Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. residues

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apriil 2016 - November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

oecd 476 (2015)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The Organisation for Economic Co-operation and Development (OECD), Guideline for the Testing of Chemicals 476, (adopted by the Council on 28th July 2015), “In vitro Mammalian Cell Gene Mutation Test”.

Deviations:

no

Principles of method if other than guideline:

Principle of the Test Method
Cells deficient in Hypoxanthine-guanine Phosphoribosyl Transferase (HPRT), due to mutation, are resistant to the cytotoxic effects of the purine analogue (6-thioguanine). HPRT proficient cells are sensitive to 6-thioguanine which causes the inhibition of cellular metabolism and halts further cell division. HPRT deficient cells are presumed to arise through mutation at the hprt locus; they cannot metabolize 6-thioguanine and thus survive and grow in its presence.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. Residues

IUPAC Name:

Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. Residues

Test material form:

semi-solid (amorphous): gel

Details on test material:

Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. residues- Physical state: black , brown semisolid- Analytical purity:100% - Storage condition of test material: room temperature- Solubility: < 10% in water, soluble in acetone, hexane and dichloromethane

Specific details on test material used for the study:

Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. residues- Physical state: black , brown semisolid- Analytical purity:100% - Storage condition of test material: room temperature- Solubility: < 10% in water, soluble in acetone, hexane and dichloromethane

Batch/Lot Number 01-37
Date of Manufacture September 2015
Storage Temperature : Room temperature
Storage Container : In original container as supplied by the Sponsor
Storage Location : Test Item Control Office (TICO), JRF

Method

Target gene:

CHO-K1 cell line (free from mycoplasma contamination), a subclone of the Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation was used for this study. The cells were grown as monolayer in disposable tissue culture flasks and were free from any contamination during the conduct of the study.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO - K1

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Treatment was performed both in the absence and presence of metabolic activation (2% v/v S9). Cultures were maintained in duplicate for each test concentration, negative and positive controls.
Cultures were exposed at concentrations of 78.125, 156.25, 312.5, 625 and 1250 µg/mL in the absence and 39.0625, 78.125, 156.25, 312.5 and 625 µg/mL in the presence of metabolic activation (2% v/v S9 mix) for a period of 4 hours. For the treatment, the first stock solution (stock A) of the test item was prepared by dissolving 500 mg (500.14 mg rounded to 500 mg) of Fatty acids, C6-24 and C6-24 unsatd., Me esters, distillation Residues in DMSO and volume was made up to 4 mL (125000 µg/mL).

Vehicle / solvent:

The test item was insoluble in sterile distilled water, while formed emulsion in DMSO at 500000 µg/mL. Therefore, DMSO was selected as the vehicle for this study.
No significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was observed at 0 and 3 h at any tested concentration (156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL of culture medium). Turbidity was observed at the tested concentrations of 2500 and 5000 µg/mL , while slight turbidity was observed at the tested concentrations of 625 and 1250 µg/mL. Precipitation was not observed up to 312.5 µg/mL of culture medium. The results of pH, osmolality and precipitation tests are provided in APPENDIX 5 of the study report.
Therefore, the guideline limit concentration of 5000 µg/mL was selected as the highest concentration for the cytotoxicity test

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Controls:
Concurrent negative (DMSO) and positive controls were maintained in duplicate, both in the absence and presence of a metabolic activation system. Ethyl Methanesulfonate (0.4 µL/mL) was used as the positive control in the absence of metabolic activation and Benzo(a)pyrene (6 µg/mL) was used as the positive control in the presence of metabolic activation.

Culture Medium:
α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was also supplemented with Penicillin (50 IU/mL of medium) and Streptomycin (50 µg/mL). At the time of selection Minimum Essential Medium Eagle -modification without nucleosides (-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.
The medium to eliminate existing mutants in the culture for treatment was prepared by addition of 2 mL of reconstituted HAT (Hypoxanthine Aminopterine Thymidine) supplement to 98 mL of α-MEM w/o NS with 5% fetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile α-MEM w/o NS. The reconstituted supplement contains 5 x 10-3 Hypoxanthine, 2 x 10-5 M Aminopterine and 8 x 10-4 M Thymidine].

