Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test on S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (OECD 471, rel.1, K): not mutagenic with and without metabolic activation.

- Chromosomal aberration (OECD 473, Rel.1, K): non-clastogenic to human lymphocytes in vitro.

- Mutagenicity in mammalian cells (OECD 476, Rel.1, K): not mutagenic with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-01-31 to 2019-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC Commission Regulation No. 440/2008. OJ L 142/262.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

EPA 721-C-98-221 (1998)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2018-01-16 / Signed on 2018-06-05

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Date received: 29 November 2018

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cells: CHO-K1 cells were obtained from the European Collection of Cell Cultures. Cells are stored at -196 to -150°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).
- Type and identity of media:
Ham’s Nutrient Mixture F12, supplemented with with 1 mM L glutamine and 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin. The resulting medium is referred to as H0.
H0 medium supplemented with 10% HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 µg/mL.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes, 4 days prior to exposure, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine for three days.
All cell cultures are maintained between 34 and 39°C in a atmosphere of 5% CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was obtained from Covance - Shardlow and stored at -90 to -70°C.
S9 mix contained: S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
Main test 1- 3hours (-S9 mix): 0, 5, 10, 20, 40 and 80 µg/mL
Main test 2 - 3hours (+S9 mix): 0, 5, 10, 20, 40 and 80 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone. The final volume of acetone added to the cultures was 1% v/v.

- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test item in vehicles compatible with this test system was assessed. Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid was found to be soluble 500 mg/mL in acetone. A 500 mg/mL solution provided a final concentration of 5000 g/mL when administered at 1% v/v

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

250 µg/mL in DMSO

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/mL in DMSO

Positive control substance:

3-methylcholanthrene

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
The cells were incubated for approximately 24 hours at between 34 and 39°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test substance on Day 1.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days, between 34 and 39°C, in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound and positive controls, four individual cultures for solvent controls.

NUMBER OF CELLS EVALUATED: 10E06 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)

Rationale for test conditions:

The maximum final concentration tested in the preliminary toxicity test was 5000 μg/mL as this is the standard limit concentration within this test system as recommended for UVCB substances in the current OECD Guideline 476 (2016)

Evaluation criteria:

The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
Demonstration of reproducibility in any increase in mutant frequency;
The mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10E6 survivors with a corresponding survival rate of 20% or greater.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 5000 µg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test substance in medium was tested at concentrations of 5000 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
The maximum final concentration tested in the preliminary toxicity test was 5000 μg/mL as this is the standard limit concentration within this test system as recommended for UVCB substances in the current OECD Guideline 476 (2016)
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation: yes
- Other confounding effects: none

PRELIMINARY TOXICITY TEST
The cell concentration was confirmed to be 1.3 x 106 cells/mL (i.e. 13 x 106 cells treated per concentration, 26 x 106 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency.
The test item was dosed at concentrations up to 5000 µg/mL. Precipitate was observed by eye at the end of treatment at concentrations of 78.1 µg/mL and above and this was, therefore, the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix.
Exposure to the test item for 3 hours at concentrations of 39.1 and 78.1 µg/mL in both the absence and presence of S9 mix caused no significant reductions in RS values. Concentrations for the main test were based upon these data.

MAIN TEST
The cell concentration was confirmed to be 1.2 x 106 cells/mL (i.e. 24 x 106 cells treated per concentration, 48 x 106 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency for the 3hours without the presence of S9-mix.
The cell concentration was confirmed to be 1.4 x 106 cells/mL (i.e. 28 x 106 cells treated per concentration, 56 x 106 cells for the vehicle control). This cell concentration was used in the calculation of the adjusted cloning efficiency for the 3hours with the presence of S9-mix.

