Denatonium benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 SSS study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

This study was performed to identify whether the test item, Denatonium benzoate (CAS No. 3734-33-6), causes any structural or numerical chromosomal aberrations (clastogenicity) in cultured human lymphocytes when exposed to both, with and without an exogenous metabolic activation using in vitro chromosomal aberration test in cultured human lymphocyte cell culture. The experiment was conducted following the OECD Guideline for the Testing of Chemicals 473.

GLP compliance:

yes

Type of assay:

other: in vitro chromosomal aberration test in cultured human lymphocyte cell culture

Test material

Test material information

Constituent 1

Reference substance name:

Denatonium benzoate

EC Number:

223-095-2

EC Name:

Denatonium benzoate

Cas Number:

3734-33-6

Molecular formula:

C21H29N2O.C7H5O2

IUPAC Name:

N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate

Test material form:

solid: granular

Details on test material:

- Name of test material: Denatonium benzoate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight : 446.58 g/mol
- Substance type: organic
- Physical state: Solid
- SMILES: CCN{+}(CC)(Cc1ccccc1)(CC(=O)Nc1c(C)cccc1C).O{-}C(=O)c1ccccc1

Specific details on test material used for the study:

- Name of test material: Denatonium benzoate
- IUPAC name: N-benzyl-2-[(2,6-dimethylphenyl)amino]-N,N-diethyl-2-oxoethanaminium benzoate hydrate
- Molecular formula: C21H29N2O.C7H5O2
- Molecular weight : 446.58 g/mol
- Substance type: organic
- Physical state: Solid
- SMILES: CCN{+}(CC)(Cc1ccccc1)(CC(=O)Nc1c(C)cccc1C).O{-}C(=O)c1ccccc1

Method

Target gene:

No data

Species / strain

Species / strain 1

Species / strain / cell type:

lymphocytes: Human

Remarks:

Short term treatment

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Young and healthy volunteers of age between 18-35 with no smoking and no recent illness records or recent exposures to genotoxic agents (e.g. chemicals, ionizing radiations) were selected as source of cells
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index: No data
- Sex, age and number of blood donors if applicable: Sex: Male, Age: 18-35 yrs, Number of donors: 3
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used for the study
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 media
- Properly maintained: Yes
- 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 2

Species / strain / cell type:

lymphocytes: Human

Remarks:

Long term/ extended treatment

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Young and healthy volunteers of age between 18-35 with no smoking and no recent illness records or recent exposures to genotoxic agents (e.g. chemicals, ionizing radiations) were selected as source of cells
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index: No data
- Sex, age and number of blood donors if applicable: Sex: Male, Age: 18-35 yrs, Number of donors: 3
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used for the study
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 media
- Properly maintained: Yes
- 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

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 0.313, 0.625 or 1.25 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI-1640 medium
- Justification for choice of solvent/vehicle: The test chemical was soluble in RPMI-1640 medium

Controls

Untreated negative controls:

yes

Remarks:

Sterilized water

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 300 mitotic cells

DURATION
- Preincubation period:
- Exposure duration: Short term treatment: 3 hrs
Long term/ extended treatment: 48 hrs
- Expression time (cells in growth medium): 96 hrs approx
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 96 hrs approx

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): 5% Giemsa stain

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were labeled and processed using ice cold methanol for fixation. A drop of resuspended cell was added to the slide (inclined approximately 45° angle) from a distance of approximately, 1 meter. To attain uniform spread of smear, the slide was gently titled. The slides were air dried and stained using 5% Giemsa stain. The stained slides were air dried and mounted using DPX mountant. The slides were coded (before scoring) and decoded (after scoring) by a technical person who was not involved in the study. Duplicate slides were used per treatment. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Breaks and gaps, Chromatid and chromosome type aberrations was recorded separately and classified by sub-types like breaks and exchanges.

