4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (2 studies: OECD 471, GLP, K, rel. 1 & eq. to OECD 471, S, Rel.2): non mutagenic up to limit concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Chromosome aberration test (OECD 473, K, rel. 1): non clastogenic up to cytotoxic concentrations.

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:

24-31 March 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation. However, the isomers ratio is not reported.

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:

Directive 2000/32/EC.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2007-07-18 / Signed on 2007-07-26

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Test item was considered at 100% for the study.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% v/v S9 fraction; rat liver microsome fraction (S9)

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Moltox. Stock plates were stored at about -80 °C and master plates at about 4 °C.
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 72 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Inhibition of growth by the test item suggests a cytotoxic activity. A cytotoxic effect at high concentrations only would require lower concentrations of the test item in the main test. Cytotoxic activity at lower concentrations could rule out the bacterial reverse mutation test for the evaluation of mutagenicity.

OTHER:
- After an incubation of about 72 hours at about 37ºC, the number of colonies per plate was counted. Data are presented as the number of colonies present per plate (mean ± standard deviation) per plate. The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. For this purpose, the highest concentration of test item and a probe of the S9 mix were added respectively to top agar preheated at about 45 °C and poured over plates. The plates were then incubated for about 72 hours at about 37 °C. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix.

Rationale for test conditions:

Tested up to limit concentration

Evaluation criteria:

Several criteria were used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or reading frame shifts in the genome of either Salmonella Typhimurium and/or Escherichia Coli.
Negative results from the test indicate that under the test conditions, the test item is not mutagenic and non-promutagenic in the tested species.

An independent confirmation test was performed with the test item. If the first assay is positive, the confirmation test is performed in the same manner. If the first assay is negative, the confirmation test is performed according to the preincubation procedure.

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY TEST
- No cytotoxic effect was observed.

MUTAGENICITY TEST
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
− No dose response was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. See "Attached background material" section for further details.

OTHER
Sterility test: Sterility test showed no contamination during the study.

None

Conclusions:

Under the test conditions, the test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol (1%) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

 

Negative and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls performed showed valid ratios (R) above 2.5.

 

No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.

 

Under the test conditions, the test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 May to 19 June 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted similarly to OECD 471 Guideline with deviations: evaluation criteria, historical negative (solvent/vehicle) and positive control data not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

evaluation criteria, historical negative (solvent/vehicle) and positive control data not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stable at room temperature

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

Test 1: 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA, with and without S9 mix
Test 2: 39, 78, 156, 313, 625, 1250, 2500 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA, with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test item in DMSO is more than 5% and stable. Also it is not soluble in water at 5%.
- Shelf time and temperature after preparation of solution: Max. 1 hour 45 minutes at 25 °C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
S. typhimurium TA98, TA1537, TA100 and TA1535 were obtained from Japan Bioassay Research Center on 30 July 2004. E. coli WP2 uvrA was obtained on 28 November 1996.

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C

- Exposure duration: Plates were inverted and incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS:
- 2 plates/dose for test item and positive control
- 3 plates/dose for vehicle control

OTHER:
Counting method: Manual and mechanical counting
Correction:
Mechanical measuring: Surface and miscounting correction
Manual measuring: Surface correction for >300
Colony counts >1500: measured manually

Rationale for test conditions:

Tested up to limit concentration

Evaluation criteria:

No data

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Other confounding effects: None

ADDITIONAL INFORMATION ON CYTOTOXICITY
- Test 1: Cytotoxicity was observed at 1250 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, with and without S9 mix; at 5000 μg/plate in E. coli WP2, with and without S9 mix
- Test 2: Cytotoxicity was observed at 1250, 2500 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, with and without S9 mix; at 2500 and 5000 μg/plate in E. coli WP2, with and without S9 mix

MUTAGENICITY
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

None

Conclusions:

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100) and E. coli (WP2uvrA) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD Guideline 471, strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA) were exposed to test item at the following concentrations using pre-incubation method:

Test 1: 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA, with and without S9 mix

Test 2: 39, 78, 156, 313, 625, 1250, 2500 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA, with and without S9 mix

S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Phenobarbital and 5,6-Benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Test 1: Cytotoxicity was observed at 1250 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, with and without S9 mix; at 5000 μg/plate in E. coli WP2, with and without S9 mix.

Test 2: Cytotoxicity was observed at 1250, 2500 and 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, with and without S9 mix; at 2500 and 5000 μg/plate in E. coli WP2, with and without S9 mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 March to 28 June 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD TG 473 without any deviation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guidelines of 31 March 2011.

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (Inspected on 2018-08-21 / Signed on 2018-11-19).

