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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Fatty acids, C16-18 (even numbered), manganese(II) salts is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified experimental conditions.

From results of the micronucleus study, it is concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, both in the absence or presence (2% v/v S9 mix) of metabolic activation, under the described experimental conditions.

Link to relevant study records

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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 materia: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: 12 June 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), 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 suspension in 95% ethanol. 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 as a suspension in 95% ethanol prior to dosing

FORM AS APPLIED IN THE TEST (if different from that of starting material) Prepared as a suspension in 95% ethanol 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 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 (95% Ethanol) 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:
In an initial toxicity-mutation test, bacterial cultures were exposed to Fatty acids, C16-18 (even numbered), manganese(II) salts at 8 concentrations from 1.5 to 5000 µg/plate, the limit guideline concentration. Normal growth and no biologically relevant increase in the number of revertant colonies (i.e. no mutagenic effect) was observed in the absence and presence of metabolic activation in the absence and presence of metabolic activation (5% v/v S9 mix) in tester strains TA1537, TA1535, TA98, TA100, and TA102.
Based on the initial toxicity mutation test, the Confirmatory Mutation Test was performed in tester strains TA1537, TA1535, TA98, TA100, and TA102 upto the limit guideline concentration of 5000 µg/plate using a modified dose spacing and an increased concentration of S9 mix. The selected test concentrations were 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate of Fatty acids, C16-18 (even numbered), manganese(II) salts in the absence and presence of metabolic activation (10% v/v S9 mix).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 95% ethanol

- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water, DMSO and acetone at 50000 µg/mL, however it formed a suspension in 95% ethanol at 50000 µg/mL. 95% ethanol was therefore selected as the vehicle for treatment. A volume of 100 µL (5000 µg/plate) was added to 2 mL of top agar to assess precipitation. No precipitation was observed at 5000 µg/plate. Therefore, 5000 µg/plate was selected as the highest tested concentration for the initial toxicity-mutation test in the absence and presence (5% v/v S9 mix) of metabolic activation.

- Justification for percentage of solvent in the final culture medium: 5% v/v (0.1 mL of ethanol was used as the vehicle into 2 ml top agar)
Untreated negative controls:
yes
Remarks:
0.2 M phosphate buffer
Negative solvent / vehicle controls:
yes
Remarks:
95% ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene +S9 (10 ug/plate: TA1537, TA1535 and TA102). -S9 (5 ug/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 an confirmatory mutation test

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate bacterial numbers 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, e.g.: 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 is considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more test 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 a over the range tested and/or a reproducible increase at one or more test concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
The biological relevance of any 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 evaluated as positive.

Negative results obtained in the initial toxicity-mutation test (II) were confirmed by 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, separately, 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:
valid
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:
valid
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:
valid
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:
valid
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:
valid
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 suspension in 95% ethanol at 50 mg/mL (stock solution). Treated a 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 (95% ethanol) in all strains were comparable with the untreated control of respective strains and also with the laboratory historical data of distilled water and other acceptable solvents, indicating that the solvent does not have any impact on bacterial growth. 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: no, as an non-standard vehicle (95% ethanol) was used. All values for the negative control (95% ethanol) in all strains were comparable with the untreated control of respective strains and also with the laboratory historical data of distilled water and other acceptable solvents, indicating that the solvent does not have any impact on bacterial growth.
Conclusions:
It was concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified experimental conditions.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2019 (Study Initiation) to 15 March 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: B.49. In Vitro Mammalian Cell Micronucleus Test (EC) No. 440/2008 of May 30, 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
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: MNST9H796L
- 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 test
- Solubility and stability of the test substance in the solvent/vehicle: The test item formed a dosable suspension in 95% ethanol at 200015 µg/mL (i.e. 200 mg/mL). Therefore, 95% ethanol was selected as vehicle for 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: none, prepared a dosable suspension in 95% ethanol.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:

