1,4-bis(p-tolylamino)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Item Identity: SANDOPLAST GREEN GSB (1,4-bis(p-tolylamino)anthraquinone)
- Lot/batch No.of test material: FRAA016121
- Expiration date of the lot/batch: October 01, 2003
- Purity test date: no data

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: 10 days in water, saline, polyethylene glycol and CMC
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item SANDOPLAST GREEN GSB was suspended in DMSO.
- Preliminary purification step (if any): no data
- Final dilution of a dissolved solid, stock liquid or gel: no data
- Final preparation of a solid: no data

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

Rat Liver S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
0; 33, 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA 1535, TA 100 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

For strains TA 1537, TA 98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For strains WP2 uvrA without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

For strains TA 1535, TA 1537, TA 98, TA100, WP2 uvrA with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 1 h
- Exposure duration:48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: evaluation of background lawn

Rationale for test conditions:

No data

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In experiment II, with metabolic activation, the number of colonies in strain TA 98 (negative control) was slightly above the range of our historical data. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.The historical range of positive controls was exceeded in strains TA 1535 (experiment I and II) and in strain TA 100 (experiment I) without metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: No mutagenic potential

The following concentrations were tested for toxicity and mutation induction with each 3 plates.

Substance	Concentration /plate (µg)	Revertants per plate (mean of 3 plates)
		TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
S-9		-	+	-	+	-	+	-	+	-	+
Negative control		19	16	9	13	35	32	128	168	53	56
Solvent control		16	14	6	11	34	36	128	132	55	54
4-NOPD	50			65
4-NOPD	100					272
MMS	4 (µl)									859
NaN3	10	1216						931
2-AA	2,5		220		115		995		1039
2-AA	10										200
Test item	3	15	12	7	14	33	36	131	145	51	53
	10	18	13	8	13	30	37	126	145	47	51
	33	16	10	7	13	26	35	128	149	45	48
	100	14	11	6	12	25	33	127	140	47	49
	333	13	12	5	13	23	31	128	139	48	48
	1000	15	11	6	13	23	32	112	137	46	48
	2500	12	9	5	10	19	27	117	132	48	48
	5000	8	8	6	10	17	27	109	125	48	47

 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutati on assay.

Executive summary:

This study was performed to investigate the potential of the given test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535,TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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: As mentioned below

Principles of method if other than guideline:

WoE for the target CAS is summarized based on 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:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

8

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture medium supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

9

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:

S9 mix

Test concentrations with justification for top dose:

8. Continuous treatment: 0, 0.3, 0.7, 1.3 µg/mL
Short term treatment method: 0, 06, 1.1, 2.2 µg/mL
9. Continuous treatment: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Short term treatment method: With S9: 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Without S9: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL

Vehicle / solvent:

8. - Vehicle(s)/solvent(s) used: 0.5% CMC Na
- Justification for choice of solvent/vehicle: The test chemical was soluble in CMC Na
9. - 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:

CMC Na

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

8

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:

9

Details on test system and experimental conditions:

8. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 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): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells for multiplicative cells were analyzed.

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
9. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 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): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group were analyzed.

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:

8. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.
9. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.

Statistics:

8. A significant difference test (p <0.05) between the negative control group and the test substance treated group and between the negative control group and the positive control group was performed on the occurrence frequency of cells having chromosomal abnormality by Fisher's exact probability test method. According to the judgment criteria of Ishikan et al, the frequency of cells with chromosomal abnormality is negative, negative 5% or higher and less than 10% positive false positive 10% or higher Respectively.
9. No data

Species / strain:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

8

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

9

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

8. 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: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. The growth inhibitory action of the test substance on CHL / IU cells was determined by measuring the proliferation degree of each group using a monolayer culture cell densitometer, and the negative control group was used as an index.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
9. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, and 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo sister chromatid exchange analysis in mice

The given test chemical was considered to be negative gene toxic to bone marrow cell of mice.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

Sister chromatid exchange (SCE)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source.

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

In vivo gene mutation assay of test chemical in C57BL/6 mice.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Jackson Laboratory, Bar Harbor, Maine.
- Age at study initiation:3-4 months, 4 days
- Weight at study initiation:22 to 30 g
- Assigned to test groups randomly: [no/yes, under following basis: No data available
- Fasting period before study:No data available
- Housing:Animals were housed 5 per cage
- Diet (e.g. ad libitum):Lab chow
(non-certified Purina), ad libitum
- Water (e.g. ad libitum):Water, ad libitum
- Acclimation period:10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-21 °C
- Humidity (%):60-68%
- Air changes (per hr):15 cycles/hr of biocleaned air
- Photoperiod (hrs dark / hrs light):12 hr light-dark cycle.

