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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that Fatty acids, C16-18 (even numbered), iron(III) salts is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102 when tested under the specified experimental conditions.

From results of this study, it is concluded that Fatty Acids, C16-18 (even numbered), Iron (III) salts did not show any potential to induce gene mutation, at the hprt locus of CHO-K1 cells, either in the absence or presence of the metabolic activation (2% v/v S9 mix), under the present experimental conditions.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2018 (Study Initiation) to 22 November 2018 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: IRST7H112A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: 18 June 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, kept in original container as supplied by the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Formed a homogenous suspension in distilled water. Assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as a homogenous suspension in distilled water prior to dosing

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Prepared as a homogenous suspension in distilled water prior to dosing

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: pH and osmolality not specified. No precipitate observed at 5000 µg/plate, the limit guideline concentration, in the absence and presence of metabolic activation.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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 : The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The Salmonella typhimurium strains used were obtained from Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, USA.

- method of preparation of S9 mix:

The prepared co-factor mix was dispensed and stored below 0 °C.
S9 mix (10 mL volume)
Constituent Initial Toxicity Mutation Test Confirmatory Mutation Test
5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)
Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL
The S9 mix was prepared fresh by adding the required quantity of S9 fraction to thawed co-factors and maintained in an ice bath. Any remaining mix was discarded.

- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for the initial toxicity-mutation assay and 10% v/v S9 mix for the confirmatory mutation assay) prepared for treatment was added aseptically to 2 mL of top agar, mixed and poured onto MGA plates.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):

Sterility Check For Initial Toxicity Mutation Test Confirmatory Mutation Test
Top agar Sterile Sterile
S9 mix Sterile # Sterile ##
Solvent (DW) Sterile Sterile
T.I. Stock Solution * Sterile Sterile
MGA Plate (Blank) Sterile Sterile
ONB Solution Sterile Sterile
0.2M Sodium Phosphate Buffer Sterile Sterile

Key: * = Highest Concentration of Test Item Stock Solution, # = 5% v/v S9 mix, ## =10% v/v S9 mix, T.I. = Test Item, DW = Distilled Water,
MGA = Minimal Glucose Agar, ONB = Oxoid Nutrient Broth
Test concentrations with justification for top dose:
Bacterial cultures were exposed to Fatty acids, C16-18 (even numbered), iron(III) salts, at 8 dose levels (two plates/dose level) from 1.5 to 5000 µg/plate in an initial toxicity-mutation test. A normal bacterial background lawn with no biologically relevant increase in the number of revertant colonies was observed in all tester strains in the absence and presence of metabolic activation up to 5000 µg/plate, when compared with that of the concurrent vehicle control.
Based on results of the initial toxicity-mutation test, a further 6 test concentrations were selected for the confirmatory mutation test. In this test bacterial cultures were exposed to Fatty acids, C16-18 (even numbered), iron(III) salts, at 6 dose levels (three plates/dose level) from 156.25 to 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water

- Justification for choice of solvent/vehicle: The test item formed a homogenous suspension in distilled water, (50000 µg/mL, Stock A,), therefore distilled water was selected as the vehicle for treatment. A volume of 100 µL from stock A (5000 µg/plate) was added to 2 mL of top agar to assess precipitation. As no precipitation was observed, 5000 µg/plate, the limit guideline concentration, was selected as the highest concentration to be tested in the absence and presence of metabolic activation.

- Justification for percentage of solvent in the final culture medium: 5% v/v (0.1 mL of distilled water was used as the vehicle into 2 mL top agar)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene +S9 (10.0 µg/plate: TA1537, TA1535 and TA102). -S9 (5.0 µg/plate: TA98 and TA100)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate plates/concentration in the initial toxicity mutation-test
Triplicate plates/concentration in the confirmatory mutation test

- Number of independent experiments : Two, an initial toxicity mutation test and a confirmatory mutation test

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 1 - 2 x 109 bacteria/mL, demonstrating the appropriate numbers of bacteria were plated.
- Test substance added in medium; in agar (plate incorporation): Treatments were performed using the plate incorporation technique in all tests.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Petri plates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the background lawn inhibition and reduction in number of colonies.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, Six analysable doses were available to evaluate assay data. Cytotoxicity was detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A result was considered positive if a concentration-related increase and/or a reproducible increase at one or more concentration in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
Rationale for test conditions:
The study was performed in accordence with the specified experimental conditions as per OECD 471 and EU METHOD B.13/14. This guidence is accepted by the OECD and other regulatory authorities.
Evaluation criteria:
A result was considered positive if a concentration-related increase and/or a reproducible increase at one or more concentration in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
The biological relavence of the result was considered.

