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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD TG 471: negative

OECD TG 473: negative

OECD TG 476: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 12 - May 28, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17).The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan: 1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate2. Series: 15.8, 50, 158, 500, and 1580 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Positive control substance:
other: Aminoacridine
Details on test system and experimental conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the in-crease in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:-----------------------------------------------------------------------------------------Mean Number of Colonies Maximal Mean Number of Colonies over the Actual(Solvent Control) Solvent Control (Test Material)-----------------------------------------------------------------------------------------<=10 <=9 >=30<=30 <=19 >=40<=80 <=29 >=80<=200 <=49 >=120<=500 <=79 >=200Assessment No increase Clear increase-----------------------------------------------------------------------------------------All further results, ranging between "no" and "clear", are assessed as "weak in-creases".Interpretations:A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)A test material is defined as mutagenic in this assay if: - a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Evaluation criteria:
cf. details in results
Statistics:
n.a.
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
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
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
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
Species / strain:
S. typhimurium TA 102
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
Species / strain:
E. coli WP2 uvr A
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:
Precipitation on agar plates: >=1580 µg/plateCytotoxicity: not observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationWith and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations larger or equal to 1580 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol (cf also "Criteria for negative and positive results"), the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 17 - November 19, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guideline 473 for the testing of Chemicals: In vitro mammalian chromosome aberration test.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium including 10% (v/v) FCS- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (induction using Aroclor 1254)
Test concentrations with justification for top dose:
S9Treatment+RecoveryVehicleConcentration -20+0 hours Ethanol17.75, 20.88, and 24.57 µg / mL-44+0 hours Ethanol15.09 and 17.75 µg / mL+3+17 hours Ethanol59.09, 65.61, and 81.00 µg / mL+3+41 hours Ethanol65.61, and 72.90 µg / mL
Vehicle / solvent:
Name:sterile ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: Continous for 20 and 44 hoursPulse for 3 + 17 and 3+41 hoursSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: Controls: 4; others: 2NUMBER OF CELLS EVALUATED: 100 metaphases (structural abberations)DETERMINATION OF CYTOTOXICITY- Method: mitotic index; relative total growthOTHER EXAMINATIONS:- Determination of polyploidy: yes- Determination of endoreplication: yes- Other::
Evaluation criteria:
A test article is considered as positive in this assay if:1. the proportions of cells with structural aberrations at one or moreconcentration exceeds the normal range in both replicate cultures, and2. a statistically significant increase in the proportion of cells with structuralaberrations (excluding gaps) occurs at these doses.Increased incidence of cells with gaps or increased proportions of cells withstructural aberrations not exceeding the normal range, or occurring only at veryhigh or very toxic concentrations are likely to be concluded as "equivocal". Fullassessment of the biological importance of such increases is likely only to bepossible with reference to data from other test systems. Evidence of a dose-relatedeffect is considered useful but not essential in the evaluation of a positive result.Cells with exchange aberrations or cells with greater than one structural aberrationoccur very infrequently in negative control cultures. Their appearance is thereforeconsidered to be of particular biological significance.
Statistics:
Standard statistical methods have been applied for data processing.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: yes (441 µg/mL)- Cytotoxicity: yes - Other confounding effects: RANGE-FINDING/SCREENING STUDIES:COMPARISON WITH HISTORICAL CONTROL DATA:ADDITIONAL INFORMATION ON CYTOTOXICITY:Due to the toxicity profiles observed (reductions in cell number), it was not possible to select the same top concentrations for analysis from treatments sampled at 44 hours as those for treatments sampled at 20 hours. The pattern of toxicity for both treatments sampled at 44 hours was such that two concentration choices were possible; one inducing below 50% cytotoxicity and one inducing 60% (or slightly above) cytotoxicity. It was considered prudent to analyse both concentrations from both treatments. Although contrary to the protocol, this was considered to add to thedata set and as such has no adverse effect on the validity of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

see attachment

Conclusions:
Interpretation of results (migrated information):negativeIt is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).
Executive summary:

Study Design

The test material was tested in an in vitro cytogenetics assay using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile ethanol (ethanol) and the highest dose level used, 400 μg/mL, was in excess of the solubility limit in culture medium. In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test material on relative cell number. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 27.49 and 83.89 μg/mL induced approximately 58% and 66% reduction in cell number and 0% and 49% mitotic inhibition (MIH) in the absence and presence of S-9 respectively. In Experiment 2, treatment in the absence of S-9 was continuous for either 20 hours or 44 hours. Treatment in the presence of S-9 was either for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17) or for 3 hours followed by a 41-hour recovery period prior to harvest (3+41). Chromosome aberrations were analysed at two or three dose levels (see overleaf) and the highest concentrations chosen for analysis, 24.57 μg/mL (20+0) or 17.75 μg/mL (44+0) in the absence of S-9 and 81.00 μg/mL (3 +17) or 72.90 μg/mL (3+41) in the presence of S-9, induced approximately 50%, 60%, 54% and 62% reduction in cell number and 81%, 29%, 55% and 7% MIH respectively. Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1 -oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Positive controls were included with both treatments in Experiment 1, but only with the 20+0 hour -S-9 and 3+17 +S-9 treatments in Experiment 2.
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 473.

Results

Treatment of cultures with the test material in the absence and the presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those observed in concurrent vehicle controls for the majority of concentrations analysed. Two exceptions to this were observed: a single culture at the intermediate concentration analysed (67.11 mg/mL) from the 3+17 hour +S-9 treatment in Experiment 1 and a single culture from the lowest concentration analysed (15.09 mg/mL) from the 44+0 hour –S-9 treatment in Experiment 2 both exhibited numbers of aberrant cells that exceeded historical negative control (normal) values. However, in both instances these increases were not observed in the replicate cultures and were not dose-related. Furthermore, the increase observed in the presence of S-9 in Experiment 1 was not observed at similar concentrations analysed in Experiment 2 (performed under identical treatment conditions). The aberrant cell frequency of all other test material treated cultures fell within normal values. It was therefore considered that the increases observed were spurious and of no biological significance.Normal frequencies of cells with numerical aberrations (within historical negative control (normal) ranges) were observed for the large majority of test material treated cultures. The only exception to this was observed in the 3+17 hour –S-9 treatment at the highest concentration tested (27.49 mg/mL) where a single replicate showed an aberrant cell frequency that exceeded the normal range. However, this increase was small and was not observed in the replicate culture. As all other cultures (for all treatments) exhibited normal frequencies of numerical aberrations, this increase was not considered of biological importance.

Conclusion

It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.03.-30.08.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD (2016). Guideline for the testing of chemicals, No 476: In vitro Mammalian
Cell Gene Mutation Tests using the Hprt and xprt genes
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: HPRT
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: N017012049
- Expiration date of the lot/batch: 28 February 2019
- Purity test date: 17 February 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The solubility limit in culture medium was in the range of 33.27 to 66.53 μg/mL. The test material was soluble in
acetone at concentrations up to at least 212.90 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: Stock solutions were prepared by formulating the test material under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required concentration.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from
Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as
frozen stocks in liquid nitrogen. Full details of the supplier are documented in central
records. Each batch of frozen cells was purged of mutants and confirmed to be
mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells
diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well,
subcultures were established in an appropriate number of flasks.
Metabolic activation:
with and without
Metabolic activation system:
S-9 from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
3.906 to 250 μg/mL,
Vehicle / solvent:
Acetone
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:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): 2 x 10 exp 5 cells/mL

DURATION
- Plating for Survival: 7 days
- Expression Period: 7 days
- Plating for Viability: 8 days
- Plating for 6TG Resistance: 13 days

SELECTION AGENT (mutation assays): 6TG
Rationale for test conditions:
According to guideline
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database
(mean MF +/- 2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of
variance.
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: limited
- Precipitation: observed at highest tested concentration
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]
Conclusions:
It is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 476. From the results of this assay it is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

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

Additional information

For this endpoint studies were performed according to GLP and the methods applied were fully compliant with relevant OECD guidelines. No effects for genetic toxicity have been observed in this testing battery.

Justification for classification or non-classification