Selective Agent:
2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of
5 µg/mL -MEM without nucleosides.

Culture Vessels:
Disposable tissue cultures flasks of 75 cm2 culture (Corning) area with canted neck were used to culture the cell line. Treatment was given in the same flask. 60 mm disposable culture dishes (Corning) were used to determine the cloning efficiency. Tissue culture dishes (Corning) of 100 mm were used to select mutant colonies.

Rationale for test conditions:

Solubility Test
Solubility was tested at 500000 µg/mL in order achieve the guideline limit dose of 5000 µg/mL. The test item was found to be insoluble in sterile distilled water, however it formed emulsion in dimethyl sulfoxide at 500000, 250000 and 125000 µg/mL. The test item was further diluted with dimethyl sulfoxide to lower concentrations to check for precipitation and changes in pH and osmolality.

Precipitation and pH Test
Prior to the cytotoxicity test, precipitation, pH and osmolality checks were performed to select the maximum achievable concentration in culture medium. The stock solution of 500000 µg/mL was serially diluted to obtain stock solutions of 250000, 125000, 62500, 31250 and 15625 µg/mL. A volume of 50 µL of relevant stock solutions was added to 4.950 mL of culture medium to obtain the final concentration of 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL of culture medium. The pH, precipitation and osmolality of test concentrations in culture medium were assessed at approximately 0 and 3 h after incubation at 37 ± 1 °C and 5% CO2 in a CO2 incubator.

Cytotoxicity Test:
Based on the results of solubility, precipitation, osmolality and pH tests, 5000 µg/mL was selected as the highest concentration to be tested for the cytotoxicity test. The cytotoxicity test was performed both in the absence and presence of metabolic activation (2% v/v S9 mix) at concentrations of 625, 1250, 2500 and 5000 µg/mL. A concurrent negative (DMSO) control was also maintained.
Based on the cytotoxicity test results a concentration of 1250 and 625 µg Fatty acids, C6-24 and C6-24 unsatd., Me esters, distillation Residues/mL were selected as the highest concentration for the main study experiment both in the absence and presence of metabolic activation system (2% v/v S9 mix), respectively.

Evaluation criteria:

Assay Acceptance Criteria
A mutation assay was considered acceptable if it met the following criteria:
a. The criteria for acceptability was a minimum 60% absolute cloning efficiency in negative controls (DMSO) and a spontaneous mutant frequency less than 20 per 106 clonable cells (Nestmann, E.R. et al,. 1991).
b. Positive controls induce a significant increase in the mutant frequency above the concurrent negative control.
2.22.2 Assay Evaluation Criteria
A test item was considered positive in the mutation assay if:
i. Test item causes a concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item causes a three-fold increase (Nestmann, E.R. et al., 1991) in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases were statistically significant and outside the laboratory historical negative (DMSO) control range.
ii. A net increase in mutant colonies of treated above the concurrent control was observed in at least two of the concentrations tested.
Clear negative results obtained in trial-I were not confirmed by a repeat test (short duration), as per revised OECD guideline.

Statistics:

Statistical Analysis
Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on fatty Acids, C6-24 And C6-24 unsatd., Me Esters, Distillation Residues treatment groups against the negative control group (excluding positive control).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS
Solubility, Precipitation, pH and Osmolality Tests
The test item was insoluble in sterile distilled water, while formed emulsion in DMSO at 500000 µg/mL. Therefore, DMSO was selected as the vehicle for this study.
No significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was observed at 0 and 3 h at any tested concentration (156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL of culture medium). Turbidity was observed at the tested concentrations of 2500 and 5000 µg/mL , while slight turbidity was observed at the tested concentrations of 625 and 1250 µg/mL. Precipitation was not observed up to 312.5 µg/mL of culture medium. The results of pH, osmolality and precipitation tests are provided in APPENDIX 5.
Therefore, the guideline limit concentration of 5000 µg/mL was selected as the highest concentration for the cytotoxicity test.

