Myrac Aldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro Ames test performed according to OECD 471 and in compliance with GLP, the registered substance is not mutagenic in the Salmonella typhimurium reverse mutation assayand in the Escherichia coli reverse mutation assay.

In an in vitro micronucleus test performed according to OECD guideline 487 and in compliance with GLP, the registered substance did not induce micronuclei.

In an in vitro HPRT test performed according to OECD guideline 476 and in compliance with GLP, the registered substance was considered to be non mutagenic.

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:

13 August 2013 - 05 September 2013

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines

Version / remarks:

31 March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine gene (his- to his+ reversions)
Escherichia coli: tryptophan gene (trp- to trp+ reversions)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1:
- All strains, with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment 2:
Without S9 mix
- Strains TA 1535 and WP2 uvrA: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Strains TA 1537, TA 98, TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
With S9 mix
- Strains TA 1537 and WP2 uvrA: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Strains TA 1535, TA 98, TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

(untreated plates)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Remarks:

see section ''Any other information on materials and methods incl. tables''

Details on test system and experimental conditions:

A pre-experiment was performed to evaluate the toxicity of the test item. The pre-experiment was performed under the same conditions as the final experiment, using all strains, and was reported as main experiment 1. The main study was performed in two experiments: experiment 1 was a plate incorporation assay, experiment 2 was a pre-incubation assay.

METHOD OF APPLICATION: in agar

DURATION (Pre-incubation assay)
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates for each straind and dose level

DATA RECORDING
Colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (v.1.21). Due to precipitation of the test item and reduced background growth the colonies were partly counted manually.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 333-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 100-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 100-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 33-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 333-5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II at 5000 μg/plate in strains TA 1535 and WP2 uvrA without S9 mix and in strains TA 1537 and WP2 uvrA with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The results of the pre-experiment were reported as experiment 1.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see table 1.

Table 1 Historical data

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max		Mean	SD	Min	Max
TA 1535	Solvent control	14	2.37	9	23		20	3.75	11	35
	Untreated control	14	2.87	7	25		20	3.82	10	32
	Positive control	1751	226.44	710	2385		367	93.12	126	703
TA 1537	Solvent control	12	3.31	5	26		16	4.34	7	30
	Untreated control	12	4.03	5	29		18	4.92	6	33
	Positive control	88	38.92	61	448		342	144.31	77	809
TA 98	Solvent control	30	5.60	17	47		40	6.08	21	58
	Untreated control	31	6.36	17	55		42	6.83	24	68
	Positive control	372	78.05	158	595		2167	717.60	249	4089
TA 100	Solvent control	142	29.42	86	243		156	29.4	99	249
	Untreated control	150	28.19	86	248		163	31.26	94	281
	Positive control	1741	488.75	569	3082		2642	769.59	825	4503
WP2uvrA	Solvent control	49	7.67	32	69		60	8.55	31	86
	Untreated control	50	7.64	31	71		60	8.06	38	83
	Positive control	697	336.39	174	1435		368	108.91	176	1534

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay performed according to OECD 471.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains (>=250 μg/plate). Adequate negative, vehicle and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in neither each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) nor in the E. coli tester strain (WP2 uvrA), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance 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:

25 July to 12 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 476 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

19 November 2018

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Modal number of chromosomes: Stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (2%); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test: 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/mL with or without S9.
The maximum concentration tested was limited by the presence of test item precipitate.

Mutagenicity Test – Main Experiment
4 h without S9: 0, 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL
4 h with S9 (2%): 0, 2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL
Justification: The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 40 μg/mL in the absence of S9 and 60 μg/mL in the presence of S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxyde (DMSO)
- Test substance preparation:
The test item is a multiconstituent substance and the maximum dose concentration was initially set at 5000 μg/mL and no correction for purity was applied to the formulations.
The solubility of the test item was investigated as part of the in vitro Micronucleus Study in Human Lymphocytes, Envigo Study Number FJ54WR on the same test item. The test item was immiscible in aqueous media at 50 mg/mL but was miscible in both DMSO and acetone at 500 mg/mL in solubility checks performed in-house. DMSO was chosen as the most suitable solvent. So, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being 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

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Cell density at seeding: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.

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

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG) at 11 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose
- Main test: 2 cultures/dose

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

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER:
- 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 cloning efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) 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 all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) 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.

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 lung fibroblasts (V79)

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: No significant change in pH when the test item was dosed into media
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Water solubility: None
- Precipitation: Yes