No significant change in pH (±1 unit) or osmolality (≥ 50 mOsm/kg H2O) was observed at any tested concentration up to 2000 µg/mL in culture medium at 0 h and 4 h after incubation at 37 ± 1 °C in 5% CO2. Precipitate was observed at tested concentrations of 500, 1000 and 2000 µg/mL in culture medium. Therefore, on consideration of the solubility and precipitation results, 500 µg/mL was selected as the highest concentration to be tested in the cytotoxicity test. Based on the results of the cytotoxicity test, the highest test concentration selected for the main study was 125 µg/mL in both the absence and presence of metabolic activation. This allowed the limit concentration to show visible precipitate (by eye and using a light microscope) at the end of treatment, in accordance with OECD 487 guideline requirements.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human peripheral blood lymphocyte
- Suitability of cells: Human peripheral blood lymphocytes in accordance with OECD 487 recommendations
- Normal cell cycle time (negative control): Not specified

For lymphocytes:
- Sex, age and number of blood donors: Blood was drawn from two healthy, female volunteers (of 23 and 28 years old), by venous puncture using a heparinised syringe (Heparin obtained from Biological E. Limited, Hyderabad).

- Whether whole blood or separated lymphocytes were used: Whole blood was cultured
- Whether blood from different donors were pooled or not: Pooled from two female donors
- Mitogen used for lymphocytes: 2% Phytohaemagglutinin (PHA-M) : Gibco (Lot # 1993847)

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

Whole blood was cultured in RPMI-1640 with L-glutamine and 25 mm HEPES (Sigma R4130) containing antibiotics and antimycotic solution (penicillin: 50 IU/mL; streptomycin: 50 µg/mL and amphotericin B: 0.25 μg/mL) supplemented with 20% heat-inactivated (56 °C; 30 min.) fetal bovine serum.

Cultures were prepared separately in a centrifuge tube containing heparin sodium. Each contained 0.5 mL of whole blood in 9.5 mL of complete medium [containing 20% heat-inactivated (56°C; 30 min.) fetal bovine serum and 2% Phytohaemagglutinin (PHA-M)] in centrifuge tubes and incubated at 37 ± 1°C and 5% CO2 in a CO2 incubator for approximately 48 hours. The same procedure was used for preparation of cultures for all the phases of the experiments.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin-B: Sigma (Lot # 087M4005V) at 6 µg/mL.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 ; The S9 fraction used in this assay was procured from Dr. G. P. Meshram, Nagpur (MWR/ARI/S9F/01/18).

- method of preparation of S9 mix In order to test these indirect acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used. The S9 fraction was buffered and supplemented with the essential co-factors β-NADP, KCl and D-Glucose-6-phosphate to form the “S9 mix”. The S9 fraction was used at a concentration of 2% in the final culture medium for the cytotoxicity test and Phase I main test.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 fraction was used at a concentration of 2% in the final culture medium.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No independent S9 controls used. The S9 was externally sourced from a known supplier, S9 CoA provided.
Test concentrations with justification for top dose:
The cytotoxicity test was performed at test concentrations of 15.625, 31.25, 62.5, 125, 250 and 500 µg Fatty acids, C16-18 (even numbered), manganese(II) salts/mL in culture medium. A concurrent negative control (untreated control) and vehicle control (95% ethanol) were also maintained.

Based on the cytotoxicity test results, Fatty acids, C16-18 (even numbered), manganese(II) salts was evaluated for its potential to induce micronuclei at tested concentrations of 3.9, 7.8, 15.6, 31.3, 62.5 and 125 µg Fatty acids, C16-18 (even numbered), manganese(II) salts /mL in culture medium in the absence and presence (2% v/v S9) of metabolic activation. 125 µg/mL, the limit test concentration, showed visible precipitate (by eye and using a light microscope) at the end of treatment in both the absence and presence of metabolic activation in accordance with OECD 487 guideline requirements.
Vehicle / solvent:
- Vehicle used: 95% ethanol

- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water (DW), and in DMSO. The test item formed a dosable suspension in 95% ethanol (200 mg/mL). Therefore, 95% ethanol was selected as vehicle for the study.

