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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance does not exert mutagenicity nor clastogenicity in in vitro bacterial reverse mutation test, mammalian cell gene mutation test and micronucleus test.

Link to relevant study records

Reference

Reference 1

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)

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 October 2021 to 18 December 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.

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:

0.0625, 0.125, 0.25 and 0.5 µL/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Cells were exposed to the test item for 3 hours and 20 minutes at 37±1°C with 5±1% CO2.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
 The increase is concentration-related when evaluated with an appropriate trend test.
 Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
 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.
 All results are inside the distribution of the historical negative/vehicle control data.

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

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.5 µL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item Esteri Metilici Acidi Grassi Da Olio Essebiochlor45 was evaluated for gene mutation test in CHO AA8 cells. The test item was found miscible in DMSO at 200 µL/mL. Precipitation test was conducted at 0.125, 0.25, 0.50, 1 and 2 µL/mL concentrations. Post 3 hours and 20 minutes of incubation, no precipitation observed at 0.125 and 0.25 µL/mL, moderate precipitation observed at 0.5 µL/mL and heavy precipitation observed at 1 and 2 µL/mL. No change in pH was observed at the tested concentrations at and up to 2 µL/mL. On the basis of precipitation results, 0.5 µL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5 µL/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 hours and 20 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10-20 % at 0.5 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.5 µL/mL was selected as highest concentration for gene mutation test. The gene mutation test was conducted at the concentrations of 0.0625, 0.125, 0.25 and 0.5 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 15 minutes). Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test. Cytotoxicity as Relative Survival was 56.84 to 61.96 % in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 0.5 µL/mL. There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with Esteri Metilici Acidi Grassi Da Olio Essebiochlor45 resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls. There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 October 2021 to 01 December 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 µL/plate of test item concentrations.
Top dose 5 µL/plate on the basis of precipitation test.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Details on test system and experimental conditions:

Salmonella typhimurium TA100 tester strain was exposed to vehicle control, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 µL/plate of test item concentrations.
The test item resulted in no cytotoxicity from 0.00625 to 5 µL/plate with lawn intensity 4+ (Thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control.

Evaluation criteria:

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item ESTERI METILICI ACIDI GRASSI DA OLIO - ESSEBIOCHLOR45 was assayed for Bacterial Reverse Mutation Test at the concentrations of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate for plate incorporation method and for preincubation method using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.
In the two trials conducted, the test item concentrations tested resulted in no appreciable increase in the number of revertant colonies over the vehicle control, while the positivecontrols tested simultaneously, resulted in 3.6 to 14.5 fold increase in the number of revertant colonies/plate under identical conditions.
This was observed for all the five tester strains.

Conclusions:

Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 µL/plate under the test conditions, when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains.

Executive summary:

The test item, ESTERI METILICI ACIDI GRASSI DA OLIO - ESSEBIOCHLOR45 was evaluated for mutagenicity in Bacterial Reverse Mutation Test. The test item found miscible in DMSO at a concentration of 50 µL/mL and resulted in mild precipitation at 5 µL/plate, minimal precipitation at 3.2 µL/plate and no precipitation from 0.00625 to 1.6 µL/plate at the tested concentrations. On the basis of precipitation test, 5 µL/plate was selected as the highest test concentration for initial cytotoxicity test. Salmonella typhimurium TA100 tester strain was exposed to vehicle control, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 µL/plate of test item concentrations. The test item resulted in no cytotoxicity from 0.00625 to 5 µL/plate with lawn intensity 4+ (Thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control. Based on the results of initial cytotoxicity test, concentrations of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate were selected for testing in the mutation test.
The test item was assessed for its mutagenic effects using Salmonella typhimurium tester strains: TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101). The test item was tested for plate incorporation method (Trial I) and for preincubation method (Trial II) in presence and absence of metabolic activation system using DMSO as the vehicle and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and 4-nitroquinoline N-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials in the presence and absence of metabolic activation. There was no appreciable increase innumber of revertant colonies at any of the tested concentrations in both the trials. Thenumber of revertant colonies in the positive controls resulted in 3.6 to 14.5 fold increase under identical conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available, the substance should not be classified for mutagenicity under CLP regulation. Since data allow to exclude genotoxicity, even carcinogenicity is not expected.