Strains TA1535, TA1537: data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strains TA98, TA100 and TA102: data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

Statistical analysis was used as an aid in the evaluation of any dose response.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not judged as positive.

The negative results obtained in the initial toxicity-mutation test were confirmed in a confirmatory mutation test using the same method as specified above, with an alteration in concentration spacing and concentration of S9 mix.
Statistics:
A simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102 seperately, to assess the dose dependent nature of any increase in revertant colonies.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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
- Data on pH: Not specified
- Data on osmolality: Not specified
- Possibility of evaporation from medium: Non-volatile
- Water solubility: Formed a homogenous suspension in distilled water at 50 mg/mL (stock solution). Treated a homogenous suspension (01 mL/2 mL plate) to acheive the limit guideline concentration of 5000 ug/plate
- Precipitation and time of the determination: No precipitate observed at 5000 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data : All values for the negative control (distilled water) were within the laboratory historical control range. Positive controls showed a clear increase in the number of revertant colonies demonstrating the efficiency of the test system.

Ames test:
- Signs of toxicity : None
- Individual plate counts : yes
- Mean number of revertant colonies per plate and standard deviation : yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes, all values for the negative control values in all strains were within the negative control historical ranges for each respective strain
Conclusions:
It was concluded that Fatty acids, C16-18 (even numbered), iron(III) salts is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102 when tested under the specified experimental conditions.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2019 (Study Initiation) to XX 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: IRST7H780L
- Expiration date of the lot/batch: August 2023
- Purity test (release) date: September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (Ambient), in original container as supplied by the Sponsor.
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Formed a homogenous suspension in ethanol after sonication for 15 minutes. Higher tested concentations were too viscous to permit further dilution. Assumed stable for the duration of the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared in ethanol as a dosable homogenous suspension.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The test item had no relevant influence on pH or osmolality in the absence or presence of metabolic activation. Based on observed results and the absence of significant cytotoxicity, 125 µg/mL (the lowest concentration with visible precipitate at the end of 4 h treatment) was selected as the highest tested concentration in the absence and presence of metabolic activation for the main study.
- other information: None
Target gene:
Hprt locus of the Chinese Hamster Ovary (CHO)-K1 cell line.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster ovary (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)
- Suitability of cells: The relatively low spontaneous mutation frequency and the stable and easily recognizable karyotype has made the CHO-K1 cell line a suitable test model for mutagen-induced cytotoxicity and gene mutation studies. The Chinese Hamster Ovary (CHO-K1) cell line is easy to maintain and culture and is recommended by the OECD and other regulatory authorities to detect mutation at the hprt locus. The Chinese hamster ovary (CHO) cell line, CHO-K1 is suitable for determining forward mutations at the hprt locus because it is a well characterized and validated system (Hsie et al., 1981).
- Normal cell cycle time (negative control): Not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 24 (Cytotoxicity test) and 25 (Main test)
- Methods for maintenance in cell culture: 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 supplemented with antibiotics and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 μg/mL). At the time of selection, Minimum Essential Medium Eagle alpha-modification without nucleosides (alpha-MEM w/o NS) with 10% dialyzed fetal bovine serum was used, as selective medium.
- Cell cycle length, doubling time or proliferation index : Not specified
- Modal number of chromosomes: Not specified
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:

α-MEM with nucleosides (Minimum Essential Medium, Eagle α-modification 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 supplemented with antibiotics and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 μg/mL). At the time of selection, Minimum Essential Medium Eagle alpha-modification without nucleosides (alpha-MEM w/o NS) with 10% dialyzed fetal bovine serum was used, as selective medium.
The medium to eliminate existing mutants in the treatment culture was prepared by addition of 2 mL of reconstituted HAT supplement with 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].