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A minimum of 300 well spread metaphases were scored per single culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity:
MI (%) = Number of mitotic cells × 100
Total number of cells scored

OTHER EXAMINATIONS:
- Determination of polyploidy: Incidence of increase in polyploidy cells was recorded separately
- Determination of endoreplication: Incidence of increase in cells with endoreduplicated chromosomes was recorded separately
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. Test item should be considered clearly positive by a concentration related and statistically significant increase in the incidence of aberrant cells for the treatment group compared with the concurrent negative control group.
2. Test item should be considered clearly negative when no concentration related and statistically significant increase in the incidence of aberrant cells for the treatment group compared with the concurrent negative control group.
3. When the results do not meet the criteria specified for a positive or negative response shall be considered as an equivocal.
4. Incidence of increase in number of polyploidy cells may be considered which indicates test substance have potential to inhibit mitotic process and also numerical aberrations. Increase in number of cells with endoreduplicated chromosomes may indicate test substances have potential to inhibit cell cycle progress.
5. All results should be inside the distribution of historical control data (e.g. Poisson based 95% control limits).

Statistics:

Data were expressed in mean ± SD. The data was analyzed with ANOVA test using Sigmaplot. Statistical analysis was performed to identify the mean difference between the control and treated groups. P value <0.05 was fixed as significance criterion.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: Human

Remarks:

Short term treatment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: Human

Remarks:

Extended / long-term treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 precipitation was observed
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Five test item concentrations were selected initially for dose or concentration range finding (5, 2.5, 1.25, 0.625, 0.313 mg/ml ). Concentrations for the main study were selected based on the level of cytotoxicity (reduction of 45±5% of mitotic index) in the dose or concentration range finding study. Duplicates treatments were used for both, dose range finding and main study.

For concentration range finding (cytotoxcity) study, a total of 12 test tubes were taken. 10 for the five test item concentration selected and two for negative control. Each test tube containing a volume of 0.8 ml of anti coagulated human blood was added with 9 ml of complete RPMI 1640 media with the presence of mitogen (PHA) of 0.2 ml volume to induce the cell division. The lymphocytes were allowed to culture for 48 h in shaker water bath at 37°C at 70 rpm. The cultured cells were exposed to test item and further incubated at 37°C for 48 h in shaker water bath at 70 rpm. Following incubation, the tubes were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and 5 ml of pre-warmed 0.075M KCl solution was added to the pellet and incubated at 37°C for 30 minutes. The tubes were centrifuged and the supernatant was discarded. The tubes were centrifuged by adding 5 ml of Carnoy’s fixative solution till the pellet colour turns white or creamy colour. 90% of the supernatant was discarded without disturbing the pellet. The pellet was re-suspended in the remaining supernatant for slide preparation and scoring of mitotic index (MI).

Cytotoxicity was observed at 5 and 2.5 mg/ml concentration with mean mitotic index value of 14.1 and 17.6 when compared with negative control. Cytotoxicity was determined using mitotic index with a value of 54.0 at test concentration 1.25 mg/ml when compared with negative control. No cytotoxicity was observed at 1.25, 0.625 and 0.313 mg/ml concentration levels. Hence, 1.25mg/ml was selected as the highest concentration for the main study.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Mitotic index
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

