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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 18, 2017 to May 18, 2017
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
CAS No.: 23054-60-6 + 23054-61-7; Batch/Lot No.: 7723329-R802; Purity: 100 %; Appearance: colourless/pale straw clear liquid (white solid was confirmed by visual inspection upon arrival at test facility at room temperature. Test substance was clear liquid after incubation at 37 °C).
Target gene:
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.
Genotypes of the used strains: S.typhimurium TA98 (hisD3052), S.typhimurium TA100 (hisG46), S.typhimurium TA1535 (hisG46), S.typhimurium TA1537 (hisC3076), WP2 (uvrA trpE).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Range finding test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate;

Salmonella typhimurium strains with and without metabolic activation:
Initial mutation test: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate
Confirmatory mutation test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate

Escherichia coli WP2 uvrA strain with and without metabolic activation:
Initial mutation test and in the confirmatory mutation test: 5000, 1581, 500, 158.1, 50 15.81, 5 and 1.581 μg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, distilled water
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; 4-nitro-1,2-phenylenediamine
Details on test system and experimental conditions:
The study included a preliminary compatibility test, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test and a confirmatory mutation test. In the preliminary concentration range finding test as well as in the initial mutation test, the plate incorporation method was used. In the confirmatory mutation test, the pre-incubation method was used.

Preliminary compatibility test:

The appropriate vehicle (solvent) and the behaviour of the test substance formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. Distilled water was used as solvent to prepare the stock solution of the test substance. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.

Preliminary concentration range finding test:

Based on the solubility test, a 100 mg/mL stock solution was prepared in distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test substance, in the absence and presence of metabolic activation.
Test substance concentrations in the mutagenicity tests (initial mutation test and confirmatory mutation test).
Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in distilled water. Maximum eight test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test substance/plate.

Procedure for exposure in the initial mutation test:

The initial mutation test followed the standard plate incorporation procedure. Bacteria (cultured in nutrient broth No.2 were exposed to the test substance both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test substance and other components were prepared freshly and added to the overlay (45 °C). The content of the tubes: top agar 2000 μL, vehicle or test substance formulation (or reference controls) 50 μL, overnight culture of test strain 100 μL, phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hours.

Procedure for exposure in the confirmatory mutation test:
The confirmatory mutation test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the initial mutation test. Bacteria (cultured in nutrient broth No.2.) were exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C. Before the overlaying, the test substance formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test substance (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hours.
Evaluation criteria:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a positive response:
A test substance was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a negative response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
The colony numbers on the untreated / negative (solvent) / positive control and test substance treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test substance and for the controls using Microsoft ExcelTM software.
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
Untreated negative 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
Untreated negative 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
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
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:
Preliminary experiment:
- In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate was observed in the preliminary concentration range finding test in both bacterial strains with and without metabolic activation.
- Slightly reduced background lawn was detected in the preliminary range finding test in Salmonella typhimurium TA98 strain on the plates at 5000 μg/plate without metabolic activation, in Salmonella typhimurium TA100 strain absent/reduced background lawn was observed on the plates at 5000, 2500 μg/plate without metabolic activation and at 5000 μg/plate concentration with metabolic activation.

The initial mutation test and confirmatory mutation test:

- No precipitate was observed in the main tests in all examined strains with and without metabolic activation.

- In the initial mutation test absent/reduced background lawn was detected in all Salmonella typhimurium strains on the plates at 5000 μg/plate concentration with and without metabolic activation.
- In the initial mutation test and confirmatory mutation test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
- In the initial mutation test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 strain at 500 and 5 μg/plate concentrations without metabolic activation (the observed mutation factor value was: MF: 1.47). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

