Fatty acids, C16 and C18-unsaturated, methyl esters, chlorinated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 October 2021 to 12 November 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, palm-oil (C16-C18), Me esters, chlorinated (35-45 % w/w)

EC Number:

953-553-3

Cas Number:

95009-45-3

Molecular formula:

It cannot be provided.

IUPAC Name:

Fatty acids, palm-oil (C16-C18), Me esters, chlorinated (35-45 % w/w)

Test material form:

liquid

Details on test material:

Batch number: SV210602R4001

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system.

Test concentrations with justification for top dose:

The test item was tested at the concentrations of 0.03125, 0.0625 and 0.125 µL/mL for short term in presence and absence of metabolic activation and long-term treatment in the absence of metabolic activation system.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The experiment was conducted with and without metabolic activation. The test item was assessed in proliferated lymphocytes in duplicates by exposing for a short term (3 to 6 hours, with and without metabolic activation) and a long term (20 to 24 hours, without metabolic activation)

Evaluation criteria:

A test item is considered to be clearly positive if:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
 A test item is considered to be clearly negative if:
None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
There is no concentration-related increase when evaluated with an appropriate trend test.

Statistics:

ANOVA

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no statistically significant increase in the percentage of micronuclei in binucleated cells were observed in any of the tested concentrations when compared to the vehicle control.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, it is concluded that the test item is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 0.125 µL/mL both in short term and long-term treatments (presence and absence of metabolic activation) as it showed no evidence of increase in the induction of micronuclei under the test conditions.

Executive summary:

 The test item, ESTERI METILICI ACIDI GRASSI DA OLIO - ESSEBIOCHLOR45 was evaluated for formation of micronuclei in the cytoplasm of interphase cells using human lymphocytes. Test item found miscible in DMSO at 200 µL/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL, post incubation mild precipitation was observed at 2 µL/mL, and no precipitation was observed in any of the concentration tested from 0.03125 to 1 µL/mL. No pH change was observed in any of the concentration tested, hence 2 µL/mL was selected as highest concentration for initial cytotoxicity test and other concentration tested were 0.125, 0.25, 0.5 and 1 µL/mL. The experiment was conducted with and without metabolic activation. The test item was assessed in proliferated lymphocytes in duplicates by exposing for a short term (3 to 6 hours, with and without metabolic activation) and a long term (20 to 24 hours, without metabolic activation). Cytokinesis was blocked using Cytochalasin B, the cells were harvested and slides were prepared. In order to assess the cytotoxicity of the test item, the Cytokinesis-Block Proliferation Index (CBPI) was calculated for cultures treated with the test item and vehicle control. The % cytotoxicity was in the range of 80.33 to 100% at 0.25 to 2 µL/mL. The % cytotoxicity ranged from of 54.10 to 55.00% at 0.125 µL/mL. As the % cytotoxicity was not more than 55±5% at 0.125 µL/mL, same was selected as the highest concentration for the micronucleus test. Other concentrations tested were 0.03125 and 0.0625 µL/mL. In micronucleus test, the test item was tested at the concentrations of 0.03125, 0.0625 and 0.125 µL/mL for short term in presence and absence of metabolic activation and long-term treatment in the absence of metabolic activation system. The test item induced cytotoxicity at 0.125 µL/mL (51.67 to 54.10%) when compared to vehicle control. There was no statistically significant increase in the percentage of micronuclei in binucleated cells were observed in any of the tested concentrations when compared to the vehicle control. Further, the micronucleus frequencies observed for test item treatments fell within acceptable ranges with regard to in-house historical control data. The positive controls resulted increase of the micronuclei frequencies with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of test system towards positive control and confirmed that the test conditions were adequate and within the range of historical control.