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: Extremely pale yellow viscous liquid
- Storage condition of test material: Room temperature in the dark.

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: female, aged 28 years (preliminary toxicity test). Male, aged 21 years (Main experiment and repeat).
- Whether whole blood or separated lymphocytes were used: for each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- Whether blood from different donors were pooled or not: no, one donor for each experiment.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

MEDIA USED :
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 ºC with 5 % CO2 in humidified air.

Additional strain / cell type characteristics:

other: Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Covance laboratory (Lot No. PB/βNF S9 31/08/18), stored at approximately -196 °C.
- method of preparation of S9 mix: S9 fraction was obtained from the liver homogenates of male rats treated with Phenobarbitone/Beta-naphthoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium : the final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test (Cell Growth Inhibition): 7.27, 14.53, 29.06, 58.13, 116.25, 232.5, 465, 930 and 1860 μg/mL, 4h exposure time with and without metabolic activation followed by a 20h recovery period (4(20)-hour with (2%) and without S9-mix), and a continuous exposure of 24h without metabolic activation (24-hour without S9-mix).
Justification: he maximum dose was the maximum recommended dose level.

- Main Experiment:
* 4-hour exposure to the test item without S9-mix, followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 58.13, 116.25, 232.5, 465, 930, 1395 and 1860 μg/mL. Justification: the maximum recommended dose level and the onset of toxicity.
* 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 29.06, 58.13, 116.25, 232.5, 465, 930, 1395 and 1860 μg/mL. Justification: the maximum recommended dose level and the onset of toxicity.
* 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 29.06, 58.13, 116.25, 232.5, 465, 697.5 and 930 μg/mL. Justification: toxicity only

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in aqueous media at 18.6 mg/mL but was fully miscible in DMSO at 186 mg/mL in solubility checks performed in-house.
- Formulation preparation: Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared. The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimehtyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimehtyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (2% S9-mix).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: quadruplicate cultures for the control; duplicate culture per dose levels
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Exp. 1: 4 hours (± S9) / Exp. 2: 24 hours (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
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. Each slide was permanently labeled with the appropriate identification data.
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 spread and analysed per concentration (number of replicate cultures and total number of cells scored): Where possible 1000 cells per culture were evaluated for the incidence of metaphase cells and expressed as the mitotic index and as a percentage of the vehicle control value.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Where possible, 300 consecutive well-spread metaphases from each concentration were counted, 600 from the vehicle control (150 per replicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides
- Determination of polyploidy / endoreplication: cells with 69 chromosomes or more were scored as polyploid cells (including endoreduplicated cells) and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Rationale for test conditions:

Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• Concurrent positive control chemicals should induce responses that are compatible with those generated in historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

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: No significant change in pH when the test item was added into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Precipitation: Yes

PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST):
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 465 μg/mL in the 4(20)-hour exposure group in the presence of metabolic activation (S9) only; no precipitate was observed in the blood-free cultures in the absence of S9.
- Hemolysis was observed following exposure to the test item at and above 930 μg/mL in the 4(20)-hour exposure groups and only at 930 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 activity towards the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1860 μg/mL in the 4(20)-hour exposures in the presence and absence of S9, however, the qualitative assessment of the slides determined that there were low numbers of metaphases present at this dose level. The maximum dose with metaphases present in the 24-hour continuous exposure was 930 µg/mL, but again in low
numbers.The selection of the maximum dose level used was based on the maximum recommended dose level and the onset of toxicity for the 4(20)hours with and without S9 and on the toxicity only for the 24 hours without S9.

MAIN STUDY RESULTS
- Concurrent vehicle negative and positive control data: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

- Results from cytotoxicity measurements:
- The qualitative assessment of the slides determined that the precipitate was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 1743.75 µg/mL in the 4(20)-hour exposure group without
S9, up to 1395 µg/mL in the 4(20)-hour exposure group with S9 and up to 697.5 µ/mL in the 24-hour exposure group without S9.
- Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 930 μg/mL in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group without S9. No precipitate was observed in the blood-free cultures in the 4(20)-hour exposure group without S9.
- Hemolysis was observed at and above 930 μg/mL in the 4(20)-hour exposure in the absence of S9, at and above 465 μg/mL in the 4(20)-hour exposure group with S9 and at and above 697.5 µg/mL in the continuous exposure group without S9.
- In the 4(20)-hour exposure group in the absence of S9, 17% and 61% mitotic inhibition was achieved at 1627.5 and 1743.75 µg/mL, respectively. Above this dose level, there were no metaphases present for analysis. Therefore, the maximum dose level selected for metaphase analysis was 1743.75 µg/mL, although this dose level was marginally above the maximum range for optimum toxicity as stated in the OECD 473 test guideline (55±5%). A non-dose related increase in the number of polyploid cells was observed (maximum less than 5%) with marked inter-culture variation but was substantially in excess of the in-house historical maxima of polyploid frequency for this exposure group at all three dose levels. Therefore, a further analysis of the frequency of polyploid cells was conducted by scoring 200 metaphase cells per culture, regardless of quality, to confirm the true incidence of polyploidy.
-In the 4(20)-hour exposure group in the presence of S9, there was only a moderate decrease in mitotic index which coincided with the onset of precipitate. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level (930 µg/mL).
- In the 24-hour exposure group in the absence of S9, 32% and 56% mitotic inhibition was achieved at 232.5 and 465 µg/mL, respectively. Above this dose level, there were either insufficient or no metaphases present. The maximum dose level selected for metaphase analysis was 465 µg/mL because this dose level achieved the optimum level of toxicity as stated in the OECD 473 test guideline (55±5%).