Cytotoxicity Test
Test item cytotoxicity was assessed by calculating the percent relative cloning efficiency following treatment.
The pH and osmolality at the beginning of treatment at 5000 µg/mL was 7.19 and 496 mOsm/kg H2O, respectively (compared to 7.18 and 474 mOsm/kg H2O in the negative control) in the absence of metabolic activation, while pH and osmolality at 5000 µg/mL was 7.11 and 491 mOsm/kg H2O, respectively (compared to 7.20 and 483 mOsm/kg H2O in the negative control), in the presence of metabolic activation (APPENDIX 6). Hence, no significant change in the pH or osmolality was observed up to the guideline limit concentration of 5000 µg/mL both in the absence and presence of metabolic activation.
The percent relative cloning efficiency observed was 39.61, 18.13 in the absence of metabolic activation and 15.25 in the presence of metabolic activation (2% v/v S9), at tested concentrations of 625, 1250 µg/mL in the absence of metabolic activation and 625 µg/mL in the presence of metabolic activation of culture medium, respectively. Excessive toxicity i.e., > 90% cells observed, were dead at the tested concentrations of 2500, 5000 and 1250, 2500 and 5000 in the absence and presence of metabolic activation of culture medium, respectively.
Based on the observed results and to achieve guidelines required cytotoxicity limit i.e. 10-20% relative survival, 1250 µg/mL was selected as the highest concentration in the absence and 625 µg/mL was selected as the highest concentration in the presence of metabolic activation for the main study experiment.

Mutagenicity Test:
No biologically relevant influence of the test item on osmolality and pH was observed in the absence and presence of metabolic activation during the main study.

Any other information on results incl. tables

Adjusted Absolute and Relative Cloning Efficiency/Survival Following Treatment

The mean adjusted absolute cloning efficiency (ACE) and percent relative cloning efficiency/survival following treatment both in the absence and presence of metabolic activation in the Main Study are summarised below:

Group (µg/mL)		Main Study
		- S9		+ S9 (2% v/v S9 mix)
-		Mean Adjusted ACE	RCE(%)	Mean Adjusted ACE	RCE(%)
NC (DMSO)		3.49	100.00	3.32	100.00
T1 (78.125)	T1 (39.0625)	3.16	90.54	3.05	91.87
T2 (156.25)	T2 (78.125)	2.95	84.53	2.92	87.95
T3 (312.5)	T3 (156.25)	2.77	79.37	2.64	79.52
T4  (625)	T4 (312.5)	1.35	38.68	1.39	41.87
T5 (1250)	T5 (625)	0.69	19.77	0.56	16.87
PC		2.66	76.22	2.48	74.7

The individual data is provided inAPPENDIX1. The dose response curves for relative cloning efficiency in the presence and absence of metabolic activation are providedFIGUR

Absolute Cloning Efficiency at Selection and Mutant Frequency

The mean absolute cloning efficiency (ACE) at selection and mean mutation frequency per 1 x 10⁶cells (MF) in the absence and presence of metabolic activation for the Main Study are provided below:

Group (µg/mL)		Main Study
		- S9		+ S9 (2% v/v S9 mix)
		Mean ACE	Mean MF	Mean ACE	Mean MF
NC (DMSO)		0.7012	16.10	0.6897	16.94
T1 (78.125)	T1 (39.0625)	0.6840	15.89	0.6637	15.76
T2 (156.25)	T2 (78.125)	0.6786	15.80	0.6559	17.18
T3 (312.5)	T3 (156.25)	0.6917	15.38	0.6679	17.23
T4  (625)	T4 (312.5)	0.6625	16.40	0.6732	16.44
T5 (1250)	T5 (625)	0.6352	17.12	0.6650	16.97
PC		0.6416	325.18	0.6530	368.80

The values of absolute cloning efficiency at selection, number of mutant colonies and mutant frequency for each test concentration are provided inTABLE2with individual data inAPPENDIX2andAPPENDIX3. The dose response curves for mutant frequency bothin the absence and presence of metabolic activation for the Main Study are provided inFIGURE2.

