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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated inSalmonella typhimuriumstrains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that The test chemical in the concentration of < 2.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Ames assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his+ gene
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA104
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 fractions prepared from Aroclor-treated rats
Test concentrations with justification for top dose:
1, 10, 50, 100, 200, 500 or 1000 μg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: Without S9 mix Strain TA97: 4-nitro-o-phenylene-diamine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Witout S9: Strain TA100: sodium azide Strain
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: TA102 tert.-butylhydroperoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: Strain TA104: methylglyoxal
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Mutagenicity test consisting of the combination of the test compound, the bacterial tester strain, and S9 mix in soft agar. Positive and negative controls are usually also included in each assay. However, negative control are not included in the current study. After incubation at 37°C for 48 h, revertant colonies are counted. A liquid preincubation procedure was also applied for some of the experiments to provide with a more sensitive assessment of mutagenic activity.

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Triplicate replications

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: N/A

OTHER EXAMINATIONS: N/A
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for no. of revertants/plate
Statistics:
No data
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Because BHA have antimicrobial properties a non-toxic dose range was established.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: No mutagenic potential

Mutagencity of the test chemical Salmonella Microsome Assay

                                            No. of his+ revertants per plate in strains

 

 

         TA97

       TA100

         TA102

         TA104

Compound

Dose

μg/plate

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

BHA

1

121 ±3

147± 9

83± 4

85± 8

243± 13

363 ±57

333 ±5

361 ±91

 

10

117 2

146 6

76 12

81 14

224 24

387 2

371 21

409 50

 

100

109 12

138 18

92 11

97 12

197 38

277 16

329 16

391 47

 

200

136 9

174 19

102 8

76 12

171 11

381 9

32811

47254

 

500

106 13

144 12

54 9

90 10

18 9

112 8

367 29

39268

 

1000

0

0

0

0

15 1

182 67

0

17 10

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
The test chemical dissolved in dimethylsulfoxide and given in the concentration of 1, 10, 100, 200, 500 or 1000 mg/plate, was not mutagenic in the Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions and hence it is not likely to be mutagenic as per the criteria mentioned in CLP regulation.
Executive summary:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated in Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosomal aberration test
Target gene:
No data
Species / strain / cell type:
mammalian cell line, other: A pseudo-diploid Chinese hamster cell line (Don)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimum essential medium supplemented with 10% fetal calf serum (pH 7.2) adjusted by HEPES6 buffer
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes, CHL cells had modal chromosome numbers of 21 and 25
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 induced with Aroclor 1254
Test concentrations with justification for top dose:
0, 0.000001, 0.00001, 0.0001 or 0.001 M
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells at the start of seeding: 1.0-1.2 X 106 cells per TD-40 culture bottle

DURATION
- Preincubation period: No data
- Exposure duration: 26 hrs
- Expression time (cells in growth medium): 26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): aqueous solution of 33258 Hoechst and Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase plates for each dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations.
Rationale for test conditions:
No data
Evaluation criteria:
The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break.
Statistics:
No data
Species / strain:
mammalian cell line, other: CHL
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.00001 and 0.0001 M
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Table: Frequencies of chromosome aberrations in Don cells after treatment with BUdR and solvent

Treatment

No. of cultures

No. of cultures observed for

Breaks/cell

Mean±SE

Range

BUdR + DMSO

6

600

0.0666±0.0010

0.02- 0.11

 

TABLE 2. Chromosome aberrations in Don cells exposed to chemical

Chemical

Dose (M)

MI

Breaks/cell

Test chemical

0.000001

-

0.02

0.00001

-

0.09

0.0001

+

0.03

0.001

++

-

Conclusions:
The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In vitro mammalian cell gene mutation assay was performed for the test chemical
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Metabolic activation:
with
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT
6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.


Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical < 2.5 mM.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that The test chemical in the concentration of < 2.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Drosophila melanogaster was tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out on Drosophila melanogaster.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Principles of method if other than guideline:
The test for sex-linked recessive lethals (SLRL) in Drosophila melanogaster is used to detect induced mutations. The advantage of the test for both screening and hazard evaluation is its objectivity in testing for transmissible mutations in various chemicals. One of the chemicals studied include the test chemical of interest.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Balancer strains: Suitably marked inverted X chromosomes Tester strains: Males from wild type strains Oregon-K, Oregon-R, Canton-S, and Berlin-K.
Sex:
male
Route of administration:
oral: feed
Vehicle:
Vehicles
- Vehicle(s)/solvent(s) used: Test chemical is dissolved in either DMSO or ethanol

- Justification for choice of solvent/vehicle: DMSO is a commonly used solvent, while ethanol has an advantage since the flies have large quantities of the alcohol dehydrogenase (ADH) enzyme and can readily metabolize ethanol.

- Concentration of test material in vehicle: No data available

- Amount of vehicle (if gavage or dermal): No data available

- Type and concentration of dispersant aid (if powder): No data available

- Lot/batch no. (if required): No data available

- Purity: No data available

OTHER INFORMATION: In a test to screen for mutagenicity of a chemical, a single concentration of the chemical may be used to test for SLRLs. The selection of the final test concentration should be preceded by one or more exposure range-finding experiments in which two points are studied:
1. Toxicity to the males during treatment and during the breeding for the mating pattern analysis; and
2. Sterilizing effect due to lethal damage to one or several germ cell stages.