 MITOTIC INDEX FORRANGEFINDING STUDY

Treatment	Slide code	Total No. of cells	Total No. of mitotic cells	Mean mitotic index (%)
Sterilized water (Negative control)	R-NC-1	1000	882	86.9
	R-NC-2	1000	856
Denatonium benzoate 0.313mg/ml	R-TC1-1	1000	714	72.2
	R-TC1-2	1000	730
Denatonium benzoate0.625mg/ml	R-TC2-1	1000	622	61.8
	R-TC2-2	1000	613
Denatonium benzoate1.25mg/ml	R-TC3-1	1000	542	54.0
	R-TC3-2	1000	538
Denatonium benzoate2.5mg/ml	R-TC4-1	1000	289	27.6
	R-TC4-2	1000	263
Denatonium benzoate5mg/ml	R-TC5-1	1000	138	14.1
	R-TC5-2	1000	144
R- Range finding; NC- Negative control; TC- Test concentration; -1,-2- R MITOTIC INDEX FORMAINSTUDY Short term treatment (withoutS9) Treatment Slide code Total No. of cells Total No. of mitotic cells Mean mitotic index (%) Sterilized water (Negative control) M-NC-1A(-) 500 496 97.8 M-NC-1B(-) 500 488 M-NC-2A(-) 500 482 M-NC-2B(-) 500 489 Mitomycin C 0.5µg/ml (Positive control) M-PC-1A(-) 500 225 47.1 M-PC-1B(-) 500 245 M-PC-2A(-) 500 233 M-PC-2B(-) 500 238 Denatonium benzoate1.25mg/ml M-TC1-1A(-) 500 432 87.8 M- TC1-1B(-) 500 445 M- TC1-2A(-) 500 440 M- TC1-2B(-) 500 439 Denatonium benzoate0.625mg/ml M- TC2-1A(-) 500 446 90.1 M- TC2-1B(-) 500 452 M- TC2-2A(-) 500 447 M- TC2-2B(-) 500 457 Denatonium benzoate 0.313mg/ml M- TC3-1A(-) 500 463 94.1 M- TC3-1B(-) 500 474 M- TC3-2A(-) 500 467 M- TC3-2B(-) 500 478 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; -1,-2- Culture replicates; A,B- replicate slides Short term treatment (withS9) Treatment Slide code Total No. of cells Total No. of mitotic cells Mean mitotic index (%) Sterilized water (Negative control) M-NC-1A(+) 500 488 97.3 M-NC-1B(+) 500 486 M-NC-2A(+) 500 480 M-NC-2B(+) 500 491 Mitomycin C 0.5µg/ml (Positive control) M-PC-1A(+) 500 286 55.1 M-PC-1B(+) 500 257 M-PC-2A(+) 500 276 M-PC-2B(+) 500 283 Denatonium benzoate1.25mg/ml M-TC1-1A(+) 500 428 86.8 M- TC1-1B(+) 500 437 M- TC1-2A(+) 500 442 M- TC1-2B(+) 500 429 Denatonium benzoate0.625mg/ml M- TC2-1A(+) 500 461 91.8 M- TC2-1B(+) 500 457 M- TC2-2A(+) 500 454 M- TC2-2B(+) 500 463 Denatonium benzoate 0.313mg/ml M- TC3-1A(+) 500 469 94.4 M- TC3-1B(+) 500 477 M- TC3-2A(+) 500 479 M- TC3-2B(+) 500 462 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; -1,-2- Culture replicates; A,B- replicate slides   Extended treatment (WithoutS9) Treatment Slide code Total No. of cells Total No. of mitotic cells Mean mitotic index (%) Sterilized water (Negative control) M-NC-1A(E) 500 473 94.9 M-NC-1B(E) 500 480 M-NC-2A(E) 500 475 M-NC-2B(E) 500 469 Mitomycin C 0.5µg/ml (Positive control) M-PC-1A(E) 500 206 41.0 M-PC-1B(E) 500 228 M-PC-2A(E) 500 198 M-PC-2B(E) 500 187 Denatonium benzoate1.25mg/ml M-TC1-1A(E) 500 304 60.9 M- TC1-1B(E) 500 311 M- TC1-2A(E) 500 297 M- TC1-2B(E) 500 306 Denatonium benzoate0.625mg/ml M- TC2-1A(E) 500 437 87.5 M- TC2-1B(E) 500 433 M- TC2-2A(E) 500 448 M- TC2-2B(E) 500 432 Denatonium benzoate 0.313mg/ml M- TC3-1A(E) 500 446 89.8 M- TC3-1B(E) 500 452 M- TC3-2A(E) 500 441 M- TC3-2B(E) 500 456 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; -1,-2- Culture replicates; A,B- replic ABERRATION SCORING-SHORT TERM TREATMENT WITHOUT S9 - INDIVIDUAL Treatment Slide code No. of mitotic cells No. of aberration