILITY OF THE ASSAY AND DISCUSSION
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay.
- Although the mutation frequency of one of the solvent control cultures in the first experiment observed in the absence of S9-mix was just above the upper control limits, these limits are 95 % control limits and a slightly higher response is within the expected response ranges.
- Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- The suspension growth over the two-day expression period for cultures treated with exposure medium was 17 and 18 (3 hour treatment) and 94 (24 hour treatment).
- In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
- In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Experiment 1: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					Total	Small	Large
Without metabolic activation - 3 hours of treatment
SC1	100	80	100	100	159	118	33
SC2		101			120	84	30
15.6	101	67	74	75	178	126	43
31.3	97	104	115	111	144	111	26
62.5	98	89	98	96	149	114	29
125	97	93	102	99	142	108	28
250	99	93	102	101	148	135	10
500	97	93	102	99	152	108	36
1000	97	85	94	91	118	84	29
2000	90	84	93	84	165	112	44
MMS	59	47	52	31	1087	677	290
With metabolic activation - 3 hours of treatment
SC1	100	98	100	100	106	73	28
SC2		86			145	111	27
15.6	100	81	88	88	129	82	41
31.3	106	91	99	105	129	86	37
62.5	104	102	111	115	118	81	32
125	108	105	114	123	105	78	22
250	98	97	105	103	125	90	29
500	102	89	96	98	167	140	20
1000	97	108	117	114	110	73	32
2000	107	85	92	99	125	78	41
CP	29	18	19	5	2990	1802	840

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (Exposure Medium)

MMS = Methylmethanesulfonate

CP = Cyclophosphamide

 

Experiment 2: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6 survivors
					Total	Small	Large
Without metabolic activation - 24 hours of treatment
SC1	100	99	100	100	97	67	26
SC2		84			107	61	41
15.6	87	105	115	100	108	59	43
31.3	93	95	104	97	125	96	25
62.5	91	90	98	89	89	60	26
125	86	108	118	102	109	76	28
250	95	85	93	88	115	73	37
500	81	86	94	76	123	75	43
1000	81	83	90	73	130	69	54
2000	78	107	116	91	94	65	25
MMS	59	75	81	48	744	404	243

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (Exposure medium)

MMS = Methylmethanesulfonate

Conclusions:

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Executive summary:

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490, under GLP conditions.

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test material was dissolved in exposure medium (R5).

In the first experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence and presence S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test material did not precipitate in the culture medium at this dose level.

In the second experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. The test material did not precipitate in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July 2017 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The objective of this study was to evaluate the test material for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Cultured peripheral human lymphocytes were used as test system.
- Peripheral human lymphocytes are recommended in the international OECD guideline.
- Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) were:
Dose-range finding study: age 28, AGT = 13.4 h
First cytogenetic assay: age 25, AGT = 14.0 h
Second cytogenetic assay: age 22, AGT = 13.2 h
- Blood samples: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

- Dose-range Finding Test: 125, 250, 500, 1000 and 2000 μg/mL
- First and Second Cytogenetic Assay: 500, 1000 and 2000 μg/mL
- The concentrations for the cytogenetic assays were selected based on the results of the dose-range finding test.

Vehicle / solvent:

- The vehicle for the test material was culture medium.
- A solubility test was performed based on visual assessment. The test material was dissolved in RPMI 1640 medium.
- Test material concentrations were used within 3 hours after preparation and were wrapped in tin-foil to protect from light.
- The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded.
- Any residual volumes were discarded.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine was used without metabolic activation at 0.1 μg/mL for a 3 hour exposure period and 0.05 μg/mL for a 24 hour exposure period.

Remarks:

HBSS was used as the solvent for the positive controls

Details on test system and experimental conditions:

DOSE-RANGE FINDING TEST
- In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test, in the absence and presence of S9-mix.
- Lymphocytes (0.4 mL blood of a healthy donor was added to 5 or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of the test material for 3 and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B was added to the cells simultaneously with the test material at the 24 hours exposure time. A vehicle control was included at each exposure time.
- The highest tested concentration was 2000 μg/mL.
- After 3 hours exposure to the test material in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 μg/mL) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
- Cytotoxicity of the test material in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).

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

SECOND CYTOGENETIC ASSAY
- To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test material with cytochalasin B (5 μg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

PREPARATION OF SLIDES
- To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1 % Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56 % (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
- Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5 % (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

CYTOTOXICITY ASSESSMENT
- A minimum of 500 cells (with a maximum deviation of 5 %) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI):
%Cytostasis = 100-100[(CBPIt – 1)/(CBPIc –1)]
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / Total number of cells
Where:
t = test material or control treatment culture
c = vehicle control culture
- Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The test material was not cytotoxic, the highest concentration analysed was the recommended 2000 μg/mL.

CYTOGENETIC ASSESSMENT/ SCORING OF MICRONUCLEI
- To prevent bias, all slides were randomly coded before examination of micronuclei and scored.
- At least 1000 (with a maximum deviation of 5 %) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5 %) mononucleated cells per culture were scored for micronuclei separately. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

- The following criteria for scoring of binucleated cells were used:
Main nuclei that were separate and of approximately equal size.
Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
Main nuclei that were linked by nucleoplasmic bridges.

- The following cells were not scored:
Trinucleated, quadranucleated, or multinucleated cells.
Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

- The following criteria for scoring micronuclei were adapted from Fenech, 1996:
The diameter of micronuclei should be less than one-third of the main nucleus.
Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
Micronuclei should have similar staining as the main nucleus.

