7-(diethylamino)-4-methyl-2-benzopyrone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The Salmonella/mammalian microsome test was performed to determine the mutagenic nature of test chemical in vitro. Preincubation assay was performed using Salmonella typhimurium TA100, TA1535, TA1537, TA98 with and without S9 metabolic activation system. To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. The doses thus selected were 0, 100, 333, 1000, 3333, 5450 µg/plate. Appropriate positive controls were also incorporated in the study. The test chemical is not mutagenic to Salmonella typhimurium TA100, TA1535, TA1537, TA98 in the preincubation assay performed with and without S9 metabolic activation system and hence does not classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

It is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

It is concluded that the given test chemical did not induce any mutations 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

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Salmonella/mammalian microsome test was performed to determine the mutagenic nature of the test chemical in vitro

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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:

Liver S-9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 5450 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Soluble and stable in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene, 4-Nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- 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): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

NUMBER OF CELLS EVALUATED: No data available

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

Rationale for test conditions:

No data

Evaluation criteria:

Increase in the number of revertants

Statistics:

Statistical analysis of Salmonella plate test data was performed as per Margolin et al, 1981

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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:

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: To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium (EGG) and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn (CWR, EGG, SRI). If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

Remarks on result:

other: No mutagenic potential

Dose(µg/plate)	TA100
	NA	RLI	HLI
0.0(Solvent control)	114±10.5	151±13.9	132±5.2
100.0	118±5.9	148±15.2	115±6.1
333.0	108±3.3	121±8.8	113±2.3
1000.0	93±3.5	107±2.5	130±4.6
3333.0	127±10.9	140±4.4	127±18.0
5450.0	122±12.3	141±9.6	150±21.5
POS	506±21.0	698±47.9	1522±90.7

Dose(µg/plate)	TA1535
	NA	RLI	HLI
0.0(Solvent control)	6±2.2	12±0.6	5±1.2
100.0	7±1.2	8±1.9	6±1.9
333.0	4±0.3	7±0.6	7±2.0
1000.0	4±1.8	5±1.2	3±1.5
3333.0	2±1.5	7±1.3	3±1.5
5450.0	3±0.3	5±1.7	2±0.6
POS	345±6.2	143±40.0	77±4.3

Dose(µg/plate)	TA1537
	NA	RLI	HLI
0.0(Solvent control)	4±1.5	8±0.0	10±1.5
100.0	5±0.9	6±0.6	11±3.2
333.0	5±1.0	11±2.6	7±1.5
1000.0	5±1.2	9±2.0	8±2.6
3333.0	3±0.7	9±1.9	8±3.2
5450.0	3±1.0	6±2.6	7±0.9
POS	829±8.5	60±8.2	69±2.7

Dose(µg/plate)	TA98
	NA	RLI	HLI
0.0(Solvent control)	16±2.0	21±1.7	23±1.2
100.0	16±0.9	16±2.0	23±1.8
333.0	13±0.7	17±2.0	21±4.7
1000.0	15±2.0	22±5.9	18±3.8
3333.0	15±4.1	23±2.3	20±4.6
5450.0	9±1.2	20±2.7	22±3.4
POS	348±29.0	414±29.4	1263±155.7

RLI: rat liver S-9 Aroclor 1254 induced;

HLI: hamster liver S-9Aroclor 1254 induced,

Mutagenic responses of Salmonella tester strains TA100, TA1535, TA1537, and TA98 (mean ± SEM) to test chemicals.

Conclusions:

The test chemical is not mutagenic to Salmonella typhimurium TA100, TA1535, TA1537, TA98 in the preincubation assay performed with and without S9 metabolic activation system and hence does not classify as a gene mutant in vitro.

Executive summary:

The Salmonella/mammalian microsome test was performed to determine the mutagenic nature of test chemical in vitro.

 

Preincubation assay was performed using Salmonella typhimurium TA100, TA1535, TA1537, TA98 with and without S9 metabolic activation system. To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix.

 

The doses thus selected were 0, 100, 333, 1000, 3333, 5450 µg/plate. Appropriate positive controls were also incorporated in the study.

The test chemical is not mutagenic to Salmonella typhimurium TA100, TA1535, TA1537, TA98 in the preincubation assay performed with and without S9 metabolic activation system and hence does not classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from 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 for the target chemical 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:

Chinese hamster Ovary (CHO)

Remarks:

5

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 μg/mL gentamycin
- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

other: CHO cells have low number of chromosomes making scoring relatively easy

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

6

Details on mammalian cell type (if applicable):

- Type and identity of media: Stocks of CHO
cells were maintained at 37°C in McCoy's 5A (modified) medium buffered with 20 mM
HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and
50 pg/ml streptomycin (Gibco. Grand Island,
NY).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, Cells were tested regularly for m
ycoplasma contamination using 4' ,6-diamidin0-2-phenyl-indole (DAPI) fluorescence and were found
to be free of mycoplasma
- Periodically checked for karyotype stability: Yes, To ensure karyotypic stability, cells were not used
beyond the fifteenth passage after cloning.
- 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:

5. No data
6. The rat liver microsomal fraction was prepared from Aroclor 1254-induced male Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.