Gaps Chromatid type Chromosome type Others (ring chromosome structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) M-NC-1A(-) 300 1 0 0 0 0 0 M-NC-1B(-) 300 0 0 0 0 0 0 M-NC-2A(-) 300 0 0 1 0 0 0 M-NC-2B(-) 300 1 0 0 0 0 0 Positive control (Mitomycin C 0.5µg/ml) M-PC-1A(-) 300 5 3 2 1 6 0 M-PC-1B(-) 300 2 9 7 6 8 0 M-PC-2A(-) 300 4 7 6 8 2 2 M-PC-2B(-) 300 3 9 5 9 6 1 Denatonium benzoate 1.25mg/ml M-TC1-1A(-) 300 1 2 1 0 1 0 M-TC1-1B(-) 300 0 1 1 0 2 0 M-TC1-2A(-) 300 0 0 1 0 1 0 M-TC1-2B(-) 300 1 0 0 0 1 0 Denatonium benzoate 0.625mg/ml M-TC2-1A(-) 300 0 0 0 1 1 0 M-TC2-1B(-) 300 1 1 0 0 1 0 M-TC2-2A(-) 300 0 1 1 0 1 0 M-TC2-2B(-) 300 0 0 1 0 0 1 Denatonium benzoate 0.313mg/ml M-TC3-1A(-) 300 0 1 0 0 0 1 M-TC3-1B(-) 300 1 0 1 0 0 0 M-TC3-2A(-) 300 0 1 0 0 0 0 M-TC3-2B(-) 300 1 1 0 0 0 0 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; 1A/B- 1^stculture replicate A/B; 2A/B‑ 2^nd culture replicate A/B; ‘-’- without S9.   ABERRATION SCORING-SHORT TERM TREATMENT WITHOUT S9- SUMMARY Treatment Total No. of aberration Total No. of aberrations per cultures without gaps Aberration without gaps (Mean ± SD) Aberration frequency without gaps % of cells with aberrations (Mean ± SD) Gaps Chromatid type Chromosome type Others (ring structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) 2 0 1 0 0 0 1 0.250 ± 0.500 0.001 0.250 ± 0.167 Positive control (Mitomycin C 0.5µg/ml) 14 28 20 24 22 3 97 24.250 ± 8.500*** 0.081 9.250 ± 2.455 Denatonium benzoate 1.25mg/ml 2 3 3 0 5 0 11 2.750 ± 1.500 0.009 1.083 ± 0.500 Denatonium benzoate 0.625mg/ml 1 2 2 1 3 1 9 2.250 ± 0.500 0.008 0.833 ± 0.192 Denatonium benzoate 0.313mg/ml 2 3 1 0 0 1 5 1.250 ± 0.500 0.004 0.583 ± 0.167 SD- Standard Deviation;***Significant criteria is fixed as P≤0.001 ABERRATION SCORING- SHORT TERM TREATMENTWITHS9- INDIVIDUAL Treatment Slide code No. of mitotic cells No. of aberration Gaps Chromatid type Chromosome type Others (ring chromosome structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) M-NC-1A(+) 300 0 0 0 1 0 0 M-NC-1B(+) 300 0 1 0 0 0 0 M-NC-2A(+) 300 0 0 0 0 1 0 M-NC-2B(+) 300 1 0 1 0 0 0 Positive control (Cyclophosphamide 3µg/ml) M-PC-1A(+) 300 3 1 4 2 1 0 M-PC-1B(+) 300 2 1 9 5 7 0 M-PC-2A(+) 300 2 6 3 9 5 7 M-PC-2B(+) 300 8 11 4 10 14 1 Denatonium benzoate 1.25mg/ml M-TC1-1A(+) 300 1 1 1 0 1 0 M- TC1-1B(+) 300 0 0 1 0 1 0 M- TC1-2A(+) 300 1 1 0 0 0 0 M- TC1-2B(+) 300 0 1 1 1 0 0 Denatonium benzoate 0.625mg/ml M- TC2-1A(+) 300 1 0 1 1 1 0 M- TC2-1B(+) 300 1 0 0 0 1 0 M- TC2-2A(+) 300 0 0 0 1 1 0 M- TC2-2B(+) 300 0 0 1 0 0 1 Denatonium benzoate 0.313mg/ml M- TC3-1A(+) 300 0 0 1 0 0 0 M- TC3-1B(+) 300 0 1 0 1 0 0 M- TC3-2A(+) 300 1 0 0 0 1 0 M- TC3-2B(+) 300 0 0 0 0 0 0 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; 1A/B- 1^stculture replicate A/B; 2A/B‑ 2^nd culture replicate A/B; ‘+’- with S9. ABERRATION SCORING-SHORT TERM TREATMENTWITHS9-SUMMARY Treatment Total No. of aberration Total No. of aberrations per cultures without gaps Aberration without gaps (Mean ± SD) Aberration frequency without gaps % of cells with aberrations (Mean ± SD) Gaps Chromatid type Chromosome type Others (ring structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) 1 1 1 1 1 0 4 1.000 ± 