PRELIMINARY CYTOTOXICITY TEST:
- At the end of the exposure period, precipitate of the test item was observed at and above 78.13 μg/mL in both the absence and presence of metabolic activation.
- There was evidence of marked concentration related reductions in relative survival in both the absence and presence of metabolic activation.
The maximum concentrations selected for the main mutagenicity experiment were therefore limited by test item-induced toxicity in the absence of metabolic activation, and a combination of toxicity and the onset of test item precipitate in the presence of metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: Ethyl methane sulphonate: 4 h without S9 at 500 µg/mL: 171-346 (261.30 ± 36.82); 4 h without S9 at 750 µg/mL: 291-574 (417.54 ± 63.80);
Dimethyl benzanthracene: 4 h with S9 at 1 µg/mL: 209-774 (334.40 ± 114.17); 4 h with S9 at 2 µg/mL: 103-925 (484.97 ± 174.83)
- Negative (solvent/vehicle) historical control data: 4 h without S9: 5-23 (12.07 ± 4.44); 4 h with S9: 5-24 (12.47 ± 3.95)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutagenicity Test – Main Experiment
- At the end of the exposure period, the onset of precipitate of the test item was observed at 50 μg/mL in the presence of metabolic activation. Therefore, following the recommendations of the OECD 476 guideline, the lowest precipitating dose level of 50 μg/mL was plated for relative survival and mutant frequency in the presence of metabolic activation. It should also be noted that excessive levels of toxicity were also observed at 60 μg/mL in the presence of metabolic activation, and at and above 20 μg/mL in the absence of metabolic activation.
- There were marked concentration-related reductions in the relative survival values in both the absence and presence of metabolic activation. Based on the relative survival values, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation toxicity (at the lowest precipitating dose level in the presence of metabolic activation). There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
MUTAGENICITY RESULTS:
- The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentration levels in the main test using a dose range that achieved optimum levels of toxicity (at the lowest precipitating dose level in the presence of metabolic activation), in either the absence or presence of metabolic activation. The vehicle control values were all considered to be within an acceptable range and the positive controls gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. The values observed were considered acceptable for addition to the historical control data.

See attached document

Conclusions:

The the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test at concentrations up to 15 μg/mL in the 4-hour exposure group in the absence of metabolic activation, associated with excessive toxicity at higher concentrations, and at concentrations up to 50 μg/mL in the 4-hour exposure group in the presence of metabolic activation, corresponding to the lowest precipitating dose level.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were exposed to test item at the following concentrations:

Preliminary Cytotoxicity Test: 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/mL with and without S9

Mutagenicity Test – Main Experiment

4 h without S9: 0, 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL

4 h with S9 (2%): 0, 2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 2%; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

 

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 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 maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 40 μg/mL in the absence of S9 and 60 μg/mL in the presence of S9.

 

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation.

 

Therefore, the test item is considered to be non-mutagenic to V79 cells at the HPRT locus in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July to 17 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.

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:

19 November 2018

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

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction prepared from liver homogenates of male rats induced with Phenobarbital/β-naphthoflavone (29/03/18)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:
0, 10, 20, 40, 80, 160 and 320 μg/mL (4 h exposure to the test item without S9-mix)
0, 10, 20, 40, 80, 160, 240 and 320 μg/mL. (4 h exposure to the test item with S9-mix (2%))
0, 10, 20, 40, 50, 60 and 80 μg/mL. (24 h continuous exposure to the test item without S9-mix)

Justification: The selection of the maximum dose level for the Main Experiment was based on both toxicity and precipitation of the test item as the onset of both coincided for the 4-hour exposure groups, but was based solely on toxicity for the 24-hour exposure group.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxyde (DMSO)
- Test item preparation: The test item was immiscible in aqueous media at 50 mg/mL but was miscible in both DMSO and acetone at 500 mg/mL in solubility checks performed in-house. DMSO was chosen as the most suitable solvent. 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 test item formulation was stable for this duration.

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:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

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 (18-35) 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. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used were:
Preliminary Toxicity Test: male, aged 26 years; Main Experiment: male, aged 27 years

METHOD OF APPLICATION:
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% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 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:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Duplicate cultures per dose for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making. The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
- Cytokinesis Block Proliferation Index (CBPI): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
- Scoring of Micronuclei:
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration) except in the 4 h exposure group without S9-mix at 64 μg/mL where only 332 and 714 cells were assessed for micronuclei from the A and B cultures, respectively. This change to the number of cells assessed was necessitated by toxicity limiting the number of binucleate cells available for assessment, despite the apparent optimum toxicity of the dose level.
Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for identifying micronuclei were 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. Binucleate cells 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.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% 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]
T = test substance treatment culture
C = vehicle control culture

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear 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 judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses 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 binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei 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 at the dose levels tested.
- Water solubility: None
- Precipitation: Yes

PRELIMINARY TOXICITY TEST:
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 156.25 μg/mL in the 4 h exposure group without metabolic activation (S9), at and above 312.5 μg/mL in the 4 h exposure with metabolic activation.
- Hemolysis was observed following exposure to the test item at and above 39.06 μg/mL in the 4-hour exposure groups and at and above 78.13 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition to hemolysis, a reduced cell pellet was observed from 78.13 to 2500 μg/mL in the 4-hour exposure groups and at 78.13 μg/mL only in the 24-hour exposure group.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 625 μg/mL in the 4 h exposure in the absence of S9, up to 2500 µg/mL in the presence of S9 and up to 39.06 µg/mL in the 24-hour exposure group. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on both toxicity and precipitation of the test item as the onset of both coincided for the 4 h exposure groups, but was based solely on toxicity for the 24-hour exposure group.