IN-LIFE DATES: From: To:No data available

Route of administration:

intraperitoneal

Vehicle:

Vehicles
- Vehicle(s)/solvent(s) used: DMSO + corn oil.
- Concentration of test material in vehicle:0, 10, 20 and 40 mg/kg
- Amount of vehicle (if gavage or dermal):0.1 ml DMSO and 0.1 ml corn oil

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS:C.T. Solvent Green No. 3 was administered in 0.1 ml DMSO + 0.1 ml corn oil.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

3 to 4 point dose

Post exposure period:

2 hour

Remarks:

0, 10, 20, 40 mg/kg bw

Dose / conc.:

15 mg/kg bw (total dose)

Remarks:

Positive control (Cyclophosphamide)

Dose / conc.:

0.2 mg/kg bw (total dose)

Remarks:

Solvent control

No. of animals per sex per dose:

Total : 24
0 mg/kg: 4 male
10 mg/kg: 4 male
20 mg/kg: 4 male
40 mg/kg: 4 male
Positive control (Cyclophosphamide)
15 mg/kg: 4 male
Solvent control
0.2 mg-l/kg: 4 male

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls
- Cyclophosphamide
- Doses / concentrations:15 mg/kg

Tissues and cell types examined:

Well-differentiated second division metaphase cells which contained the diploid ± 2 chromosomal complement. Cell replication kinetics were also assessed in 200 marrow cells/ animal and the proportions of first (MI), second (M2) and third (M3) division cells were determined from chromosome stain patterns.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:No data available

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):The dye injections were given 1/2 hr after BrdU tablet implantation. Approximately 23 hrlater, all control mice were injected I.P. with 0.6 mg/kg of colchicine (Sigma) in order to collect metaphases. Treated mice were injected with colchicine after an additional 3-4 hr since preliminary chemical injection trials had indicated that cell-cycle delays were occurring. Two hours after colchicine injection, animals were sacrificed by cervical dislocation,

DETAILS OF SLIDE PREPARATION:Bone marrow cells were harvested and processed through hypotonic (0.075 M KC1) and fixative (3:1 methanol: glacial acetic acid) steps, and slides were prepared in accordance with standard cytogenetic methodology.

METHOD OF ANALYSIS:cells/animal

OTHER:No data available

Evaluation criteria:

Second division metaphase cells and Cell replication kinetics were evaluated

Statistics:

No data available

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Conclusions:

The given test chemical was considered to be negative gene toxic to bone marrow cell of mice.

Executive summary:

In an in vivo gene mutation assay, C57BL/6 male mice were treated with the given test chemical at the concentration of 0, 10, 20 and 40 mg/kg by single intraperitoneal exposure. No Sister chromatid exchange (SCE) induction were observed in bone marrow of treated mice. Therefore, the given test chemical was considered to be negative gene toxic to bone marrow cell of mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames assay:

In vitro gene mutation study in bacteria was performed to investigate the potential of the given test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535,TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

In another study, Salmonella typhimurium reverse mutation assay was performed to determine the mutagenic response of the given test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 3, 10, 33, 100, 333, 1000 3330 or 5000 µg/plate. Triplicate plates were used per dose level. Concurrent positive control chemicals were also included in the study. The test substance precipitated in the top agar at concentrations of 100 µg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. Both bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants. The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the given test chemical is not mutagenic in the Salmonella typhimurium reverse mutation assay using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

The above study is supported with the gene mutation toxicity study performed to determine the mutagenic nature of the given test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 with and without S9 metabolic activation system. 100-200 µg of the test material dissolved in 10-20µL DMSO/disc was applied to 6mm paper concentration discs and used for mutagenicity testing. From 1 to 4 discs were applied per plate. Concurrent solvent and negative control chemicals were also included in the study. The given test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

These studies are supported with the in vitro gene mutation test was performed to evaluate the mutagenic nature of the given test compound. Plate incorporation method was the method of choice. The test compound failed to induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 and hence is negative for gene mutation in vitro.

 

Another study on Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the given test compound. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test compound failed to induce mutation in Salmonella typhimuriumTA98, TA1537, TA100, TA1535) and hence is negative for gene mutation in vitro.

 

All the above studies are further supported with the Salmonella/ mammalian-microsome test (Spot test) performed to evaluate the mutagenic nature of the given test compound. The spot test was used to screen the test material for potential mutagenicity. The test material was placed in the center of the plate. The test compound was tested with and without the S9 mix. Inhibition of the bacterium was indicated by a clearing of the background lawn in a zone surrounding the test material. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. A known mutagen, Captan, was used as a reference mutagen. The test compound failed to induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535) in the spot test performed and hence is negative for gene mutation in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 

In vitro mammalian chromosome aberration study:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, and 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

In vivo sister chromatid exchange analysis in mice

In an in vivo gene mutation assay, C57BL/6 male mice were treated with the given test chemical at the concentration of 0, 10, 20 and 40 mg/kg by single intraperitoneal exposure. No Sister chromatid exchange (SCE) induction were observed in bone marrow of treated mice. Therefore, the given test chemical was considered to be negative gene toxic to bone marrow cell of mice.

 

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay and In vitro mammalian chromosome aberration study and in vivo sister chromatid exchange analysis in mice. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.