Cultures were exposed to the test item at concentrations from 5 to 80 µg/mL. Precipitate was observed by eye at the end of treatment at 80 µg/mL.
No significant reductions in mean RS at any concentration tested without S9-mix was observed and a mean RS values from 89 to 68% were observed with S9-mix . All cultures were plated out for determination of cloning efficiency and mutant frequency.
No statistically significant increases in mean mutant frequency at any concentration was observed. Tests for both a linear dose-concentration relationship and non-linearity were applied across all treatment groups, neither of which was statistically significant. The mean mutant frequencies for all the vehicle and test item treated groups were within the laboratory 95% confidence limits. These results fulfilled the criteria for a clearly negative result.

EMS, the positive control, induced a significant increase in mutant frequency. 3MC, the positive control, induced a significant increase in mutant frequency.

Table 7.6.1/2: Summary table

cf. attached backgroung material

Treatment	Concentration (µg/mL)	3-hour Treatment -S9 mix		3-hour Treatment +S9 mix
		Mean RS (%)	Mean MFa	Mean RS (%)	Mean MFa
Acetone	0	100	9.29	100	6.59
TI	5	83	12.68	70	12.76
TI	10	103	13.72	68	8.25
TI	20	102	9.61	89	8.78
TI	40	112	8.33	82	5.54
TI	80b	104	14.36	68	10.67
Ethyl methanesulphonate	250	93	99.28***	NT	NT
3-methylcholanthrene	5	NT	NT	74	41.07**

a. Mutant frequencies expressed per 10^6 viable cells

b. Precipitate observed at the end of treatment

RS: Relative Survival

MF: Mutant Frequency

NT: Not tested

*** p<0.001; ** p <0.01 all other cultures p≥0.05. Treated groups were compared to the vehicle control using one-tailed Dunnett’s tests for an increase and the positive control was compared to the vehicle control using a one-tailed t-test for an increase

Conclusions:

Under the test conditions, the test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation. The test material did not demonstrate mutagenic potential.

Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary cells (CHO-K1) were exposed to the test item diluted in acetone, in duplicate in the presence and absence of metabolic activation (S9-mix).Two independent tests are performed, one in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix.Three-hour exposures were used both with and without activation (S9) in all tests.

 

The highest final concentration used in the preliminary toxicity test was 80μg/mL. This is the standard limit concentration within this test system as recommended in the regulatory guidelines. Precipitate was observed by eye at the end of treatment at 80 µg/mL, and these were therefore the highest concentrations plated for determination of toxicity.

No significant reductions in mean RS at any concentration tested without S9-mix was observed and a mean RS values from 89 to 68% were observed with S9-mix . All cultures were plated out for determination of cloning efficiency and mutant frequency.

No statistically significant increases in mean mutant frequency at any concentration was observed. Tests for both a linear dose-concentration relationship and non-linearity were applied across all treatment groups, neither of which was statistically significant. The mean mutant frequencies for all the vehicle and test item treated groups were within the laboratory 95% confidence limits. These results fulfilled the criteria for a clearly negative result.

The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test item did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation,under the experimental conditions described. The test material did not demonstrate mutagenic potential.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 February to 18 March 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:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

EPA 712-C-98-223 (1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Ec Commission Regulation No.2017/735

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (Inspected on 2018-01-16 / Signed on 2018-06-05).

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Date received: 29 November 2018

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: two healthy, non-smoking, adult (between 18-35 years of age) donors
- 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: yes, pooled (in equal volumes from each donor) and diluted with HML media.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

MEDIA USED :
Cells (whole blood cultures) were grown in the following media:
HML media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L-glutamine.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- method of preparation of S9 mix: S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, and stored at -90 to -70°C.
- S9 contains : S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilised before use.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test (Cell Growth Inhibition): 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 1250, 2500 and 5000 μg/mL, 3h exposure time with and without metabolic activation followed by a 18h recovery period (3(18)-hour with S9-mix (2%) and without S9-mix), and a continuous exposure of 21h without metabolic activation (21-hour without S9-mix).
Justification: the maximum dose was the maximum recommended dose level.