0.000 0.003 0.417 ± 0.167 Positive control (Cyclophos-phamide 3µg/ml) 15 19 20 26 27 8 100 25.000 ± 13.515*** 0.083 9.583 ± 5.159 Denatonium benzoate 1.25mg/ml 2 3 3 1 2 0 9 2.250 ± 0.957 0.008 0.917 ± 0.319 Denatonium benzoate 0.625mg/ml 2 0 2 2 3 1 8 2.000 ± 0.816 0.007 0.883 ± 0.333 Denatonium benzoate 0.313mg/ml 1 1 1 1 1 0 4 1.000 ± 0.816 0.003 0.417 ± 0.319 SD- Standard Deviation;*, **, ***Significant criteria is fixed as P≤0.05,P ≤0.01 & P≤0.001 respectively ABERRATION SCORING- EXTENDED TREATMENT WITHOUTS9- INDIVIDUAL Treatment Slide code No. of mitotic cells No. of aberration Gaps Chromatid type Chromosome type Others (ring chromosome structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) M-NC-1A(E) 300 0 1 1 0 0 0 M-NC-1B(E) 300 0 0 1 0 0 1 M-NC-2A(E) 300 0 0 1 0 0 0 M-NC-2B(E) 300 1 0 0 1 0 0 Positive control (Mitomycin C 0.5µg/ml) M-PC-1A(E) 300 3 3 9 5 6 0 M-PC-1B(E) 300 5 8 9 7 5 0 M-PC-2A(E) 300 5 7 8 6 3 2 M-PC-2B(E) 300 3 9 5 9 6 1 Denatonium benzoate 1.25mg/ml M-TC1-1A(E) 300 1 2 1 0 1 0 M- TC1-1B(E) 300 0 1 1 0 2 0 M- TC1-2A(E) 300 0 0 1 0 1 0 M- TC1-2B(E) 300 1 0 0 0 1 0 Denatonium benzoate 0.625mg/ml M- TC2-1A(E) 300 0 0 0 1 1 0 M- TC2-1B(E) 300 1 1 0 0 1 0 M- TC2-2A(E) 300 0 1 1 0 1 0 M- TC2-2B(E) 300 0 0 1 0 0 1 Denatonium benzoate 0.313mg/ml M- TC3-1A(E) 300 0 1 0 0 0 1 M- TC3-1B(E) 300 1 0 1 0 0 0 M- TC3-2A(E) 300 0 1 0 1 0 0 M- TC3-2B(E) 300 1 1 0 0 0 0 M- Main study; NC- Negative control; PC- Positive control; TC- Test concentration; 1A/B- 1^stculture replicate A/B; 2A/B‑ 2^nd culture replicate A/B; E- Extended treatment without S9. ABERRATION SCORING-EXTENDEDTREATMENT WITHOUTS9- SUMMARY Treatment Total No. of aberration Total No. of aberrations per cultures Aberration without gaps (Mean ± SD) Aberration frequency without gaps % of cells with aberrations (Mean ± SD) Gaps Chromatid type Chromosome type Others (ring structure) Breaks Exchanges Breaks Exchanges Sterilized water (Negative control) 1 1 3 1 0 1 6 1.500 ± 0.577 0.005 0.500 ± 0.192 Positive control (Mitomycin C 0.5µg/ml) 16 27 31 27 20 3 108 27.000 ± 3.162*** 0.090 10.333 ± 1.186 Denatonium benzoate 1.25mg/ml 2 3 3 0 5 0 11 2.750 ± 1.500 0.009 1.083 ± 0.500 Denatonium benzoate 0.625mg/ml 1 2 2 1 3 1 9 2.250 ± 0.500 0.008 0.833 ± 0.192 Denatonium benzoate 0.313mg/ml 2 3 1 1 0 1 6 1.500 ± 0.577 0.005 0.583 ± 0.167 SD- Standard Deviation;*, **, ***Significant criteria is fixed as P = ≤0.05,P≤0.01 & P≤0.001 respectively HISTORICAL CONTROL DATA Control Type Aberration frequency +S9 (short term treatment) Negative control - DMSO 0.00-0.01 Positive control -Cyclophosphamide,12.5mg/ml 0.15-0.20 -S9 (short term treatment) Negative control - DMSO 0.00-0.01 Positive control -Mitomycin C, 5mg/ml 0.10-0.20 -S9 (extended treatment) Negative control - DMSO 0.00-0.02 Positive control -Mitomycin C, 5mg/ml 0.10-0.15 Note: Historical control data for negative control with water is not available. Positive control concentration used in the study differs from the historical control data. Hence, negative control and positive controls data cannot be directly compared with the historical control data. However, available historical control data for negative control and positive control is included.

