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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the bacterial reverse mutation study with and without metabolic activation (reference 7.6.1 -1).

The test item was non-mutagenic in mouse lymphoma cells with this screening test system under conditions that exerted potent mutagenic effects for the positive controls (reference 7.6.1 -2).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 02 -April 04, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine for S. thyphimurium
tryptophan for E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Bioscience Research Laboratory, Kikkoman Corporation (S9; produced on Jan. 11, 1991,Lot No:RAA-248)
- method of preparation of S9 mix: A 9,000 g supernatant (S9), prepared from liver tissue of male Sprague-Dawley rats (7 weeks old) induced with phenobarbital and 5,6-benzoflavone was used. For 1 mL: 0.1 mL S9 + 8 µmol MgCl2 + 33 µmol KCl + 5 µmol G-6-P + 4 µmol NADH + 4 µmol NADPH + 0.5 mL 0.2 M Sodium phosphate buffer (pH 7.4)
- concentration of S9 mix: 0.1 mL S9 in 1 mL S9 mix
- volume of S9 mix added: 0.5 mL per plate with metabolic activation
Test concentrations with justification for top dose:
0, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
The test substance was dissolved up to 50 mg/mL in acetone, and was used for the assay immediately after diluting with the same solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

FOR GENE MUTATION:
- Plates were incubated for 48 h at 37 °C.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- not examined
Evaluation criteria:
The results were judged to be positive when the revertants were twice or more that of the negative control, and when dose dependency or reproducibility were observed in the increase of revertants at least in one strain.
Statistics:
The mean of two plates counted was calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
but tested up to the recommended limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
but tested up to the recommended limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
but tested up to the recommended limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
but tested up to the recommended limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Ames test:
Please refer to "Attached background material" for:
- Individual plate counts
- Mean number of revertant colonies per plate
Conclusions:
The test item was not mutagenic in the bacterial reverse mutation study with and without metabolic activation.
Executive summary:

An in vitro bacterial reverse mutation study was conducted similar to OECD 471. In one plate incorporation test the test item was applied at concentrations of 0, 150, 500, 1500 and 5000 µg/plate to Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA) strains with and without metabolic activation using S9 mix. There were no increasing number of revertants observed for any strains and any concentration of the test item. The positive control substances increased the number of mutants twice or more the negative control in each strains. Under the test conditions the test item did not show mutagenic potential in the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jun 16 - Dec 08, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Principles of method if other than guideline:
The methodology, originally complying with the ICH guidelines, OECD Test Guideline 476 and Annex V, EEC Directive 79-831, was modified in this study in order to screen a large number of test materials. In brief, the main modification is the use of fewer concentrations of the test material, use of only single cultures per concentration and a simplified data reporting. No quality assurance inspection according to GLP guidelines has been performed.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK)
mutation in TK locus remove the 5-trifluorothymidine (TFT) resistance
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The original cultures were obtained from Dr. W. Muster, Hoffmann-La Roche, Basel, Switzerland on March 24, 1995. Cells were stored as frozen stocks in liquid nitrogen.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: flask cultivation
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media: growth medium: RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum)
- Conditions: CO2 concentration 5 % v/v, humiditfied atmosphere, temperature 37 °C
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver homogenate (S9 mix) with standard co-factors
Type and composition of metabolic activation system:
- source of S9 : Wistar Chbb:THOM rats
- method of preparation of S9 mix: Rats were treated with Aroclor 1254 (500 mg/kg body weight) 5-7 days before sacrifice. Livers were collected, homogenised, centrifuged and the supernatant was collected and stored in liquid nitrogen. On the day of the experiment, glucose-6-phosphate (590 mM), NADP (30 mM), KCI (150 mM) and rat liver S9 were mixed at the ratio of 1:1:1:2.
- cconcentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 in the cell culture medium was 2 %. For all cultures treated in the presence of S9 mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL.
- quality controls of S9: The bacterial mutagenicity (according to Ames et al., 1975) of 2-amino anthracene, benzo[a]pyrene, and 3-methyl-cholanthrene is determined once for every S9 batch.
Test concentrations with justification for top dose:
2.81, 8.89 and 28.1 µg/mL
The highest concentration was selected based on precipitation in the culture medium observed in the range-finding test at >= 8.89 µg/mL and cytotoxicity characteristics of the test material.
Vehicle / solvent:
- Solvent used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single for positive control and test item, duplicate for solvent control
- Number of independent experiments : 1 without S9, 2 with S9