NUMBER OF CELLS WITH MICRONUCLEI
Mean % binucleate cells with micronuclei:
4 h exposure without S9: 0.60, 0.40, 0.55, 0.45 and 0.65 at 0, 20, 40, 80 and 160 μg/mL
4 h exposure with S9: 0.90, 0.45, 1.20, 0.90 and 0.55 at 0, 80, 160, 240 and 320 μg/mL
24 h exposure without S9: 0.38, 0.40, 0.20, 0.20 and 0.30 at 0, 40, 50, 60 and 80 μg/mL

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Vehicle historical control data:
4 h exposure without S9: 0.05-1.20 (0.56 ± 0.29)
4 h exposure with S9: 0.05-1.30 (0.51 ± 0.29)
24 h exposure without S9: 0.15-0.90 (0.47 ± 0.19)

- Positive control historical control data:
4 h exposure without S9 (Mitomycin C): 1.33-11.80 (5.51 ± 2.43)
4 h exposure with S9 (Cyclophosphamide): 1.75-8.15 (3.79 ± 1.39)
24 h exposure without S9 (Demecolcine): 1.8-6.70 (3.41 ± 1.04)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Micronucleus Test – Main Experiment
- The qualitative assessment of the slides determined that the toxicity was not the same as expected from the results of the Preliminary Toxicity Test. In addition, the expected precipitate of test item was not observed in the 4-hour exposure groups. There were binucleate cells suitable for scoring at the maximum dose level of test item in the presence of S9 and the 24-hour exposure group. In the 4-hour group in the absence of S9, the maximum dose level of the test item with binucleate cells suitable for scoring was 160 μg/mL.
- Hemolysis was observed following exposure to the test item at and above 40 μg/mL in the 4-hour exposure groups and at and above 80 μg/mL in the 24-hour continuous exposure group. Additionally, a reduced cell pellet was observed at and above 80 μg/mL in the 4-hour exposure groups only. No reduced cell pellet was observed in the 24-hour exposure group.
-In the 4-hour exposure group in the absence of S9, only a very moderate dose-related inhibition of CBPI was observed: 12% and 16% inhibition of cell proliferation (cytostasis) was achieved at 80 and 160 μg/mL, respectively. Above this dose level, there were no scorable binucleates present for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 160 μg/mL because it was the highest scorable dose level.
In the presence of S9, no dose-related inhibition of CBPI was observed other than a plateau of toxicity where 30%, 16%, 28%, and 21% cytostasis was achieved at 80, 160, 240 and 320 μg/mL, respectively. The maximum dose level selected for analysis of binucleate cells was the maximum dose level (320 μg/mL).
In the 24-hour exposure group, 18%, 17% and 72% of cytostasis was achieved at 50, 60 and 80 μg/mL, respectively. The maximum dose level selected for analysis of binucleate cells was, therefore, 80 μg/mL, even though this dose level exceeded optimum toxicity as defined in the OECD 487 Test Guideline.

See attached document

Conclusions:

The test item 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 item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4 h exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24 h exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

Preliminary Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:

0, 10, 20, 40, 80, 160 and 320 μg/mL (4 h exposure to the test item without S9-mix)

0, 10, 20, 40, 80, 160, 240 and 320 μg/mL (4 h exposure to the test item with S9-mix (2%))

0, 10, 20, 40, 50, 60 and 80 μg/mL (24 h continuous exposure to the test item without S9-mix)

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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced approximately 72% reduction in Cytokinesis Block Proliferation Index (CBPI).

Therefore, the test item 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

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains (>=250μg/plate). Adequate negative, vehicle and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in neither each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) nor in the E. coli tester strain (WP2 uvrA), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assayand in the Escherichia coli reverse mutation assay.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the registered substance in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4 h exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24 h exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

Preliminary Toxicity Test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:

0, 10, 20, 40, 80, 160 and 320 µg/mL (4 h exposure to the test item without S9-mix)

0, 10, 20, 40, 80, 160, 240 and 320 µg/mL (4 h exposure to the test item with S9-mix (2%))

0, 10, 20, 40, 50, 60 and 80 µg/mL (24 h continuous exposure to the test item without S9-mix)

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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The registered substance was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced approximately 72% reduction in Cytokinesis Block Proliferation Index (CBPI).

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP,Chinese hamster (V79) cellswere exposed to test item at the following concentrations:

Preliminary Cytotoxicity Test:4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/mLwith and without S9

Mutagenicity Test – Main Experiment

4 h without S9:0, 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL

4 h with S9 (2%): 0, 2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL

 Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 2%; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone. 

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 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 maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 40 μg/mL in the absence of S9 and 60 μg/mL in the presence of S9.

 The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation.

Justification for classification or non-classification

In an in vitro Ames test performed according to OECD 471 and in compliance with GLP, the registered substance is not mutagenic in the Salmonella typhimurium reverse mutation assayand in the Escherichia coli reverse mutation assay.

In an in vitro micronucleus test performed according to OECD guideline 487 and in compliance with GLP, the registered substance did not induce micronuclei.

In an in vitro HPRT test performed according to OECD guideline 476 and in compliance with GLP, the registered substance was considered to be non mutagenic.

According to these results the registered substance can be considered as non genotoxic according to CLP Regulation (EC) n° 1272/2008.