Applicant's summary and conclusion

Conclusions:

Denatonium benzoate (CAS No. 3734-33-6) was considered non-cytotoxic and non clastogenic at 1.25mg/ml for in vitro chromosomal aberration test and has no clastogenicity effect on human lymphocyte cells under the experimental conditions.

Executive summary:

This study was performed to identify whether the test item, Denatonium benzoate (CAS No. 3734-33-6), causes any structural or numerical chromosomal aberrations (clastogenicity) in cultured human lymphocytes when exposed to both, with and without an exogenous metabolic activation using in vitro chromosomal aberration test in cultured human lymphocyte cell culture.The experiment was conducted following the OECD Guideline for the Testing of Chemicals 473.

Clastogenicity ofDenatonium benzoate (CAS No. 3734-33-6)was conducted in cultured human lymphocytes with negative control (0 mg/ml) and positive control.Five test item concentrations (5, 2.5, 1.25, 0.625 and 0.313 mg/ml)were selected for concentration range finding study to determine the cytotoxicity. Cytotoxicity was determined using mitotic index with a value of 54.0 at test concentration 1.25 mg/ml when compared withnegative control (0 mg/ml).Denatonium benzoate (CAS No. 3734-33-6) at5 mg/ml and 2.5 mg/ml was found to be cytotoxic with mean mitotic index value of 14.1 and 17.6 when compared withnegative control (0 mg/ml).Denatonium benzoate (CAS No. 3734‑33-6) at1.25, 0.625 and 0.313 mg/mlwere selected as the concentrations for the main study. Duplicate lymphocyte cultures were used for both, concentration range finding and main study. Sterilized water is used asnegative control, and positive controls such as Mitomycin C (0.5 µg/ml) for without S9 and Cyclophosphamide (3 µg/ml) for with S9 were used, respectively.