Test concentrations with justification for top dose:

5. 0, 1511, 2159, 3084 µg/mL
6. Without S9: 0.0, 49.8000, 150.0000, 499.0000 μg/mL
With S9: 0.0, 500.0000, 1500.0000, 5000.0000 μg/mL

Vehicle / solvent:

5. - Vehicle(s)/solvent(s) used: Sterile ROHP water for test material and negative control; DMSO for positive control
- Justification for choice of solvent/vehicle: No data available

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile ROHP water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-l-oxide (in absence of UV light) 8-methoxypsoralen (8-MOP) (in presence of UV light)

Remarks:

5

Untreated negative controls:

yes

Remarks:

Medium control

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

5. METHOD OF APPLICATION: in medium
DURATION- Preincubation period: 15 mins
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 17 hrs
- Selection time (if incubation with a selection agent): 17 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hrs
SELECTION AGENT (mutation assays): gentamycin
SPINDLE INHIBITOR (cytogenetic assays): colchicines 1µg/mL
STAIN (for cytogenetic assays): 4%(v/v) filteredGiemsa stain in pH 6 .8 buffer
NUMBER OF REPLICATIONS: Duplicate (with and without irradiation)
NUMBER OF CELLS EVALUATED: 100D
ETERMINATION OF CYTOTOXICITY- Method: mitotic index
OTHER EXAMINATIONS:- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Other:

6. METHOD OF APPLICATION: in medium
Cells seeded: 1.75 X 106 cells/75 cm2 flask
DURATION
- Preincubation period: No data available
- Exposure duration:
Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium):
Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): Standard harvest time: 10-14 hr af
ter addition of BrdUrd
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: Only one trial was performed
NUMBER OF CELLS EVALUATED: 100-200 cells were scored
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

Evaluation criteria:

5. The test article was to be considered as positive in this assay if-1)A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and 2) The proportion of cells with structural aberrations at such doses exceeded the normal range.3) Statistically significant increases in cells with chromosome aberrations were induced in the presence of UV but not in itsabsence, and4) Cells with chromosomal aberrations occurred at lower doses in the presence of UV, or with significantly higher frequencies than the total of aberration frequencies observed in the irradiated solvent control plus the aberration frequency in the non-irradiated sample at that concentration.

6. A positive response was defined as one for which the adjusted P value was <0.05. A positive response at a single dose was designated “+ W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘+ W*”. A test was designated “+” if at least two doses gave significantly increased responses.

Statistics:

5. The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.The proportion of cells for each test treatment condition is compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.

6. All categories of aberrations (simple, complex, and other) were combined for the statistical analysis, which was based on the percent of total cells with aberrations. The percent of cells with ABs (i.e.,percent of aberrant cells) was used for the analysis, rather than the average number of aberrations per cell.

A binomial sampling assumption as described by Margolin et al. [1983] was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

5

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

6

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:

5. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: Yes
- Precipitation: No precipitation observed in culture medium
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The highest dose level used in the range-finder, 3083 μg/mL, was a concentration of 10 mM in culture medium. The doses of UV used were 1500 and 750 mJ/cm2.In the phototoxicity range-finder, there was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The concentration of 3084 μg/mL (approximately 10 mM) was chosen as the top dose for the main study and a range of doses from this concentration were used in the absence and presence of UV light. The test article dose levels for chromosome analysis from the irradiated cultures were selected by evaluating the effect of 2-hydroxy-4 methoxybenzophenone-5-sulfonic acid (sulisobenzone) on mitotic index. Chromosome aberrations were analysed at 3 consecutive dose levels. The equivalent doses were taken for analysis from the cultures treated in the absence of UV light.The highest concentration chosen for analysis, 3084 μg/mL, did not induce mitotic inhibition in either the absence or presence of UV light.

COMPARISON WITH HISTORICAL CONTROL DATA:Historical solvent controls were incorporated in the study and a comparison was made to check whether the proportion of cells with structural aberrations fell within the solvent control ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:No data available

6. 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: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

It is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

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

The given test chemical was used to detect whether it is capable of causing chromosomal aberrations. In vitro mammalian chromosome aberration test was carried out on cells obtained from Chinese hamster Ovary. As some test chemicals may become potent clastogens when photosensitised, the clastogenic potential of the test article in this study was assessed by its effects on the chromosomes of CHO cells treated in the absence and presence of UV light. A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The doses of UV used were 1500 and 750 mJ/cm2. There was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The highest concentration of the test material chosen for analysis was 3084mg/Land a range of doses from this concentration were used in the absence and presence of UV light. Appropriate negative (solvent) and positive controls were included in the test system in the presence and absence of UV light. Treatment of cultures with chemical in the absence of UV light resulted in frequencies of cells with structural aberrations that were similar to and not significantly different from the frequency seen in concurrent, non irradiated, solvent control cultures . Aberrant cell frequency was within the historical negative control range. Treatment of cultures with test chemical in the presence of UV light resulted in frequencies of cells with structural aberrations that were not increased compared to the concurrent, irradiated, solvent control cultures. It is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells in the absence or presence of UV light. In accordance with the CLP classification, the test material does not classify a mutagen in vitro.