- Main Experiment:
* 3-hour exposure to the test item without S9-mix, followed by 18-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 10, 20, 40 and 80 μg/mL. Justification: the maximum recommended dose level and the onset of toxicity.
* 3-hour exposure to the test item with S9-mix (2%v/v), followed by 18-hour recovery period in treatment-free media prior to cell harvest. The dose range of test item used was 0, 10, 20, 40 and 80 μg/mL. Justification: the maximum recommended dose level and the onset of toxicity.
* 21-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 10, 20, 40 and 80 μg/mL. Justification: toxicity only

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle:The test item was found to be soluble in acetone at 500 mg/mL.
- Formulation preparation: A 500 mg/mL solution provided a final concentration of 5000 µg/mL when administered at 1% v/v. The test item was dissolved and formulated in acetone (analytical reagent grade), shortly before dosing. The final volume of acetone added to the cultures was 1% v/v (preliminary toxicity test) or 0.5% v/v (main test).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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:

Acetone

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: duplicate culture per dose levels
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Exp. 1: 3 hours (± S9) / Exp. 2: 21 hours (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 18 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 2-h 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 fifteen 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 two 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 10 % Giemsa prepared in buffered water (pH 6.8), 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):From each culture 150 metaphase figures were examined, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 cells with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. The vernier readings of all aberrant metaphase figures were recorded. A peer review of the metaphase analysis was performed by the analysis of 10 metaphases for the vehicle, highest concentration selected and positive control for each exposure condition.
Traditionally gaps have been excluded from the quantitation of chromosome aberrations. Some gaps, however, have been shown to be real discontinuities in DNA (Heddle and Bodycote, 1970, Satya-Prakash et al., 1981). In this study the total number of cells containing aberrations both with and without gaps has been calculated.
- Determination of polyploidy / endoreplication: The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded as a percentage of the 150 metaphases analysed per slide, independently from the analysis for chromosome aberrations.

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 number of aberrant metaphase cells in each test item group was compared with the vehicle control value using the mid-p one-tailed Fisher exact test for an increase (Richardson et al., 1989). Statistical significance was declared at 5%.

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test item groups. If this was significant at the 1% level, the test was reiterated excluding the highest concentration group - this process continued until the trend test was no longer significant.
The data were analysed using the SAFEStat Chromosome Aberrations application.

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
-Osmolarity and pH Measurements: The osmolality and pH of the test item in medium were measured by analysing samples of HML media, dosed at 1%
(v/v), with either the vehicle (acetone) or a test item formulation at 500 mg/mL (to provide a final concentration of 5000 μg/mL). For medium dosed with test item at 5000 μg/mL; no increases in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 5000 μg/mL as this is the standard limit concentration within this test system as recommended for UVCB substances in the current OECD Guideline 473 (2016).
- Precipitation: Yes

PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST):
- In all exposure conditions the highest concentration tested was 5000 µg/mL and precipitate was observed by eye at 78.13 µg/mL and above as assessed in concurrently treated HML media-only cultures. 78.13 µg/mL was, therefore, the highest concnetration carried forward for assessment of toxicity.
- No significant reductions in the mitotic index at any analyzed concentration was observed, when compared with the vehicle control, in any exposure condition.

The selection of the maximum dose level used was based on the maximum recommended dose level and the onset of toxicity for the 3(18)hours with and without S9 and on the toxicity only for the 21 hours without S9.

MAIN STUDY RESULTS
- Concurrent vehicle negative and positive control data: Due to the low mitotic indices observed in the vehicle control cultures following the 21-hour exposure in the preliminary toxicity test, the vehicle and test item formulations were administered at a reduced dose volume of 0.5% (v/v) in all exposure conditions in the main test. Positive control formulations were administered at a dose volume of 1% (v/v).

