Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

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:

29 July to 12 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline 490 without deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2021-04-26

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

EC Number:

252-335-9

EC Name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

Cas Number:

35044-59-8

Molecular formula:

C12H18O2

IUPAC Name:

ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Physical appearance: colourless to pale yellow liquid
- Stored in a dry, well ventilated and dark location at room
temperature (i.e. 10–30°C)

Method

Target gene:

Thymidine kinase (tk) gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine
kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C were obtained from the American Type
Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells
was tested and found to be free of mycoplasma contamination. This test system has been
demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

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: MolTox (Boone, NC), Lot No. 4141, Exp. Date: 05 Sep 2021 stored at -60°C or colder until used.
- method of preparation of S9 mix: S9 fraction, prepared from male Sprague-Dawley rats injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- S9 mix contained final concentration in cultures of: S9 homogenate (10 µL/mL), DL-isocitric acid (17.4 mM) and NADP (3.0 mM). Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

Short treatment without S9 mix: 4-hour treatment:
The selected dose levels were 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121 μg/mL
Short treatment with S9 mix: 4-hour treatment:
The selected dose levels were 4.0, 8.0, 16.1, 32.2, 64.3, 121, 133, 147, 164, 182 and 243 μg/mL
Continuous treatment without S9 mix: 24-h treatment:
The selected dose levels were 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- DMSO was the vehicle of choice based on information provided by the Sponsor and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With S9

Details on test system and experimental conditions:

CELL CULTURE: L5178Y TK+/- cells are an established cell line recommended by international regulations for the in vitro mammalian cell gene mutation test. These cells have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency.
Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK-/- cells. Using the procedure described by Clive and Spector (1975), L5178Y cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.

TREATMENT OF CELLS
The preparation and addition of the test substance dose formulations was carried out under filtered lighting during the exposure period. Treatment was carried out by combining 100 μL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 10^6 L5178Y/TK+/- cells in a total volume of 10 mL.
All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24-hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows: 4 hour treatment – 1 and 2 days after treatment and 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.

SELECTION OF MUTANT PHENOTYPE
Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 11 to 12 days.

The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

EXTENDED TREATMENT AND/OR CONFIRMATORY ASSAY
Verification of a clear positive response was not required (OECD Guideline 490). For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.

Rationale for test conditions:

In the preliminary toxicity test, L5178Y/TK+/- cells was exposed to the vehicle alone in duplicate cultures and nine concentrations of test substance using single cultures. The maximum concentration evaluated the limit dose (10mM) for this assay. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment. Precipitation was determined with the unaided eye at the beginning and end of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control).
In the mouse lymphoma assays, eight to eleven concentrations were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was determined with the unaided eye at the beginning and end of treatment.

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency which occurred only at highly
toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant.
All conclusions were based on scientific judgment; however, the following criteria are presented
as a guide to interpretation of the data (Moore et al., 2006).
- A result was considered positive if a concentration-related increase in mutant frequency
was observed in the treated cultures and one or more treatment conditions with 10% or
greater total growth exhibited induced mutant frequencies of ≥90 mutants/10^6 clonable
cells (based on the average mutant frequency of duplicate cultures). If the average
vehicle control mutant frequency was >90 mutants/10^6 clonable cells, a doubling of
mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
- A result was considered negative if the treated cultures exhibited induced mutant
frequencies of less than 90 mutants/10^6 clonable cells (based on the average mutant
frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
There are some situations in which a chemical would be considered negative when there was no
culture showing between 10 to 20% survival (Office of Food Additive Safety, 2001).
- There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/10^6) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20 and 25% survival.
- There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/10^6) in a series of data points between 100 to 25% survival and there was also a negative data point between 10 and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

Statistics:

The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated
control for suspension growth over two days post-treatment and for total growth (suspension
growth corrected for plating efficiency at the time of selection). The mutant frequency for each
treatment condition was calculated by dividing the mean number of colonies on the TFT-plates
by the mean number of colonies on the VC-plates and multiplying by the dilution factor
(2 x 10-4), and was expressed as TFT-resistant mutants/106 surviving cells. The induced mutant
frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant
frequency of the vehicle control cultures. The International Workshop on Genotoxicity
established a Global Evaluation Factor (GEF) for a positive response at an IMF of
≥90 mutants/106 clonable cells at the Aberdeen meeting in 2003 (Moore et al., 2006)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested in 4-hour treatment with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY ASSAY
The test item was evaluated at concentrations of 7.58, 15.2, 30.3, 60.6, 121, 243, 485, 970 and 1940 μg/mL. The maximum concentration evaluated the limit dose for this assay. Visible precipitate was observed as indicated in the Table 7.6.1/01.

The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Relative suspension growth (RSG) was 34, 94 and 102% at concentrations of 121 μg/mL (4-hour treatment with S9), 60.6 μg/mL (4-hour treatment without S9) and 60.6 μg/mL (24-hour treatment without S9), respectively. RSG was 0% at higher concentration using all treatment conditions.