- Justification for percentage of solvent in the final culture medium: 1% v/v
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
95% ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
Cyclophosphamide at 30 µg/mL in the presence of metabolic activation
Positive control substance:
cyclophosphamide
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Two replicates per treatment concentration
- Number of independent experiments : The test item was initially evaluated for cytotoxicity in the absence and presence of metabolic activation (2% v/v S9 mix). The main study was conducted in two phases with lymphocytes exposed for 4 h with and without metabolic activation (2% v/v S9) as Phase I, and for 24 h exposure without S9 as Phase II.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The main study was conducted in two phases with lymphocytes exposed for 4 h with and without metabolic activation (2% v/v S9) as Phase I, and for 24 h exposure without S9 as Phase II.

- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. Phase I - Cytochalasin B (6 µg/mL) at 6 µg/ml for 4h. The cells were harvested and processed for slide preparation approximately 24 h from the beginning of treatment.
Phase II - Cytochalasin B (6 µg/mL) at 6 µg/ml for 24h. The cells were harvested and processed for preparation of slides after the 24 h exposure period.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were prepared from each culture tube by pouring approximately 0.5 mL of fixed cell suspension, drop by drop on two, pre-cleaned, ice-chilled slides. The slides were dried over a slide warmer and labelled with the study number, treatment code, absence/presence of metabolic activation and slide number. Two slides were prepared per replicate culture per test concentration (i.e. four slides per test concentration). Out of these two slides, one was used for scoring and the other served as a reserve or used for additional scoring, if required. The dried slides were stained with 5% Giemsa in phosphate buffer for 30 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. To prevent bias in the scoring procedure for micronuclei, the slide numbers were masked with coded numbers provided by Department of Bio-statistics and Systems Information, JRF. All slides were coded before microscopic analysis and decoded after scoring was completed.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): All slides, including those of positive, negative control (untreated control), treatment group and vehicle control (95% ethanol), were independently coded prior to microscopic analysis for replicative index (during the cytotoxicity test and main study), and for concentrations selected for micronuclei analysis (during the main study). The slides were examined under a light microscope and a minimum of 500 cells per slide (culture) were counted, the number of binucleated cells, multi nucleated cells and mono nucleated cells were recorded in different fields of view to determine the replicative index.

Two replicate slides per concentration were used to record the number of micronucleated binucleated cells whereas the other two slides were kept in reserve, for scoring if required (i.e. if insufficient binucleated cells were observed on two slides). The slides were examined for the presence of micronuclei in binucleated cells under a light microscope. A minimum of 1000 binucleated cells were screened per concentration to evaluate the incidence of micronuclei. The masked labels were removed and all slides were decoded after scoring.

Rationale for test conditions:
This assay measures the ability of test item to induce micronucleus formation in binucleated cells of cultured human lymphocytes, known for their reliability and reproducibility in mutagenicity assays and also recommended by the OECD and other regulatory authorities. The results of the study are of value in predicting the potential of the test item to induce micronuclei in humans.
Evaluation criteria:
The test item was considered to have clastogenic activity in this study if the following criteria were met:

• At least one of the test concentrations exhibits a statistically significant increase in micronucleus frequency when compared with the concurrent vehicle control.
• The increase micronucleus frequency is concentration-related (biologically significant) when evaluated with an appropriate trend test e.g. Chi-square trend analysis.
• Increases in micronuclei are not associated with large changes in pH or osmolality of the treated cultures.
• Any of the results are outside the distribution of the historical vehicle control data
When all of these criteria were met, the test item was considered as able to induce the formation of micronuclei in this test system.

The test item was considered clearly negative in this study if the following criteria were met:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test (e.g. Chi-square trend analysis).
• All results were inside the distribution of the historical control data.