- In the confirmatory mutation test absent/reduced/slightly reduced background lawn was observed in all Salmonella typhimurium TA98 strain and in Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations with and without metabolic activation. The same effect was detected in Salmonella typhimurium TA100, TA1535, TA1537 strains at 5000, 1581 μg/plate concentrations with metabolic activation and 5000, 1581, 500 μg/plate concentrations without metabolic activation.
- In the confirmatory mutation test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.39). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Conclusions:
Under the study conditions, the reported data of this mutagenicity assay show that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test substance has no mutagenic activity on the growth of the bacterial strains.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and OPPTS 870.5100, in compliance with GLP. The experiments were carried out using strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and WP2 uvrA strain of Escherichia coli in the presence and absence of a post mitochondrial supernatant (S9 fraction). The test substance was dissolved in distilled water at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of this test, the test substance concentrations in the initial mutation test were within range of 5000 - 5 μg/plate and in the confirmatory mutation test were in the range of 5000 - 1.581 μg/plate at the Salmonella typhimurium strains with and without metabolic activation. In the initial mutation test and in the confirmatory mutation test tested concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation were in between 5000 and 1.581 μg/plate. No precipitate was observed in the main tests in all examined strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test substance (absent / reduced / slightly reduced background lawn development) was observed in the main tests in all examined strains with and without metabolic activation at several concentrations (expect in the initial mutation test at Escherichia coli WP2 uvrA strain). In the initial mutation test and confirmatory mutation test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid. Under the study conditions, the reported data of this mutagenicity assay show that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test substance has no mutagenic activity on the growth of the bacterial strains (Orovecz, 2017).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/mL respectively) and 30 U/mL heparin.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range finding study:
At 3 h exposure time: 3, 10, 33, 100 and 333 µg/mL culture medium with and without S9-mix.
At 24 and 48 h continuous exposure time: 3, 10, 33, 100, 333 and 1000 µg/mL culture medium without S9-mix


Experiment 1:
Without and with S9-mix: 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (3 h exposure time, 24 h fixation time)


Experiment 2:
- Without S9-mix: 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time)
10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix 50, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10 µg/mL 3 h exposure period ( 24 h fixation time)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.1 µg/mL (48 h exposure), 0.2 (24 h exposure) and 0.5 µg/mL (3 h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 48 h (Experiment 2 with S9 mix)


SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µ g/mL medium)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED: 1000 cells


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:
A test substance was considered positive (clastogenic) if:
a) A dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations
b) A significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship
A test substance was considered non-clastogenic if:
a) None of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
One sided, Chi-square test to calculate dose-related statistically significant increase in the number of cells with chromosome aberrations
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: At the 24 and 48 h exposure time, test material was tested beyond the limit of solubility to obtain adequate toxicity data.
- Precipitate of the test material was seen at 333 µg /mL


COMPARISON WITH HISTORICAL CONTROL DATA: Yes, within the laboratory historical control data range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Increased number of polyploid cells in the absence of S9-mix in a dose dependent manner in the first cytogenetic assay indicating potential to inhibit mitotic processes and to induce numerical chromosome aberrations.

The doses selected for scoring of chromosome aberrations:

Without S9-mix: 10, 50 and 100 µg/mL culture medium (24 h exposure time, 24 h fixation time).

50, 100 and 150 µg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix:50, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Conclusions:
Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro.
Executive summary:

A study was conducted to assess the ability of the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473. In experiment 1, 3 h exposure with a 24 h fixation in the absence and presence of S9-mix (1.8%) were used. In experiment 2, 24 h exposure with a 24 h fixation time and 48 h exposure with a 48 h fixation time in the absence of S9 mix and 3 h exposure with a 48 h fixation in presence of S9 mix were tested. Vehicle control cultures had frequencies of cells with aberrations within the historical control data range. Both of the positive control materials induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence, in either of the two independently repeated experiments. Dose-dependent increase in polyploid cells (absence of S9-mix) in the first cytogenetic assay was noted, indicating the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip CM, 2009).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) , 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 h exposure cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium) and for 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction (Rat liver microsomal enzymes prepared from adult male Wistar rats)
Test concentrations with justification for top dose:
First mutagenicity test:
Without S9-mix: 0.1, 0.3, 1, 3, 10, 15, 20, 25, 30, 35, 40 and 45 µg/mL exposure medium
With 8% (v/v) S9-mix: 1, 3, 10, 30, 70, 100, 125, 150, 175, 200, 225 and 250 µg/mL exposure medium

Second mutagenicity test:
Without S9-mix: 0.1, 0.3, 1, 3, 10, 15, 20, 25, 27.5, 30, 32.5 and 35 µg/mL exposure medium
With 12% (v/v) S9-mix: 1, 3, 10, 50, 100, 125, 150, 160, 170, 180, 190 and 200 µg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 15 and 5 µg/mL for a 3 and 24 h treatment period
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3 h (Experiment 2 with S9 mix)
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 d after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CE Day 2) and the mutation frequency (MF).