A weighted regression analysis was performed to evaluate any significant dose-related effect in mutation frequency of cultures treated with fatty acids, C6-24 and C6-24 unsatd., Me esters, distillation Residues with the concurrent negative control group. Statistical analysis was not performed for the positive controls.Themean mutant frequencyof the positive control exhibited a clear increase over the mean value of the negative control demonstrating that positive controlhad potential to induce gene mutations at thehprtlocus of CHO-K1 both in the absence and presence of metabolic activation.This also demonstrated that the S9 mix was capable of metabolizing a pro-mutagen to its mutagenic form(s), thus, demonstrating integrity of the S9 mix.

The regression equation for Main Study is given below:

Main Study	Regression Equation
	Absence of metabolic activation	Presence of metabolic activation
	Y = 0.001 X + 15.699	Y = 0.0004 X + 16.653

The absolute cloning efficiency in the negative control was above 60% during main study. The mutant frequency in the negative control group was less than 20 per 10⁶clonable cells during the main study validating the acceptability of the test system (Li, A.P.et al., 1987). A significant dose-related increase in the mutation frequency was not observed in any of the treated concentrations and the mutation frequency was comparable to that from the negative control group. The increased mutant frequency observed in the positive controls in main study demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Applicant's summary and conclusion

Conclusions:

Based on the results of cytotoxicity test, proposed concentrations for Mutagenicity experiment are:           
78.125, 156.25, 312.5, 625, 1250 in the absence of metabolic activation
39.0625, 39.0625, 78.125, 156.25, 312.5 and 625 µg/mL in the presence of metabolic activation of culture medium.

According to the main study experiment of in vitro cell gene mutation test, the substance is found to be non mutagenic up to the tested concentration of 1250 µg/ml in the absence and 625 µg/ml in the presence of metabolic activation system.
From these results, it is concluded that Fatty acids, C6-24 and C6-24 unsatd., Me esters, Distillation Residues does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the experimental conditions described.

Executive summary:

In a mammalian cell gene mutation assay [hprtlocus], CHO-K1 cells culturedin vitrowere exposed toFatty Acids, C6-24 And C6-24 unsatd., Me Esters, Distillation Residuesat different concentrations, both in the absence and presence of metabolic activation (2% v/v S9 mix) for a period of 4 hours.

Cultures were exposed toFatty Acids, C6-24 And C6-24 unsatd., Me Esters, Distillation Residuesat 5 concentrations (two cultures/dose-level) between 78.125 and 1250 µg/mL of culture medium, in the absence of metabolic activation and between 39.0625 and 625 µg/mL in the presence of metabolic activation (2% v/v S9 mix), selected from a preliminary cytotoxicity test for a period of 4 hours.

No significant dose-related increase in mutation frequency was observed in any treatment concentration between 78.125 and 1250 µg/mL of culture medium in the absence and 39.0625 and 625 µg/mL of culture medium in presence of metabolic activation system (2% v/v S9 mix). The induced mutation frequency was comparable to that of the negative control group. All negative controls were within the historical limits and positive controls showed a clear increase in mutant frequency. No biologically relevant influence of the test item on pH or osmolality was observed both in the absence and presence of metabolic activation during the main study.

All criteria for a valid study were met as described in the study plan. Based on the results from this study, it is concluded thatFatty Acids, C6-24 And C6-24 unsatd., Me Esters, Distillation Residuesdoes not have potential to induce gene mutations at thehprtlocus of CHO-K1 cells both in the absence and presence of metabolic activation system under the experimental conditions described.