DEFINITIVE MUTAGENICITY ASSAYS
No visible precipitate was observed at the beginning or end of treatment.
The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

Cultures treated at concentrations of 8.0, 16.1, 32.2, 64.3, 121 and 133 µg/mL (4-hour treatment with S9), 64.3, 71.4, 79.4, 88.2, 98.0 and 109 µg/mL (4-hour treatment without S9) and 64.3, 71.4, 79.4, 88.2 and 98.0 µg/mL (24-hour treatment without S9) exhibited 12 to 45%, 18 to 98% and 75 to 113% RSG, respectively, and were cloned. Cultures treated at other concentrations were not selected due to toxicity and due to sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 10 to 41% (4-hour treatment with S9), 20 to 96% (4-hour treatment without S9) and 72 to 124% (24-hour treatment without S9). At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested in 4-hour treatment with and without S9. In the 24-hour without activation portion, highest concentration was not between 10 to 20% RTG. Thus this portion of the assay was repeated. No increases in induced mutant frequency ≥ 90 mutants/10^6 clonable cells were observed under any 4 hour treatment conditions. There was no positive statistical trend based in 4-hour treatment without S9. Although there was a positive dose dependent trend in the statistical analysis for 4-hour treatment with S9 condition, no increases in induced mutant frequency ≥ 90 mutants/10^6 clonable cells were observed. Therefore, this condition is not considered as biologically relevant.

In the retest of initial definitive mutagenicity assay, the concentrations tested were 90.9, 92.7, 94.6, 96.6, 98.5, 101, 103, 105, 107 and 109µg/mL with 24-hour treatment without S9.
No visible precipitate was observed at the beginning or end of treatment.
The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Cultures treated at concentrations of 90.9, 92.7, 94.6, 96.6, 98.5, 101 and 103 μg/mL (only replicate at dose levels 101 and 103 μg/mL was cloned due to toxicity) exhibited 11 to 47% RSG, and were cloned. Cultures treated at other concentrations were not selected due to toxicity. Relative total growth of the cloned cultures ranged from 10 to 46%. At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested. No increases in induced mutant frequency ≥ 90 mutants/10^6 clonable cells were observed under any treatment condition. There was no positive statistical trend.

Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Any other information on results incl. tables

Table 7.6.1/01: Preliminary Toxicity Test results of visible precipitate

Treatment condition	Treatment time	Visible precipitate
		At the beginning of treatment period	At the end of treatment period
Non-activated	4-h	≥ 243 µg/mL	≥ 1940 µg/mL
	24-h	≥ 243 µg/mL	None
S9-activated	4-h	≥ 243 µg/mL	≥ 1940 µg/mL

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay, Ethyl Safranate was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The objective of this study was to evaluate the genotoxic potential of the test substance based on quantitation of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells and the sizing of the resulting colonies.

The study was performed according to international guidelines (OECD guideline No. 490) and in compliance with the principles of Good Laboratory Practice.

 

Methods

The test substance, Ethyl Safranate, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Treatment was carried out by combining 100 μL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 10^6 L5178Y/TK+/- cells in a total volume of 10 mL All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24-hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.

The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10-4), and was expressed as TFT-resistant mutants/106 surviving cells.

 

Results

In the preliminary toxicity assay, the concentrations tested were 7.58, 15.2, 30.3, 60.6, 121, 243, 485, 970 and 1940 μg/mL. The maximum concentration evaluated the limit dose (10mM) for this assay. Visible precipitate was observed at concentrations ≥243 μg/mL at the beginning of treatment and at concentrations ≥1940 μg/mL (4-hour treatment with S9), ≥1940 μg/mL(4-hour treatment without S9) and no precipitate for 24-hour treatment without S9 by the end of treatment. Relative suspension growth (RSG) was 34, 94 and 102% at concentrations of 121 μg/mL (4-hour treatment with S9), 60.6 μg/mL (4-hour treatment without S9) and 60.6 μg/mL (24-hour treatment without S9), respectively. RSG was 0% at higher concentration using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 4.0, 8.0, 16.1, 32.2, 64.3, 121, 133, 147, 164, 182 and 243 μg/mL (4-hour treatment with S9), 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121 μg/mL (4-hour treatment without S9) and 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121 μg/mL (24-hour treatment without S9).

In the initial definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 8.0, 16.1, 32.2, 64.3, 121 and 133 μg/mL (4-hour treatment with S9), 64.3, 71.4, 79.4, 88.2, 98.0 and 109 μg/mL (4-hour treatment without S9) and 64.3, 71.4, 79.4, 88.2 and 98.0 μg/mL (24-hour treatment without S9) exhibited 12 to 45%, 18 to 98% and 75 to 113% RSG, respectively, and were cloned. Cultures treated at other concentrations were not selected due to toxicity and due to sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 10 to 41% (4-hour treatment with S9), 20 to 96% (4-hour treatment without S9) and 72 to 124% (24-hour treatment without S9). At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested in 4-hour treatment with and without S9. In the 24-hour without activation portion, highest concentration was not between 10 to 20% RTG. Thus this portion of the assay was repeated. No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any 4 hour treatment conditions. There was no positive statistical trend based in 4-hour treatment without S9. Although there was a positive dose dependent trend in the statistical analysis for 4-hour treatment with S9 condition, no increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed. Therefore, this condition is not considered as biologically relevant.

In the retest of initial definitive mutagenicity assay, the concentrations tested were 90.9, 92.7, 94.6, 96.6, 98.5, 101, 103, 105, 107 and 109μg/mL with 24-hour treatment without S9. No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 90.9, 92.7, 94.6, 96.6, 98.5, 101 and 103 μg/mL (only replicate at dose levels 101 and 103 μg/mL was cloned due to toxicity) exhibited 11 to 47% RSG, and were cloned. Cultures treated at other concentrations were not selected due to toxicity. Relative total growth of the cloned cultures ranged from 10 to 46%. At least one replicate had 10 to 20% RTG compared to the vehicle control in the highest concentration tested. No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition. There was no positive statistical trend.

 

Conclusion

Under the conditions of the assay, Ethyl Safranate was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.