2-methylanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES assay

Test substance did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test

The test chemical did not induce chromosomal aberrations in the CHO cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

PCB-induced rat liver S9

Test concentrations with justification for top dose:

0, 5, 10, 50 or 250 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The 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 agar (plate incorporation)DURATION- Preincubation period: No data- Exposure duration: 2 days (48 hrs)- Expression time (cells in growth medium): 2 days (48 hrs)- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell growth was notedOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of revertants/plate

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic to the strain TA97 at 250 µg/plate

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 effect were observed

Conclusions:

Test substance did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of 2 -methylanthraquinone. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system at dose levels of 0, 5, 10, 50 or 200µg/plate. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. Test substance did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL / 1

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

2

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5A
medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, At least once per year representative cells were sent to Flow Laboratories (McLean, VA) for mycoplasma testing using the Hoechst stain test followed by the Agar and Hyorhinis test. Results from all tests for mycoplasma contamination were negative.
- Periodically checked for karyotype stability: No data
- 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:

S9 fractions (livers of Aroclor 1254-treated male Sprague-Dawley rats.)

Test concentrations with justification for top dose:

1. -S9 (Harvest time: 10 hrs): 0, 2500, 3850, 5000 µg/mL
+S9 (Harvest time: 12 hrs): 0, 2500, 3850, 5000 µg/mL

2. 0, 1000, 1600, 3000 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

2. - Vehicle(s)/solvent(s) used: Serum-free culture medium
- Justification for choice of solvent/vehicle: The test chemical was soluble in Serum-free culture medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Serum-free culture medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration:
- S9: 8 hrs
+ S9: 2 hrs
- Expression time (cells in growth medium): 8 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): -S9: 10 hrs, +S9: 12 hrs

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: One hundred to 200 cells from each of the three highest scorable doses were analyzed

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

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

OTHER: No data available

2. METHOD OF APPLICATION: in medium

Cells at the start of experiment: 1.2 x 106 cells

DURATION
- Preincubation period: Not applicable
- Exposure duration:
Without S9: 2 hrs
With S9: 2 hrs
- Expression time (cells in growth medium):
Without S9: 10 hrs
With S9: 11 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): 6% Giemsa stain

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: One hundred cells were scored for each dose. Only metaphase cells in which the chromosome number was between 19 and 23 were scored

Details on slide preparation:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation. Gaps and endoreduplications were recorded but were not included in the statistical analyses.

2. The cell line was observed for chromosome aberrations. The chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).

Statistics:

1. Trend test.

2. The percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL / 1

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:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 – 7.5
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Chemicals were tested up to 5 mg/ml or as limited by solubility and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent (water, dimethyl sulfoxide, acetone, or ethanol, in that order of preference). In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). The doses used were generally the TD, 0.75 TD, 0.50 TD, 0.25 TD, 0.1 TD, 0.075 TD, 0.05 TD, and 0.025 TD. The highest three doses with a sufficient number of cells were analyzed for chromosomal aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The dose levels for ABS studies were chosen based on the toxicity of the test chemical observed in the SCE studies.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberrations in the CHO cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data from various test chemicals was reviewed to determine the mutagenic nature of the test chemicals. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in serum free culture medium and used at dose level of0, 1000, 1600, 3000 or 5000 µg/plate. Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 10⁶cells were seeded per 75 cm2 flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. Acid red 14 did not induce ABS at doses up to 5000µg/ml in either the presence or the absence of S9. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the data available for the various test chemicals, the test chemical did not induce chromosomal aberrations in the CHO cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available for the target chemical, 2 -methylanthraquinone does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.