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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Mutation Test (OECD 471, 2007, GLP, K, rel.1): not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 & TA100, and E.coli strain WP2uvrA.

 

- in vitro Micronucleus test (OECD 487, 2021, GLP, K, rel. 1): negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

 

- in vitro Mouse lymphoma assay  (OECD 490, 2021, GLP, K, rel. 1): 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.

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:

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

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

Target gene:

Thymidine kinase (tk) gene

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.

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

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)

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

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.

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

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 July to 03 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 487 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

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 2021-04-30

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable (in vitro micronucleus test of: clastogenic and aneugenic potential)

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, male and female adult (31 years of age for the preliminary toxicity assay and 26 years of age for the minronucleus assay) donors, without recent history of radiotherapy, viral infection or the administration of
drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals.

Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

After the 4-hour treatment in the non-activated and the S9-activated studies or for the 24-hour treatment in the non-activated study, cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: MolTox (Boone, NC), Lot No. 4203, Exp. Date: 04 Feb 2022 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 culture medium (RPMI 1640 serum-free medium) of: S9 homogenate (20 µL/mL), MgCl2 (2 mM), KCl (6 mM), glucose-6-phosphate (1 mM) and NADP (1 mM). Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test (4-hour with and without S9 mix) and (24-hour without S9 mix): 0.193, 0.579, 1.93, 5.79, 19.3, 57.9, 193, 579 and 1930 µg/mL

- Main tests:
* Without S9 mix (4-hour): 59, 81.6, 96.1, 113, 156 and 184 µg/mL
* With S9 mix (4-hour): 69.4, 133, 156, 184, 217, 255 and 300µg/mL
* Without S9 mix (24-hour): 59, 69.4, 81.6, 96.1, 113, 133 and 156 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Lot No. SHBL2820, Exp. Date 08-2022, supplier Sigma-Aldrich)
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

vinblastine

Remarks:

Without metabolic activation.

Details on test system and experimental conditions:

CULTURE OF LYMPHOCYTES: Peripheral blood lymphocytes were obtained from healthy non-smoking individuals (31 years of age for the preliminary toxicity assay and 26 years of age for the minronucleus assay) donors, without recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals.
As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen.
Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

SELECTION OF DOSE LEVELS
In the preliminary toxicity test, precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).
In the micronucleus assay, precipitation of the test substance dosing solution in the treatment medium was also determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the 55 ± 5% cytotoxicity (CBPI relative to the vehicle control). Two additional doses were included in the evaluation of micronuclei. The pH of the highest dose of dosing solution in the treatment medium was measured using test tape.

METHOD OF APPLICATION: in medium
RPMI-1640 containing 15% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin.

DURATION
- Exposure duration: 4-h (± S9) and 24-h continuous exposure (-S9) in preliminary toxicity test; 4-h (± S9) and 24-h continuous exposure (-S9) in main tests
- Fixation time (start of exposure up to fixation or harvest of cells): 24-h

SPINDLE INHIBITOR (cytogenetic assays): After the 4-hour treatment in the non-activated and the S9-activated studies, cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL for 20 hours (± 30 minutes). After the 24-hour treatment in the non-activated study, cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL for 24 hours (± 30 minutes).

STAIN (for cytogenetic assays): Slides were stained with Acridine orange solution.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: HPBL were exposed to vehicle alone and to nine concentrations of test substance with halflog dose spacing using single cultures.
- Main test: Duplicate cultures were prepared for each treatment level, vehicle and positive control cultures;

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 µg/mL and returned to the incubator under standard conditions. For the 24-hour treatment in the non-activated study, cytoB (6.0 µg/mL) was added at the beginning of the treatment. Cells were collected by centrifugation after being exposed to cyto B, 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells and swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest.

Slide Preparation: To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

NUMBER OF CELLS EVALUATED:
- Cell Cycle Kinetics Scoring: For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control.
- Scoring of Micronuclei: The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and
scored for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus.
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic
bridges).
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
CBPI = [(1 x No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells scored]
T = test item treatment culture
C = vehicle control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Evaluation criteria:

The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the
criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest dose of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality of the test substance doses in treatment medium was considered acceptable (< 120% of vehicle).

- Precipitation: Visible precipitate and hemolysis were observed in treatment medium at the doses of the preliminary toxicity test presented below in the Table 7.6.1/01.
Visible precipitate was observed in treatment medium at the doses of the micronucleus assay presented below in the Table 7.6.1/02.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) was observed at doses ≥ 193 µg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 579 µg/mL in the S9-activated 4-hour exposure group.
These data were used to select concentrations for the main test.