Evaluation criteria:

ACCEPTABILITY CRITERIA
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
- The concurrent negative control data are considered acceptable when they are within the 95 % control limits of the distribution of the historical negative control database.
- The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
- The positive control material colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control materials MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei.

EVALUATION OF THE RESULTS
A test material is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
- At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
- The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
- Any of the results are outside the 95 % control limits of the historical control data range.

A test material is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
- None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
- All results are inside the 95 % control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 was used for statistical analysis of the data with:
- Chi-square test
- Cochran Armitage trend test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

FIRST CYTOGENETIC ASSAY
- All dose levels were selected for scoring of micronuclei.
- Both in the absence and presence of S9-mix, the test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei .

SECOND CYTOGENETIC ASSAY
- All dose levels were selected for scoring of micronuclei.
- The test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

EVALUATION OF THE RESULTS
- The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95 % control limits of the distribution of the historical negative control database.
- The positive control materials, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- The test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Table 1: Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with The Test Material in the First Cytogenetic Assay

Concentration μg/mL	Without metabolic activation (-S9-mix) 3 hours exposure time, 27 hours harvest time			With metabolic activation (+S9-mix) 3 hours exposure time, 27 hours harvest time
	CBPI	Mean CBPI	% Cytostasis	CBPI	Mean CBPI	% Cytostasis
0	1.56 - 1.57	1.57	0	1.46 - 1.50	1.48	0
500	1.43 - 1.46	1.44	22	1.42 - 1.45	1.44	10
1000	1.42 - 1.43	1.43	25	1.49 - 1.50	1.49	-2
2000	1.46 - 1.46	1.46	19	1.39 - 1.52	1.45	7
0.25 MMC-C	1.27 - 1.32	1.30	48	-	-	-
0.38 MMC-C	1.23 - 1.26	1.24	57	-	-	-
0.1 Colch	1.05 - 1.07	1.06	90	-	-	-
15 CP	-	-	-	1.17 - 1.20	1.19	62
17.5 CP	-	-	-	1.10 - 1.15	1.13	74

 

Table 2: Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with The Test Material in the First Cytogenetic Assay Without Metabolic Activation (-S9-mix) (3 hours exposure time, 27 hours harvest time )

Concentration μg/mL	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0	0	0	2	2	0	4	4
500	22	0	1	1	3	2	5
1000	25	1	0	1	2	1	3
2000	19	0	0	0	1	2	3
0.25 MMC-C	48	1	1	2	9	10	19***
0.38 MMC-C	57	2	4	6	20	12	32***
0.1 Colch	90	15	17	32***	25	20	45***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

 

Table 3: Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with The Test Material in the First Cytogenetic Assay With Metabolic Activation (-S9-mix) (3 hours exposure time, 27 hours harvest time )

Concentration μg/mL	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0	0	2	0	2	4	2	6
500	10	0	0	0	2	2	4
1000	-2	0	0	0	1	1	2
2000	7	0	1	1	3	3	6
15 CP	62	1	3	4	2	15	17*
17.5 CP	74	2	4	6	8	18	26***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

 

Table 4: Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with The Test Material in the Second Cytogenetic Assay (Without metabolic activation (-S9-mix) 24 hours exposure time, 24 hours harvest time)

Concentration μg/mL	CBPI	Mean CBPI	% Cytostasis
0	1.81 - 1.83	1.82	0
500	1.78 - 1.80	1.79	4
1000	1.74 - 1.79	1.77	7
2000	1.71 - 1.76	1.74	10
0.15 MMC-C	1.51 - 1.55	1.53	36
0.23 MMC-C	1.40 - 1.41	1.40	51
0.05 Colch	1.04 - 1.06	1.05	94

 

Table 5: Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Thiamine hydrochloride in the Second Cytogenetic Assay (Without metabolic activation (-S9-mix) 24 hours exposure time, 24 hours harvest time)

Concentration μg/mL	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0	0	0	2	2	0	2	2
500	4	1	0	1	4	2	6
1000	7	0	2	2	1	2	3
2000	10	0	0	0	0	0	0
0.15 MMC-C	36	1	1	2	2	3	5
0.23 MMC-C	51	1	2	3	10	14	24***
0.05 Colch	94	26	34	50***	7+	9+	16***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

+) 486 and 408 binucleated cells were scored for the presence of micronuclei, respectively.

Conclusions:

Under the conditions of this study, the test material was not clastogenic or aneugenic in human lymphocytes.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 487, under GLP conditions.

The test material was evaluated for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test material was tested in two independent experiments.

The test material was dissolved in culture medium. In the first cytogenetic assay, the test material was tested up to the recommended dose level of 2000 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, the test material was tested again up to 2000 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. The positive control materials, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control material colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Under the conditions of this study, the test material was not clastogenic or aneugenic in human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames

The potential of the test material to cause genetic toxicity to bacteria was determined in accordance with the standardised guidelines OECD 471 and EU Method B13/14, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

MLA

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test material was dissolved in exposure medium (R5).

In the first experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence and presence S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test material did not precipitate in the culture medium at this dose level.

In the second experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. The test material did not precipitate in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

In vitro Micronucleus

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 487, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was evaluated for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test material was tested in two independent experiments.

The test material was dissolved in culture medium. In the first cytogenetic assay, the test material was tested up to the recommended dose level of 2000 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, the test material was tested again up to 2000 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. The positive control materials, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control material colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Under the conditions of this study, the test material was not clastogenic or aneugenic in human lymphocytes.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.