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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid
Cas Number:
2166089-27-4
Molecular formula:
Non applicable (UVCB)
IUPAC Name:
Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid
Test material form:
liquid
Details on test material:
- Appearance: Limpid liquid
- Storage condition of test material: Keep container tightly closed. Preferably store in the original packaging. Store at room temperature, protect from humidity.
Specific details on test material used for the study:
Date received: 29 November 2018

Method

Target gene:
HPRT locus
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.

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