- Genotoxicity results:
- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item induced a statistically significant increase in the numbers of polyploid cells at all dose levels in the 4(20)-hour exposure group without S9. There was indication of endoreduplication noted. The additional evaluation confirmed that the polyploidy response was not an artefact of toxicity. However, there was a marked difference in response between duplicate cultures of the two lower dose levels and an apparent association with a decrease in mitotic index. No increase in polyploid cell frequency was observed in either of the other exposure groups.

HISTORICAL CONTROL DATA (mean ± standard deviation)
- Positive historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 25.13 ± 13.14
4(20)-hour exposure with S9 (2%): 16.22 ± 7.00
24-hour exposure without S9: 26.81 ± 12.28
% cells with polyploids:
4(20)-hour exposure without S9%: 0.01 ± 0.06
4(20)-hour exposure with S9 (2%): 0.03 ± 0.12
24-hour exposure without S9: 0.02 ± 0.11

- Negative (solvent/vehicle) historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 0.48 ± 0.40
4(20)-hour exposure with S9 (2%): 0.54 ± 0.53
24-hour exposure without S9: 0.36 ± 0.43

% cells with polyploids:
4(20)-hour exposure without S9%: 0.04 ± 0.13
4(20)-hour exposure with S9 (2%): 0.03 ± 0.10
24-hour exposure without S9: 0.02 ± 0.07

None.

Conclusions:

Under the test conditions, test item did not induce any statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:

 

Preliminary Toxicity Test (Cell Growth Inhibition Test) : 7.27, 14.53, 29.06, 58.13, 116.25, 232.5, 465, 930 and 1860 μg/mL., 4h exposure time with and without metabolic activation followed by a 20h recovery period (4(20)-hour with (2%) and without S9-mix), and a continuous exposure of 24h without metabolic activation (24-hour without S9-mix).

 

Main experiment

4(20)-hour without S9-mix: 0,58.13, 116.25, 232.5, 465, 930, 1395 and 1860 μg/mL.

4(20)-hour with S9 (2%): 0, 29.06, 58.13, 116.25, 232.5, 465, 930, 1395 and 1860 μg/mL.

24-hour without S9-mix: 0, 29.06, 58.13, 116.25, 232.5, 465, 697.5 and 930 μg/mL.

 

Mitotic activity was arrested by addition of colcemid at 0.1 μg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

 

All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9- mix were validated.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was either the lowest precipitating dose level as in the 4(20)-hour exposure group in the presence of S9 or induced near optimum toxicity with 32 and 56% mitotic inhibition as in the 24-hour exposure group.

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

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   VIVOTECNIA, 2008	Ames Test (OECD 471) K, rel. 1)	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to limit concentration (in ethanol)	-S9 : non mutagenic +S9 : non mutagenic
2 Koei Techno, 2009	Similar toAmes Test (OECD 471) S, rel. 2)	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to limit concentration (in DMSO)	-S9 : non mutagenic +S9 : non mutagenic
3 COVANCE, 2019	CAT (OECD 473) K, rel.1	Chromosomal aberration	Human lymphocytes	-S9 +S9	Up to cytotoxicty	-S9: non clastogenic +S9: non clastogenic

Gene mutation Assay (Test n° 1 - 2):

A Key study was identifed (Vivotecnia Research, 2008). A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the registered substance(See Table 7.6/1).No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test conditions, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. These results were confirmed in a supporting study performed similarly to the OECD guideline No. 471 (See Table 7.6/1).The substance is therefore considered as non-mutagenic according to the Ames test.

Chromosomal aberration (Test n°3)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no classification is proposed regarding genetic toxicity according to the CLP and to the GHS.