MAIN STUDY RESULTS
- The results of cytotoxicity and micronucleus data for the untreated controls were comparable
to that of the vehicle control. Therefore, use of organic solvent (DMSO) had no adverse effect.
The results for the positive and vehicle controls indicate that all criteria for a valid assay were
met indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

- Results from cytotoxicity measurements:
Cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) was observed at doses ≥ 133 µg/mL in the non-activated 4-hour exposure groups, and at doses ≥ 156 µg/mL in the S9-activated 4-hour
exposure group; and at doses ≥ 96.1 µg/mL in the non-activated 24-hour exposure group.

- Genotoxicity results:
Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.

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

Treatment condition	Treatment time	Visible precipitate		Hemolysis at the conclusion of treatment period
		At the beginning of treatment period	At the conclusion of treatment period
Non-activated	4-h	≥ 579 µg/mL	≥ 579 µg/mL	≥ 579 µg/mL
	24-h	≥ 579 µg/mL	≥ 579 µg/mL	≥ 193 µg/mL
S9-activated	4-h	≥ 579 µg/mL	≥ 579 µg/mL	≥ 579 µg/mL

 

Table 7.6.1/02: Micronucleus assay results of visible precipitate

Treatment condition	Treatment time	Visible precipitate
		At the beginning of treatment period	At the conclusion of treatment period
Non-activated	4-h	None	None
	24-h	None	None
S9-activated	4-h	≥ 217 µg/mL	300 µg/mL

Conclusions:

Under the conditions of the assay described in this report, Ethyl Safranate was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Therefore, it was concluded that Ethyl Safranate is not clastogenic or aneugenic and has no genotoxic potential in this test system.

Executive summary:

The micronucleus study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls.

 

The study consisted of a preliminary toxicity test and a main micronucleus test where HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle and positive control cultures were included in all appropriate test conditions.

In the preliminary toxicity assay, the doses tested ranged from 0.193 to 1930 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity [≥ 55 ± 5% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 193 µg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 579 µg/mL  in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 579 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 59 to 184 µg/mL for the non-activated 4-hour exposure group, from 69.4 to 300 µg/mL for the S9-activated
4-hour exposure group, and from 59 to 156 µg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) was observed at doses ≥ 133 µg/mL in the non-activated 4-hour exposure groups, and at doses≥ 156 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 96.1 µg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the three exposure groups.

 

The doses selected for evaluation of micronuclei were 59, 96.1, and 133 µg/mL for the nonactivated 4-hour exposure group; 69.4, 133, and 156 µg/mL for the S9-activated 4-hour exposure group; and 59, 69.4, and 96.1 µg/mL for the non-activated 24-hour exposure group.

 

Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.

These results indicate Ethyl Safranate was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. Therefore, it was concluded that Ethyl Safranate is not clastogenic or aneugenic and has no genotoxic potential in this test system.

This study is considered as acceptable and satisfies the requirement for the mammalian cell chromosomal aberration endpoint.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 24 April and 15 July 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (Inspected on 30th August 2005).

Type of assay:

bacterial gene mutation assay

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

Cytokinesis block (if used):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 was prepared in house on 03 February and 20 May 2007.
- method of preparation of S9 mix : the S9 is prepared from 6-8 weeks old male SD rats, weight approx. 250 g induced by applications of 80/100 mg/kg bw. Phenobarbital i.p and beta-naphtoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 was stored at -196°C. The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)

Test concentrations with justification for top dose:

Pre-experiment/Cytotoxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2 uvrA with and without S9 under the direct plate incorporation method.

Mutagenicity test:
Experiment I: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 without S9 under the direct plate incorporation method.
15, 50, 150, 500, 1500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 with S9 and WP2 uvrA with and without S9 under the direct plate incorporation method.
Experiment II: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 without S9 under the direct plate incorporation method.
15, 50, 150, 500, 1500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 with S9 and WP2 uvrA with and without S9 under the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: the test material was immiscible in sterile water and only partially miscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed in house.
- Justification for percentage of solvent in the final culture medium: no data.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2 AA

Remarks:

With S9-mix

Details on test system and experimental conditions:

TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne Liquid Nitrogen Freezer, Model SXR 34.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approximately 48 hours at 37 °C

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

Rationale for test conditions:

The most widely used assays for detecting gene mutations are those using bacteria.
They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Statistics:

A statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: In solubility testing, the test item was found to be fully miscible in acetone. Hence, Acetone was selected as a vehicle for the study.
- Precipitation: no test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

CYTOTOXICITY TEST:
The test material was toxic initially at and above 1500 µg/plate to the strains of bacteria used (TA 100 and WP2uvrA), (See Table 7.6.1/1).