Based on the results obtained in such studies, a convenient exposure may be selected, usually at the LD50 level for treated males, provided sterility does not create problems. If sterility is induced, a lower exposure may have to be used. If no adverse effects are detected, the highest technically feasible exposure at which the flies will feed should be used.
Details on exposure:
For oral route
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available

- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available
Duration of treatment / exposure:
No data
Frequency of treatment:
No data
Post exposure period:
No data
Remarks:
No data
No. of animals per sex per dose:
No data
Control animals:
yes, historical
Positive control(s):
Yes, nonconcurrent positive control with ethylmethanesulphonate (EMS) or diethylnitrosamine (DEN)

Justification for choice of positive control(s): Nonconcurrent positive control are satisfactory for routine recessive lethal tests

- Route of administration: No data available

- Doses / concentrations: No data available
Tissues and cell types examined:
Eye shape and eye color
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No data available

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No data available

DETAILS OF SLIDE PREPARATION: No data available

METHOD OF ANALYSIS:
Hazard evaluation has been made using dose to the germ cells of ionizing radiation, and similar use of this test system with chemical mutagens is possible with the development of dosimetry of chemical mutagens (Comment: I don’t think this statement is correct, however, I am not sure what to state here since there is no real directions on methods of analysis in the report text.)

OTHER: No data available
Evaluation criteria:
Statistical evaluation
Statistics:
1. Comparison of control and treated frequencies
The most common problem is a comparison of the mutation frequencies obtained in a control and in a treated group. As long as the total number of mutations recovered (control and treated) is below 100, the Kastenbaum-Bowman test should be used. If more than 100 mutations are obtained, the chi square test is appropriate.

2. Analysis of the pattern of successive matings
The pooling of data from submatings is one by adding the number of chromosomes tested and the number of mutations found. In this way a weighted mean is obtained. For each of the samples, the statistical significance of a difference between the mutation frequency found in the control and in the treated groups should be checked as described above.
In a second step, the same procedure is repeated to obtain the analysis of the data pooled from the two samples.

3. Dose-response data
Dose-response data can be analyzed using standard regression techniques when germ cell dosimetry is available.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
other: Yes, solvent (If used) without the test chemical (administered in the same way as the treatment) is used for the negative control.
Positive controls validity:
other: Nonconcurrent positive control
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: No data available
- Solubility: No data available
- Clinical signs of toxicity in test animals: No data available
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): No data available
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available
Conclusions:
The test chemical is regarded to be negative for any mutagenic response when a SLRL test carried out on male Drosophila melanogaster.
Executive summary:

Drosophila melanogaster was tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out on Drosophila melanogaster.

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

Additional information

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation in vitro:

In a Salmonella/microsome assay, the mutagenic activity of the test chemical was evaluated in Salmonella typhimurium strains TA97, TA100, TA102 and TA104 with and without metabolic activation by S9 liver fractions from Aroclor-induced rats. At doses of 100 mg/plate, the phenolic antioxidant BHA exhibited toxic effects. However, a modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Therefore, exposure of the test chemical in Salmonella typhimurium, at concentrations below 500 mg/plate with or without metabolic activation by S9 liver fractions, is not regarded to be mutagenic.

This is further supported by data another publication. Bacterial reverse mutation assay was performed for the test chemical. The study was performed using E.coli WP2 (PKM101) with metabolic activation. The test chemical gives negative results for gene mutation conducted on E.coli WP2 (PKM101) with metabolic activation.

In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with DMSO and used at dose level of 0, 0.000001, 0.00001, 0.000, or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and with and without S9-induced metabolic activation for 3 hours. The results showed evidence of cytotoxicity when treated with 2.5 or 5.0 mM for 24 and 48 hours with S9 and 1.0, 2.5 or 5.0 mM for 24 and 48 hours without S9. Independently of tested concentrations, the results showed no evidence of gene toxicity when exposed to < 2.5 mM with S9 and < 1.0 mM without S9. The number of cells and colonies at 2.5 and 5.0 mM were insufficient to say that these two concentration are non-genotoxic. Therefore, it is considered that the test chemical in the concentration of < 2.5 mM with S9 and < 1.0 mM without S9 does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Sister chromatid exchange assay was also performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in HBSS and used at dose levels of 0, 0.000001, 0.00001, 0.0001 or 0.001 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). Sister chromatids were differentiated by the fluorescence or Giemsa staining techniques. The acridine orange technique was used for fluorescence, and a modified FPG technique was used for Giemsa staining. Taking the existence of a dosage effect greater than two fold background as a criterion, the frequency of SCE remained at about twice the background value for all concentrations shown. Based on these considerations, the test chemical did not induce SCEs in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In an evaluation of acute cytotoxicity of metabolic endpoints in V79 fibroblasts, the cells was exposed to the test chemical in the concentrations of 50 or 100mM. The results show that the test chemical was lethal and may also induce a 50% loss of cellular energy charge when V79 cells are exposed to the test chemical. It may also act as an inhibitor of oxygen consumption. It however did not induce DNA damage in the V79 fibroblast cell line and hence it is not likely to be mutagenic in vitro.

Gene mutation in vivo:

Drosophila melanogasterwas tested in a test for sex-linked recessive lethals (SLRL) to detect induced mutations. Ten day germ cell-staged adult male flies were fed with a solvent containing the test chemical to detect if it induced any mutagenic responses. No mutagenic responses were detected when a SLRL test was carried out onDrosophila melanogaster.

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.