Culture flasks prepared for treatment were incubated in a humid atmosphere at 37 ± 1 °C in 5% CO2 (standard conditions).

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 ; The S9 fraction procured from M/s G.P. Meshram, Nagpur (Lot N° MWR/ARI/S9F/01/18).
- method of preparation of S9 mix: To test indirect acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction is used. The S9 fraction is buffered and supplemented with the essential co-factors beta-NADP, KCl and Glucose-6- phosphate to form the “S9 mix”.
- concentration or volume of S9 mix and S9 in the final culture medium: For treatment in the presence of metabolic activation, S9 fraction was used at a final concentration 2% v/v in the main study.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability); In line with the historical data, the mean mutation frequency of the positive control exhibited a clear increase over the mean value of the negative control, thus demonstrating that positive control had potential to induce gene mutations, at the hprt locus of CHO-K1, in the absence and presence of the metabolic activation. This also demonstrated that the S9 mix was capable of metabolising a pro-mutagen to its mutagenic form(s), thus, demonstrating the integrity of the S9 mix. Maintained under sterile conditions using sterile media and suppliments where possible. An S9 efficiency check report is included within the final report.
Test concentrations with justification for top dose:
Main test: 7.81, 15.63, 31.25, 62.5 and 125 µg/mL in the absence and presence of metabolic activation (2% v/v S9 mix) for a period of 4 hours.
Doses were selected based on cytotoxicity test results, 125 µg/mL was selected as the limit test concentration as visible precipitation was present at the end of
4 h treatment in the absence and presence of metabolic activation.

Vehicle / solvent:
- Vehicle used: 99.9% ethanol

- Justification for choice of solvent/vehicle: The test item solubility was tested in sterile distilled water, DMSO, and ethanol. The test item was insoluble in distilled water, and DMSO. However, a thick suspension was observed at 200 mg/mL (i.e. 200005 µg/mL) in ethanol (99.9%), which was too thick to process for further dilution. A lower stock of 50 mg/mL (50001.25 µg/mL) of the test item in ethanol has provided homogenous suspension after 15 minute sonication. Hence, ethanol was selected as the suitable vehicle.

- Justification for percentage of solvent in the final culture medium: 1% in the final treatment medium, as generally organic solvents should not exceed 1% v/v.
Untreated negative controls:
yes
Remarks:
α-MEM
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration; Two cultures/dose-level
- Number of independent experiments : One cytotoxicity test and one main mutagenicity test

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 24 hours prior to treatment, 32 culture flasks were prepared by seeding 2014500 cells per flask.
- Test substance added in suspension : yes, administered as a homogenous suspension.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Culture flasks were maintained for each test concentration, negative, vehicle and positive controls. The cultures were incubated at 37 ± 1 °C in 5% CO2 in humidified air for 4 hours.
- Harvest time after the end of treatment (sampling/recovery times): Treated (Day 1), Subculture for Mutation Expression (Days 3 and 5), Expression period (Days 7 to 9) Selection of Mutants (Day 8).

FOR GENE MUTATION:
- Expression period: 2 days (days 7 to 9)
- Selection time (if incubation with a selective agent): 2-amino-6-mercaptopurine (6-thioguanine) was used as the selective agent at 5 µg/mL alpha-MEM without nucleosides. No time period specified.
- Fixation time (start of exposure up to fixation or harvest of cells): Main test: On day 16 (Eneumeration of colonies), the plates were removed from the incubator and the medium decanted, colonies were then fixed using 3.7% formaldehyde for 10 minutes. The fixative was removed and the colonies stained using 0.4% methylene blue for 10 minutes. The number of colonies formed on each replicate plate was counted and recorded. The number of colonies was used to calculate the absolute cloning efficiency at the time of selection, the number of clonable cells and the mutation frequency per 1 x 10-6 clonable cells.
- Method used: Cells were grown as a monolayer in disposable tissue culture flasks.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure. 2-amino-6-mercaptopurine (6-thioguanine) was used as the selective agent at a concentration of 5 µg/mL alpha-MEM without nucleosides. For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C in 5% CO2 in humidified air for 8 days.

- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Culture Preparation (Day 0), approximately 24 hours prior to treatment, 32 culture flasks were prepared by seeding 2014500 cells per flask.
140, the number of cells seeded for Individual Plate Count, Absolute an Relative Cloning Efficiency Following Treatment and Individual Plate Counts and Absolute Cloning Efficiency at Selection
Ca. 203300 for the number of cells inoculated/plate.

- Criteria for small (slow growing) and large (fast growing) colonies: Not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Plates were incubated for 8 days to determine relative survival and demonstrate the cytotoxic effect of selected test concentrations.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
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 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.
Rationale for test conditions:
The relatively low spontaneous mutation frequency and the stable and easily recognizable karyotype has made the CHO-K1 cell line a suitable test model for mutagen-induced cytotoxicity and gene mutation studies. The Chinese Hamster Ovary (CHO-K1) cell line is easy to maintain and culture and is recommended by the OECD and other regulatory authorities to detect mutation at the hprt locus. The Chinese hamster ovary (CHO) cell line, CHO-K1 is suitable for determining forward mutations at the hprt locus because it is a well characterized and validated system (Hsie et al., 1981). Culture flasks prepared for treatment were incubated in a humid atmosphere at 37 ± 1 °C in 5% CO2 (standard conditions).
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item was considered to be clearly positive if all the following criteria are met, in any of the experimental conditions examined:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is concentration-related when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit).

When all of these criteria are met, the test item will be then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:

d. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
e. There is no concentration-related increase when evaluated with an appropriate trend test.
f. All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).

The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

There is no requirement for verification of a clearly positive or negative response.
Statistics:
Weighted regression analysis was performed to evaluate any significant dose-related effect in the mutation frequency of cultures treated with the concurrent negative control group. Statistical analysis was not performed for positive controls (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981).

Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: None
- Data on osmolality: None
- Possibility of evaporation from medium: Not applicable
- Water solubility: Insoluble in distilled water. Formed a homogenous suspension at 50 mg/mL in ethanol after 15 minute sonication. Main test doses prepared as a homogenous suspension in ethanol to permit a doasable suspension acceptable for test system administration.
- Precipitation and time of the determination: Precipitation at 125, 250 and 500 µg/mL in the cytotoxicity test. 125 µg/mL (the lowest concentration with visible precipitate at the end of 4 h treatment) was selected as the highest tested concentration in the absence and presence of metabolic activation for the main study.

RANGE-FINDING/SCREENING STUDIES (if applicable): Doses selected based on cytotoxicity test results, 125 µg/mL was selected as the limit test concentration based on visible precipitate at the end of 4 h treatment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data . All negative controls were within historical limits and positive controls showed a clear increase in mutation frequency.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible . No dose response relationship was observed on treatment groups against the negative control group (excluding positive controls).

Gene mutation tests in mammalian cells: - Results from cytotoxicity measurements:

In the Absence of Metabolic activation
Treatment Conc.
(µg/mL) No. of cells plated Number of colonies MEAN ACE Adjusted ACE Mean Adjusted ACE % RCE
P1 P2
NC 140 93 91 92.00 0.6571 2.2570 2.2375 100
140 95 100 97.50 0.6964 2.2180
VC 140 95 96 95.50 0.6821 2.3854 2.1421 95.74
140 101 97 99.00 0.7071 1.8988
T1 (3.91) 140 95 86 90.50 0.6464 2.0184 1.8924 88.34
140 101 97 99.00 0.7071 1.7663
T2 (7.81) 140 95 96 95.50 0.6821 1.7039 1.7351 81.00
140 101 97 99.00 0.7071 1.7663
T3 (15.63) 140 87 89 88.00 0.6286 1.6095 1.5275 71.31
140 92 88 90.00 0.6429 1.4454
T4 (31.25) 140 86 91 88.50 0.6321 1.5395 1.5658 73.10
140 84 82 83.00 0.5929 1.5921
T5 (62.5) 140 71 75 73.00 0.5214 1.2699 1.0950 51.12
140 62 63 62.50 0.4464 0.9200
T6 (125) 140 59 65 62.00 0.4429 0.9128 0.8188 38.22
140 63 62 62.50 0.4464 0.7248
In the Presence of Metabolic activation (2% v/v S9)
NC 140 98 96 97.00 0.6929 2.5098 2.3701 100
140 101 99 100.00 0.7143 2.2304
VC 140 95 92 93.50 0.6679 2.2107 2.1815 92.04
140 99 94 96.50 0.6893 2.1523
T1 (3.91) 140 95 92 93.50 0.6679 1.9604 1.9703 90.32
140 99 94 96.50 0.6893 1.9802
T2 (7.81) 140 89 86 87.50 0.6250 1.5613 1.8499 84.80
140 93 95 94.00 0.6714 2.1384
T3 (15.63) 140 94 91 92.50 0.6607 2.0630 1.8317 83.97
140 95 89 92.00 0.6571 1.6004
T4 (31.25) 140 71 72 71.50 0.5107 1.4990 1.4169 64.95
140 85 86 85.50 0.6107 1.3348
T5 (62.5) 140 84 79 81.50 0.5821 1.5631 1.3706 62.83
140 75 64 69.50 0.4964 1.1780
T6 (125) 140 68 62 65.00 0.4643 1.1598 0.9714 44.53
140 52 65 58.50 0.4179 0.7829
Key: NC = Negative control (α-MEM), VC = Vehicle Control (Ethanol), ACE = Absolute cloning efficiency, RCE = Relative cloning efficiency, P1 = Number of colonies in plate 1, P2 = Number of colonies in plate 2, R = Replicate.

- Genotoxicity results:

Group
(µg/mL) Absence of Metabolic Activation Presence of Metabolic Activation
(2% v/v S9 mix)

R ACE N° CP N° CC N° MC MF ACE N° CP N° CC N° MC MF
NC 1 0.7179 2439720 1751475 30 17.13 0.7179 2440512 1752044 32 18.26
2 0.7071 2440476 1725661 34 19.70 0.6964 2440236 1699380 27 15.89
VC 1 0.7107 2439840 1733994 27 15.57 0.7000 2439456 1707619 29 16.98
2 0.6893 2440476 1682220 35 20.81 0.6964 2439600 1698937 32 18.84
T1
(7.81) 1 0.6571 2439840 1603219 28 17.46 0.6500 2440320 1586208 27 17.02
2 0.6893 2439672 1681666 32 19.03 0.6071 2439600 1481081 29 19.58
T2 (15.63) 1 0.6500 2440200 1586130 26 16.39 0.6393 2439672 1559682 27 17.31
2 0.6321 2439600 1542071 29 18.81 0.5964 2440320 1455407 25 17.18
T3 (31.25) 1 0.5750 2440356 1403205 20 14.25 0.5536 2440068 1350822 27 19.99
2 0.6107 2439804 1489988 25 16.78 0.5750 2439840 1402908 19 13.54
T4
(62.5) 1 0.5857 2439996 1429106 21 14.69 0.4893 2440356 1194066 22 18.42
2 0.5786 2440356 1411990 19 13.46 0.5000 2439600 1219800 17 13.94
T5
(125) 1 0.4571 2439996 1115322 20 17.93 0.4464 2440200 1089305 12 11.02
2 0.4536 2440140 1106848 15 13.55 0.4214 2439600 1028047 16 15.56
PC 1 0.6643 2440200 1621025 446 275.13 0.6393 2440200 1560020 443 283.97
2 0.6464 2439804 1577089 501 317.67 0.6714 2440164 1638326 482 294.20

Key: NC = Negative control (α-MEM), VC = Vehicle Control (Ethanol), ACE = Absolute cloning efficiency, N° CP = N° of cells plated, N° CC = N° of Clonable cells, N° MC = N° of mutant colonies, MF = Mutant frequency per 106 clonable cells, T = Treatment concentration, PC = Positive control [0.4 µL Ethyl methanesulfonate/mL in the absence of metabolic activation and 6 µg Benzo(a)pyrene/mL in the presence of metabolic activation].

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes
Conclusions:
It is concluded that Fatty acids, C16-18 (even numbered), iron(III) salts did not show any potential to induce gene mutations, at the hprt locus of CHO-K1 cells, in the absence or presence of metabolic activation (2% v/v S9 mix), under the described experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification