[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

of August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Molecular formula:

C32H58O10

IUPAC Name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Constituent 2

Reference substance name:

not yet assigned

Molecular formula:

C50H88O16 C51H90O16 C52H92O16

IUPAC Name:

not yet assigned

Constituent 3

Reference substance name:

2,3-epoxypropyl neodecanoate

EC Number:

247-979-2

EC Name:

2,3-epoxypropyl neodecanoate

Cas Number:

26761-45-5

Molecular formula:

C13H24O3

IUPAC Name:

(oxiran-2-yl)methyl 2,2-dimethyloctanoate

Test material form:

liquid: viscous

Details on test material:

Former EC 615-318-0 / CAS: 716336-43-5: changed to EC 825-846-5 / CAS 876528-25-5

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempterature
- Stability under test conditions: given
- Solubility and stability of the test substance in the solvent/vehicle: stable in acetone for 4 and 24 hours
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

The dose range of the main experiment was set according to the stability and homogeneity data generated within the Envigo Study Number HH16TC. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

The maximum test item concentration of the pre-experiment (250 μg/mL) was based on the solubility properties of the test item in DMSO as determined in the solubility test. Since the stability in DMSO could not be analytically confirmed the solvent was changed to acetone. Based on the result of an additional solubility test with acetone, 1000 μg/mL was chosen as maximum concentration in the main experiment. The stability of the test item formulated in acetone was analytically determined and the data were provided prior to the start of the main experiment.

Main experiment (4 hours exposure) without and with S9-mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
Phase separation was observed at 31.3 µg/mL and above without S9 and at 62.5 µ/mL and above with S9 mix. The cultures at 62.5 μg/mL and above without metabolic activation, and at 125.0 μg/mL and above with metabolic activation were not continued to avoid analysis of too many phase separating concentrations.
In the main experiment there was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item dissolved in acetone.

Vehicle / solvent:

- solvent(s) used: for the pre-experiment DMSO was used; for the main experiment acetone was used
- final concentration of solvent in the culture medium: 0.5 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen based on its compatibility with the test item, its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation. The homogeneity and stability was confirmed for the test item in acetone formulations (within Envigo Study no. HH16TC).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Culture Medium:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10 % FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.

Pre-Experiment:
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH-value and the osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.

In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 2.0 μg/mL and 250.0 μg/mL were used. The highest concentration of the pre-experiment was based on the solubility properties of the test item in DMSO.

No relevant cytotoxic effects indicated by a relative cloning efficiency of 50 % or below were noted up to the maximum concentration with and without metabolic activation.

The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 15.6 μg/mL and above without metabolic activation, and at 250.0 μg/mL with metabolic activation after 4 hours treatment.

Main Experiment:
Since the stability of the test item in acetone was determined analytically, acetone was used as solvent in the main experiment. The highest concentration of 1000.0 μg/mL used in the main experiment was based on the result of an additional solubility test performed with acetone.
In the pre- and main experiment there was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item in acetone.

Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5-10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10 E7 were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.

Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90 % cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled (see 3.5.2 Dose selection).

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95 % control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
A t-Test was not performed since all mean mutant frequencies were well within the 95 % confidence interval of our laboratory’s historical negative control data.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no, but tested up to and including phase separating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See Table 1 - Summary of results.
The cultures at 62.5 μg/mL and above without metabolic activation, and at 125.0 μg/mL and above with metabolic activation were not continued to avoid analysis of too many phase separating concentrations.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in the absence nor in the presence of metabolic activation.
No biologically relevant increase in mutation frequency as the mean of both parallel cultures was observed in the main experiment up to the maximum concentrations scored for gene mutations.
The mean mutant frequency obtained in the solvent controls was 18.2 without S9 mix and 20.8 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 12.2 up to 27.7 mutants per 106 cells.
The linear regression analysis showed no significant dose dependend trend of the mutation frequency at any of the experimental groups.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Any other information on results incl. tables

Table 1: Summary of results (main experimen, 4 hour treatment)

	conc. µg/mL	PS	S9 mix	RACE (%)	mutant colonies/10 E6 cells
solvent control	-	-	-	100.0	18.2
positive control (EMS)	300.0	-	-	73.5	242.0
test item	2.0	-	-	83.3	27.7
test item	3.9	-	-	69.9	20.0
test item	7.8	-	-	82.5	20.8
test item	15.6	-	-	83.3	19.1
test item	31.3	PS	-	78.2	12.2
test item	62.5	PS	-	#	#
solvent control	-	-	+	100.0	20.8
positive control (DMBA)	2.3	-	+	99.6	132.9
test item	2.0	-	+	133.1	18.8
test item	3.9	-	+	114.1	19.1
test item	7.8	-	+	105.9	19.5
test item	15.6	-	+	101.5	17.5
test item	31.3	-	+	102.2	16.6
test item	62.5	PS	+	93.8	22.9
test item	125.0	PS	+	#	#

solvent control: 0.5 % acetone

PS: phase separation at the beninning and at the end of treatment

RACE: Relative Adjusted Cloning Efficiency I

# these and higher cultures were not continued to avoid analysis of too many phase separating concentrations

95 % confidence interval for all evaluable cultures without S9 mix: 2.7 -31.2

95 % confidence interval for all evaluable cultures with S9 mix: 2.9 -30.9

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster following OECD 476. The main experiment was performed with a treatment time of 4 hours with and without metabolic activation. Concentrations of 2.0 to 31.3 μg/mL with and 2.0 to 62.5 μg/mL without metabolic activation were evaluated in the main experiment due to phase separation at higher concentrations. No relevant cytotoxicity and no substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.