Human lymphocytes were cultured in RPMI-1640 media and exposed toDenatonium benzoate (CAS No. 3734-33-6)and reference items both, with and without S9. The experiment was designed to expose human lymphocyte cells to short term (with and without metabolic activation (S9)) and long term or extended (without metabolic activation) treatment. In short term treatment, human lymphocyte cells were treated in the presence and absence of S9 mix for 3 h. After 3 h, old medium was replenished with fresh medium and harvested for 48 h. In extended treatment, human lymphocyte cells were continuously exposed to theDenatonium benzoate (CAS No. 3734-33-6)and reference items for 48 h. At the end of cell cycle (45 h), Colchicine was added to arrest the cell division at metaphase. Cells were harvested and stained using 5% Giemsa stain and then analyzed microscopically for cytotoxicity and the presence of 300 metaphase cells to assess the clastogenicity (numerical and structural chromosomal aberrations).

In short term treatment without S9, the total number of chromosomal aberration was observed 1 in negative control; 97 in positive control (Mitomycin C - 0.5µg/ml); 11 inDenatonium benzoate (CAS No. 3734-33-6) at1.25mg/ml; 9 inDenatonium benzoate (CAS No. 3734-33-6) at0.625mg/ml; and 5 inDenatonium benzoate (CAS No. 3734‑33‑6) at0.313mg/ml. No statistically significant difference total number of chromosomal aberration was observed in test concentrations at 1.25, 0.625, 0.313 mg/mlwhen compared withnegative control (0 mg/ml). The percentage of chromosomal aberration was found high in Mitomycin C (0.5µg/ml) 9.250% when compared with negative control(0 mg/ml).

In short term treatment with S9, the total number of chromosomal aberration was observed 4 in negative control; 100 in positive control (Cyclophosphamide - 3µg/ml); 9 inDenatonium benzoate (CAS No. 3734-33-6) at1.25mg/ml; 8 inDenatonium benzoate (CAS No. 3734-33-6) at0.625mg/ml; and 4 inDenatonium benzoate (CAS No. 3734-33-6) at0.313mg/ml. No statistically significant difference in total number of chromosomal aberration was observed in test concentrations at 1.25, 0.625, 0.313 mg/mlwhen compared withnegative control (0 mg/ml). The percentage of aberration was found high in Cyclophosphamide (3µg/ml) 9.583% when compared withnegative control (0 mg/ml).

In extended treatment without S9, the total number of chromosomal aberration was observed 6 in negative control (sterilized water); 108 in positive control (Mitomycin C - 0.5µg/ml); 11 inDenatonium benzoate (CAS No. 3734-33-6) at1.25mg/ml; 9 inDenatonium benzoate (CAS No. 3734-33-6)at 0.625mg/ml; and 6 inDenatonium benzoate (CAS No. 3734-33-6)at 0.313mg/ml. No statistically significant difference in total number of chromosomal aberration was observed in test concentrations at 1.25, 0.625, 0.313 mg/mlwhen compared withnegative control (0 mg/ml). The percentage of aberration was found to be high in Mitomycin C (0.5µg/ml) 9.000% when compared with negative control(0 mg/ml).

Ring aberration observed in test and control groups is insignificant and common. However, the ring aberration in test or treatment group is not because of the test item. Positive control results obtained was as expected and fell within the range, confirming the sensitivity of the test system, the effectiveness of the S9 mix, and the validity of the assay.

From the above results, concentration dependent increase in chromosomal aberration frequency was observed in all the three (1.25, 0.625, 0.313 mg/ml) concentrations of Denatonium benzoate (CAS No. 3734-33-6). However, there was no statistical significant difference in the chromosomal aberration frequency was observed between the negative control (0 mg/ml) and all three concentrations of Denatonium benzoate (CAS No. 3734‑33-6) at 1.25, 0.625, 0.313 mg/ml. Based on the observations, it is concluded that Denatonium benzoate (CAS No. 3734-33-6) was considered to be non-clastogenic at 1.25 mg/ml.