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^7
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h with S9, 24 h without S9 (in medium, afterwards washing and seeding of cells)
- Harvest time after the end of treatment: survival: 5-8 days, viability: 6-9 days (with subculturing if required), TFT resistance: 9-13 days

FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: 7-11 days
- Selective agent: 5-trifluorothymidine, concentration: 3 µg/mL, duration: 7-11 days (days 3 to 10 or up to day 14 of experiment)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: viability: 1.6 cells per wells (afterwards growing for 4-7 days), resistance: 2*10^3 cells per well (afterwards growing for 7-11 days); stained with MTT for viable cells

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
- The mean mutant frequencies in the negative (solvent) control cultures fell within the normal range for this test system and
- at least one concentration of each of the positive control chemicals relevantly increased the mutation frequency as compared to the actual negative control (at least a 2-fold increase).

Test materials are assessed as negative or non-mutagenic in this test system if
- the assay is considered valid and
- no relevant increase in the mutation frequency (at least a 2-fold) occurs.
Test materials are assessed as positive or mutagenic in this test system if
- the assay is considered valid and
- a clear increase in the mutation frequency (at least a 2-fold) occurs dose-dependently (over at least two test material concentrations) or reproducibly at identical concentrations in two independent experimental series performed.
Statistics:
Not performed
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: observed at 28.1 µg/mL:with S9 at beginning and disolved during exposure time, during the whole time without S9; observed at 15.8 µg/mL: during the whole time with S9

RANGE-FINDING/SCREENING STUDIES: Was condcuted but no details provided.

STUDY RESULTS
please refer to "Attached background material"

HISTORICAL CONTROL DATA: not specified
Conclusions:
The test item was non-mutagenic in mouse lymphoma cells with this screening test system under conditions that exerted potent mutagenic effects for the positive controls.
Executive summary:

The test item was screened for its ability to induce mutations at the TK locus (5- trifluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test item was non-toxic to the mouse lymphoma cells in the absence and presence of S9 mix. Under the different experimental conditions in a range finding test the test item precipitated in the cell culture medium at concentrations between 8.89 and 28.9 µg/mL. Concentrations ranging from 2.81 to 28.1 µg/mL were therefore tested. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test item, neither in the absence nor presence of S9 mix. It is therefore concluded that the test item is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the key study an in vitro bacterial reverse mutation study was conducted similar to OECD 471. In one plate incorporation test the test item was applied at concentrations of 0, 150, 500, 1500 and 5000 µg/plate to Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA) strains with and without metabolic activation using S9 mix. There were no increasing number of revertants observed for any strains and any concentration of the test item. The positive control substances increased the number of mutants twice or more the negative control in each strains. Under the test conditions the test item did not show mutagenic potential in the presence and absence of metabolic activation.

In the supporting study the test item was screened for its ability to induce mutations at the TK locus (5- trifluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test item was non-toxic to the mouse lymphoma cells in the absence and presence of S9 mix. Under the different experimental conditions in a range finding test the test item precipitated in the cell culture medium at concentrations between 8.89 and 28.9 µg/mL. Concentrations ranging from 2.81 to 28.1 µg/mL were therefore tested. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and benzo[a]pyrene (with S9 mix). Therefore the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test item, neither in the absence nor presence of S9 mix. It is therefore concluded that the test item is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.