In another in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 10.50 hr, washed to remove the test chemical, and treated with colcemid for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 12 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, and the cells were harvested 2 hr later. Thus the total durations of the non-activated and activated AB experiments were 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. For early studies; one hundred cells were scored for each of three concentrations: the highest test concentration in which sufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap, break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomes or cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic

activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on data available for the various test chemicals, it is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

The purpose of this study was to assess toxic and genotoxic effects of the given test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 1.0, 2.5, 5.0 or 10.0 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test chemical was easily dissolved in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU) (2.5 mM) without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation
DURATION- Preincubation period: 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 (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

not determined

Remarks:

The metabolic activation system could not be ascertained as the positive control failed to produce a significant increase in the number of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Table 1A. Effect of 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	835/4	0/4	683/4
Pos. control	1/4	814/4	31/4	831/4
1.0 mM	0/4	777/4	0/4	820/4
2.5 mM	0/4	927/4	0/4	881/4
5.0mM	0/4	922/4	0/4	889/4
10.0 mM	0/4	843/4	0/4	839/4

 

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	-2.61x10^-4	3.84x10^-4
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0mM	N/A	N/A
10.0 mM	N/A	N/A

 

Conclusions:

Without metabolic activation, the test chemical tested negative for mutagenicity in Chinese hamster ovary (CHO) cells at doses up to 10 mM. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

Executive summary:

The chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

Ames assay:

The Salmonella/mammalian microsome test was performed to determine the mutagenic nature of test chemical in vitro. Preincubation assay was performed using Salmonella typhimurium TA100, TA1535, TA1537, TA98 with and without S9 metabolic activation system. To select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. The doses thus selected were 0, 100, 333, 1000, 3333, 5450 µg/plate. Appropriate positive controls were also incorporated in the study. The test chemical is not mutagenic to Salmonella typhimurium TA100, TA1535, TA1537, TA98 in the preincubation assay performed with and without S9 metabolic activation system and hence does not classify as a gene mutant in vitro.

The above study for the target chemical is further supported by data from various closely related test chemicals. The studies are as given below:

Gene mutation study was performed to evaluate the mutagenic nature of the test chemicals 1 and 2 using Salmonella typhimurium strain TA1538, TA98 and TA100 at dose level of 0.1 or 1 mg/plate (100 or 1000 µg/plate). Bacteria were grown overnight in Oxoid nutrient broth, then refrigerated at 4-5^OC for a few hours before use. 0.1 ml of bacterial culture was added to 2 ml of 45°C molten top agar containing 0.01 mg histidine HCI and 0.012 mg biotin/ml, followed by the test sample in ≤0.2 ml DMSO. Finally, 0.5 ml of sodium phosphate buffer, pH 7.4 (no activation), or 0.5 ml of Aroclor-induced rat S9 mixture was added, and the mixture was poured on minimal glucose agar plates. Histidine revertant colonies were counted on a Biotran II automated colony counter after 2-day incubation at 37°C. A sample was judged mutagenic if it produced greater than twice the spontaneous background colonies at more than one dose or at the highest dose tested. In the above mentioned study, the test chemicals 1 and 2 did not induce gene mutation in the Salmonella typhimurium strains TA1538, TA98 and TA100 with and without metabolic activation. Hence it is not likely to be a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The given test chemical was used to detect whether it is capable of causing chromosomal aberrations. In vitro mammalian chromosome aberration test was carried out on cells obtained from Chinese hamster Ovary. As some test chemicals may become potent clastogens when photosensitised, the clastogenic potential of the test article in this study was assessed by its effects on the chromosomes of CHO cells treated in the absence and presence of UV light. A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The doses of UV used were 1500 and 750 mJ/cm2. There was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The highest concentration of the test material chosen for analysis was 3084mg/Land a range of doses from this concentration were used in the absence and presence of UV light. Appropriate negative (solvent) and positive controls were included in the test system in the presence and absence of UV light. Treatment of cultures with chemical in the absence of UV light resulted in frequencies of cells with structural aberrations that were similar to and not significantly different from the frequency seen in concurrent, non irradiated, solvent control cultures . Aberrant cell frequency was within the historical negative control range. Treatment of cultures with test chemical in the presence of UV light resulted in frequencies of cells with structural aberrations that were not increased compared to the concurrent, irradiated, solvent control cultures. It is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells in the absence or presence of UV light. In accordance with the CLP classification, the test material does not classify a mutagen in vitro.

In another in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 10.50 hr, washed to remove the test chemical, and treated with colcemid for 2 -2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 12 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, and the cells were harvested 2 hr later. Thus the total durations of the non-activated and activated AB experiments were 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. For early studies; one hundred cells were scored for each of three concentrations: the highest test concentration in which sufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap, break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomes or cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line 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 vitro mammalian cell gene mutation assay:

The chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data.No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

Based on data available for the various test chemicals, it is concluded that the given test chemical did not induce chromosome aberrations in cultured CHO cells and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on all the available data, it was concluded that the test chemical does not induce any mutation and thus is not likely to classify as per the CLP criteria of classification and labeling.