SELECTION AGENT (mutation assays): Trifluorothymidine


NUMBER OF REPLICATIONS: Two


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more than MF (controls) + 126 in a dose-dependent manner; or
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF (controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 33 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 µg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 333 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

EVALUATION OF THE MUTAGENICITY:
No significant increase in the mutation frequency at the TK locus was observed after treatment with test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material-treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 74% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 77% compared to the total growth of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 95% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 72% compared to the total growth of the solvent controls.

The growth rate over the two-day expression period for cultures treated with DMSO was between 16 and 24 (3 h treatment) and 41 (24 h treatment). Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS in the absence of S9-mix, and by 16 and 9.7-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Conclusions:
Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476. The study was performed in two independent experiments with L5178Y mouse lymphoma cells in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 35 and 175µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. The substance was tested up to cytotoxic levels of 85 and 80 % in the absence and presence of S9-mix, respectively. In the second experiment, the substance was tested up to concentrations of 25 and 200 µg/mL in the absence and presence S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 77% (presence of S9-mix). The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS (absence of S9-mix), and by 16 and 9.7 fold for CP (presence of S9-mix). The test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned appropriately. In both the presence and absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment and this result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip CM, 2009).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and OPPTS 870.5100, in compliance with GLP. The experiments were carried out using strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and WP2 uvrA strain of Escherichia coli in the presence and absence of a post mitochondrial supernatant (S9 fraction). The test substance was dissolved in distilled water at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of this test, the test substance concentrations in the initial mutation test were within range of 5000 - 5 μg/plate and in the confirmatory mutation test were in the range of 5000 - 1.581 μg/plate at the Salmonella typhimurium strains with and without metabolic activation. In the initial mutation test and in the confirmatory mutation test tested concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation were in between 5000 and 1.581 μg/plate. No precipitate was observed in the main tests in all examined strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test substance (absent / reduced / slightly reduced background lawn development) was observed in the main tests in all examined strains with and without metabolic activation at several concentrations (expect in the initial mutation test at Escherichia coli WP2 uvrA strain). In the initial mutation test and confirmatory mutation test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid. Under the study conditions, the reported data of this mutagenicity assay show that the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test substance has no mutagenic activity on the growth of the bacterial strains (Orovecz, 2017).

Mouse lymphoma study

A study was conducted to determine the mutagenic potential of the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476. The study was performed in two independent experiments with L5178Y mouse lymphoma cells in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 35 and 175 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. The substance was tested up to cytotoxic levels of 85 and 80 % in the absence and presence of S9-mix, respectively. In the second experiment, the substance was tested up to concentrations of 25 and 200 µg/mL in the absence and presence S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 77% (presence of S9-mix). The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS (absence of S9-mix), and by 16 and 9.7 fold for CP (presence of S9-mix). The test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned appropriately. In both the presence and absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment and this result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip CM, 2009).

Chromosome aberration study

A study was conducted to assess the ability of the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473. In experiment 1, 3 h exposure with a 24 h fixation in the absence and presence of S9-mix (1.8%) were used. In experiment 2, 24 h exposure with a 24 h fixation time and 48 h exposure with a 48 h fixation time in the absence of S9 mix and 3 h exposure with a 48 h fixation in presence of S9 mix were tested. Vehicle control cultures had frequencies of cells with aberrations within the historical control data range. Both of the positive control materials induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence, in either of the two independently repeated experiments. Dose-dependent increase in polyploid cells (absence of S9-mix) in the first cytogenetic assay was noted, indicating the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip CM, 2009).

Justification for classification or non-classification

Based on the results of in vitro testing on the substance itself or on the read across substance, amides, C8-18 (even-numbered) and C18-unsatd., N-(2-hydroxypropyl), the test substance does not require classification for mutagenicity according to CLP (EC 1272/2008) criteria.