When all of these criteria were met, the test item was considered as unable to induce micronuclei formation i.e. chromosome breaks and/or the gain or loss of chromosomes in this test system.
Statistics:
Micronuclei data containing binucleated cells was statistically analysed for normality using Shapiro-Wilk’s test, and for homogeneity of variance the Bartlett test was conducted before appropriate parametric test (ANNOVA, t-test).
Key result
Species / strain:
primary culture, other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Limit concentration (125 µg/mL) showed visible precipitate (by eye and using a light microscope) at the end of treatment.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: None, no significant change in pH (±1 unit)
- Data on osmolality: None, no significant change in osmolality (≥ 50 mOsm/kg H2O) s
- Possibility of evaporation from medium: Not applicable, test item non-volatiile
- Water solubility: < 0.17 mg.L-1 (estimated), insoluble at concentrations required for OECD 487. 95% ethanol therfore selected as the test vehicle.
- Precipitation and time of the determination: Precipiate at 500 µg/mL (cytotoxicity test) after 4 hour incubation (cytotoxicity test). Based on the results of the cytotoxicity test, the highest test concentration selected for the main study was 125 µg/mL in both the absence and presence of metabolic activation. This allowed the limit concentration to show visible precipitate (by eye and using a light microscope) at the end of treatment, in accordance with OECD 487 guideline requirements.
- Definition of acceptable cells for analysis: Not specified

RANGE-FINDING/SCREENING STUDIES (if applicable): yes, before conducting the main study, Fatty acids, C16-18 (even numbered), manganese(II) salts was evaluated for cytotoxicity in the absence and presence of metabolic activation(2% v/v S9 mix).

STUDY RESULTS
- Concurrent vehicle negative and positive control data , yes, concurrent negative control (Untreated control), vehicle control (95% ethanol) and appropriate positive controls used for 4 hour exposure (phase I.) and 24 hr exposure (Phase II)

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible . No statistically significant or biologically relevant response for test item treated cultures, against control cultures.
- Statistical analysis; p-value if any . Micronuclei data containing binucleated cells was statistically analysed for normality using Shapiro-Wilk’s test, and for homogeneity of variance the Bartlett test was conducted before appropriate parametric test (ANNOVA, t-test). No statistically significant or biologically relevant response for test item treated cultures, against control cultures.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells

Concentration
of test item In the Absence of Metabolic Activation In the Presence of Metabolic Activation
(2% v/v S9 mix)
% RI (Mean) Cytostasis % RI (Mean) Cytostasis
NC (Untreated control) 100 0.00 100 0.00
VC (95% ethanol) 100 0.00 100 0.00
T1 (15.625 µg/mL) 95.53 4.47 93.09 6.91
T2 (31.25 µg/mL) 84.54 15.46 76.68 23.32
T3 (62.5 µg/mL) 67.78 32.22 68.81 31.19
T4 ( 125 µg/mL) 42.04 57.96 43.35 56.65
T5 (250 µg/mL) 29.30 70.70 36.01 63.99
T6 (500 µg/mL) 20.97 79.03 27.07 72.93

Key: R = Replicate, T = Treatment group, NC = Negative control, VC = Vehicle Control, RI = Replicative index.

Concentration
of test item
Replicate N° of Mononucleated cells N° of Binucleated Cells Scored N° of Multinucleated cells Total N° of cells
NC (Untreated control) R1 92 273 138 503
R2 122 280 103 505
VC (95% ethanol) R1 105 295 105 505
R2 124 275 102 501
T1 (15.625 µg/mL) R1 95 349 60 504
R2 102 380 68 550
T2 (31.25 µg/mL) R1 132 324 48 504
R2 129 333 39 501
T3 (62.5 µg/mL) R1 234 244 34 512
R2 193 266 49 508
T4 (125 µg/mL) R1 325 176 15 516
R2 301 188 12 501
T5 (250 µg/mL) R1 298 123 6 427
R2 321 104 3 428
T6 (500 µg/mL) R1 342 76 6 424
R2 370 85 4 459

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, the positive controls, vinblastine produced a statistically significant increase in the frequency of micronuclei containing binucleated cells in phase II (continuous exposure) in absence of metabolic activation. Cyclophosphamide produced a statistically significant increase in the frequency of micronuclei containing binucleated cells in Phase I (short term exposure) in the presence of metabolic activation.
- Negative (untreated and vehicle) historical control data: yes, the number of binucleated cells with micronuclei within the negative and vehicle control cultures were within the published range (Fenech, M., 2007).
Conclusions:
From results of this study, it is concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, both in the absence or presence (2% v/v S9 mix) of metabolic activation, under the described experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

All studies returned a negative result. This substance will not be classified for genetic toxicity.