MUTAGENICITY TEST:
The test material caused a visible reduction in the growth of the bacterial background lawn initially at and above 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or its toxic limit.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.
HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

Table 7.6.1/3: Preliminary Toxicity Test

 

+/- S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	74	87	85	77	124	79	70	86	76	55*	52*
+	TA100	87	65	81	78	63	75	75	77	78	64	0*
-	WP2uvrA-	25	28	23	27	11	24	22	13	24	28	0*
+	WP2uvrA-	27	31	31	30	26	37	26	39	27	24	24*

 * Partial absence of bacterial background lawn.

Prior to the use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all found to be satisfactory). The amino acid supplemented to agar and the S9-mix used in both experiments was shown to be sterile.

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and 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 to the test material after dilution in acetone, both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the first experiment (range-finding test) was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial background lawn initially at and above 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

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

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and 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 No.	Test / Guideline Reliability	Endpoint	Strains/Cell Type	Metabolic Activation (S9)	Test Concentration	Result and Comment
1	Ames Test (OECD 471) K, rel. 1, 2007	Gene mutation in bacteria	S. typhimurium strains TA1535, TA1537, TA98 & TA100, and E.coli strain WP2uvrA	With and without S9	All Salmonella strains without S9: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate All Salmonella with S9 and E.Coli with and without S9: 15, 50, 150, 500, 1500 and 5000 µg/plate	Negative with and without S9
2	Micronucleus (OECD 487) K, rel.1, 2021	in vitro mammalian cell micronucleus test	Human lymhocytes	With and without S9	*4-hour, -S9: 59, 81.6, 96.1, 113, 156 and 184 µg/mL *4-hour, +S9: 69.4, 133, 156, 184, 217, 255 and 300µg/mL *24-hour, -S9: 59, 69.4, 81.6, 96.1, 113, 133 and 156 µg/mL	-S9: non clastogenic +S9: non clastogenic
3	L5178Y cells/MLA (OECD 490) K, rel.1	Gene mutation in mammalian cells	L5178Y mouse lymphoma cells	With and without S9	*4-hour, -S9: 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121 μg/mL *4-hour, +S9: 4.0, 8.0, 16.1, 32.2, 64.3, 121, 133, 147, 164, 182 and 243 μg/mL *24-h, -S9: 57.9, 64.3, 71.4, 79.4, 88.2, 98.0, 109 and 121μg/mL.	Negative with and without S9

 

Gene mutation Assay (Test N° 1):

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

 

Micronucleus test (Test n°2)

The micronucleus study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls.

The study consisted of a preliminary toxicity test and a main micronucleus test where HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle and positive control cultures were included in all appropriate test conditions.

In the preliminary toxicity assay, the doses tested ranged from 0.193 to 1930 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity [≥ 55 ± 5% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 193 µg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 579 µg/mL  in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 579 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 59 to 184 µg/mL for the non-activated 4-hour exposure group, from 69.4 to 300 µg/mL for the S9-activated
4-hour exposure group, and from 59 to 156 µg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 55 ± 5% CBPI relative to the vehicle control) was observed at doses ≥ 133 µg/mL in the non-activated 4-hour exposure groups, and at doses≥ 156 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 96.1 µg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the three exposure groups.

The doses selected for evaluation of micronuclei were 59, 96.1, and 133 µg/mL for the nonactivated 4-hour exposure group; 69.4, 133, and 156 µg/mL for the S9-activated 4-hour exposure group; and 59, 69.4, and 96.1 µg/mL for the non-activated 24-hour exposure group.

Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.

These results indicate Ethyl Safranate was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. Therefore, it was concluded that Ethyl Safranate is not clastogenic or aneugenic and has no genotoxic potential in this test system.

This study is considered as acceptable and satisfies the requirement for the mammalian cell chromosomal aberration endpoint.

 

Mouse lymphoma assay (Test n°3)

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.

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.

 

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.

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.

Justification for classification or non-classification

Harmonized classification:

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

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.