- Results from cytotoxicity measurements:
- In all exposure conditions the highest concentration tested was 80 µg/mL and precipitate was observed by eye at the end of treatment at 80 µg/mL as assessed in concurrently treated HML media-only cultures.
No reductions in the mitotic index at any analyzed concentration and any experiment were observed, when compared with the vehicle control. The concentrations selected for metaphase analysis were 20, 40 and 80 µg/mL.
In the 3(18)-hour exposure group in the absence and the presence of S9 and in the 21-hour exposure, no mitotic inhibition was achieved at 40 and 80 µg/mL, respectively.

- 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.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed when compared with the vehicle control.
- No evidence of a linear dose-concentration relationship and all mean values (excluding gaps) for the vehicle control and test item treated cultures were within the laboratory historical 95% confidence limits. These results fulfilled the criteria for a clearly negative result.
- The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.

HISTORICAL CONTROL DATA (mean ± standard deviation)
- Positive historical control data:
cells with aberrations (-gaps):
3(18)-hour exposure without S9%: 18.2 ± 5.4
3(18)-hour exposure with S9 (2%): 29.7 ± 15.7
21-hour exposure without S9: 37.3 ± 18.4


- Negative (solvent/vehicle) historical control data:
cells with aberrations (-gaps):
3(18)-hour exposure without S9%: 0.9 ± 0.6
3(18)-hour exposure with S9 (2%): 0.9 ± 0.5
21-hour exposure without S9: 0.9 ± 0.4

cf. attached background material

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) : 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 1250, 2500 and 5000 μg/mL, 3h exposure time with and without metabolic activation followed by a 18h recovery period (3(18)-hour with (2%) and without S9-mix), and a continuous exposure of 21h without metabolic activation (21-hour without S9-mix).

 

Main experiment

3(18)-hour without S9-mix: 0, 10, 20, 40 and 80 μg/mL.

3(18)-hour with S9 (2%): 0, 10, 20, 40 and 80 μg/mL.

21-hour without S9-mix: 0, 10, 20, 40 and 80 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 (Acetone) 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 either in the absence or presence of metabolic activation.

No statistically significant increases in polyploid or endoreduplicated metaphases were observed when compared with the vehicle control.

No evidence of a linear dose-concentration relationship and all mean values (excluding gaps) for the vehicle control and test item treated cultures were within the laboratory historical 95% confidence limits. These results fulfilled the criteria for a clearly negative result.

The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 31, 2019 to February 16, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP

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:

EC Commission Regulation No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 16/01/2018/signed on 05/06/2018

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Date received: 29 November 2018

Target gene:

Histidine gene for Salmonella and tryptophan gene for E.coli

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:

Type and composition of metabolic activation system:
- source of S9: prepared from male Sprague Dawley rats induced with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver. S9 fraction was obtained from a suitable source and stored at -90 to -70°C.
- composition of S9 mix: S9 fraction (10% - test 1; 20% v/v – test 2 and test 2 repeat), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in water. All the cofactors were filter-sterilized before use.

Test concentrations with justification for top dose:

Initial: 5.00, 15.0, 50.0, 150, 500, 1500, and 5000 µg/plate in each strains.
Confirmatory: 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg/plate in each stains. The pre-incubation procedure is not suitable for acetone, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In a solubility assessment with DMSO and Acetone, the test item did not dissolve in DMSO but did in acetone. Acetone (analytical reagent grade) was, therefore, used as the vehicle for this study.

Untreated negative controls:

yes

Remarks:

Without S9-mix

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Solvent DMSO

Untreated negative controls:

yes

Remarks:

With S9-mix

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

Solvent DMSO

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 3
Due to contamination of WP2 uvrA (pKM101) plates and insufficient viability counts for TA98, TA100 and TA1537, only TA1535 plates were scored for the second test. Therefore, a
repeat of the second experiment for WP2 uvrA (pKM101), TA98, TA100 and TA1537 was performed in the absence and presence of metabolic activation.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 70-72 hours at 34 to 39°C.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

- OTHER: ACCEPTANCE CRITERIA
The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥3-fold (in strains TA98, TA100, TA1535, TA1537, or WP2uvrA,) the concurrent vehicle control confirming discrimination between different strains, and an active S9 preparation.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2.0-fold (in strains TA98, TA100 or WP2uvrA) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Biological relevance was taken into account on a case-by-case basis, for example consistency of response within and between concentrations and between experiments, where applicable. Other criteria were used in reaching a conclusion about the study results (e.g., comparison to historical control values, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
Initial: Precipitate was observed on plates containing test item at 1500 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate only in the presence of metabolic activation.
Repeat: In the repeat experiment, precipitate was observed on plates containing test item at 1500 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate only in the presence of metabolic activation.

STUDY RESULTS (cf. attached background material)
- Concurrent vehicle negative and positive control data: The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that the mean vehicle control counts fell within the laboratory’s historical ranges. All the positive control chemicals induced increases in revertant numbers of ≥3-fold in all tester strains when compared with the concurrent vehicle control. The study therefore demonstrated assay functionality and was accepted as valid.
- Signs of toxicity:
In the initial experiment, no evidence of toxicity was obtained following exposure.
In the repeat experiment, no evidence of toxicity was obtained in strain TA1535 following exposure (Test 2), nor in tester strains TA98, TA100, TA1537 or WP2 uvrA (pKM101) (repeat Test 2).
- Individual plate counts: cf attachment
- Mean number of revertant colonies per plate and standard deviation (cf. attachment): No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix in each experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf attachment
- Negative (solvent/vehicle) historical control data: cf attachment

Conclusions:

The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in Acetone both in the presence and absence of metabolic activation system (10% - test 1; 20% v/v – test 2 and test 2 repeat) using the plate incorporation method.

The test material was evaluated in the initial mutagenicity assay in all five tester strains at concentrations of 5.00, 15.0, 50.0, 150, 500, 1500, and 5000 μg/plate in the presence and absence of S9. Precipitate was observed on plates containing test item at 1500 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate only in the presence of metabolic activation. No evidence of toxicity was obtained following exposure.

Based on the results of the initial mutagenicity assay, an independent confirmatory mutagenicity assay was conducted in tester strains TA98, TA100, TA1535, and TA1537 at the same concentrations in the presence and absence of S9, but as the pre-incubation procedure is not suitable for acetone, which is toxic under such conditions, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v was performed. Precipitate was observed on plates containing test item at 1500 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate only in the presence of metabolic activation. No evidence of toxicity was obtained in strain TA1535 following exposure (Test 2), nor in tester strains TA98, TA100, TA1537 or WP2 uvrA (pKM101) (repeat Test 2).

No increase in the mean number of revertant colonies was observed at any tested concentration in any tester strain in the presence or absence of S9. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation 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  COVANCE, 2019	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	5.00, 15.0, 50.0, 150, 500, 1500, and 5000 µg/plate (in acetone)	-S9: non mutagenic +S9: non mutagenic
2 COVANCE, 2019	CAT (OECD 473) K, rel.1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	0, 10, 20, 40 and 80 μg/mL	-S9: non clastogenic +S9: non clastogenic
3 COVANCE, 2019	HPRT (OECD 476) K, rel.1	Gene mutation	Chinese hamster ovary cells	-S9 +S9	0, 5, 10, 20, 40 and 80 µg/mL	-S9: non mutagenic +S9: non mutagenic

 

Gene mutation Assays (Tests n° 1-3):

A Bacterial Reverse mutation Assay (Ames test) were performed according to OECD 471 test guideline with the test material (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in the test, with any dose of the test material, either with or without metabolic activation. The test material did not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.

The substance is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO/HPRT test) (Test n°3). None of the dose levels, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material did not induce forward mutations at the hprt locus in CHO cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases.

The substance is therefore considered as negative for inducing forward mutations at the hprt locus in CHO cells under activation and non-activation.

 

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using anin vitrochromosome 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 dis 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 test material has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification

No additional classification is proposed regarding in vitro genetic toxicity according to the criteria of Annex I to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.