5-amino-o-cresol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Study period:

From Nov. 07, 2005 to Nov. 17, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed basic scientific principle, not in compliance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The in vitro comet assay was performed to determine the potential of test material to induce DNA damage in cultured V79 Chinese hamster lung cells. The detection of a significantly elevated level of DNA damage was considered as an indicator for genotoxic effects. DNA damage (indicated by DNA breaks) in single cell by the test material was evaluated from the migration pattern of DNA fragments from the nucleus toward the positive electrode in an agarose gel during electrophoresis. The migrating DNA fragments appear in the form of a comet with head and tail. The percentage of DNA fragments in the tail (% tail DNA) was assessed for genotoxicity potential of the test material. The DNA damage observed in the treated cells was compared with the untreated control cells.

GLP compliance:

no

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

5307-07-3

Cas Number:

5307-07-3

IUPAC Name:

5307-07-3

Constituent 2

Reference substance name:

Acetylpurpur

IUPAC Name:

Acetylpurpur

Constituent 3

Reference substance name:

4-acetylamino-2-hydroxytoluene

IUPAC Name:

4-acetylamino-2-hydroxytoluene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 4-acetylamino-2-hydroxytoluene; Acetylpurpur (Code # A007329)
- TSIN: 803389
- Substance type: Pure active substance
- Physical state: White powder
- Stability under test conditions: The substance is considered to be stable for more than 3 years if stored dry and in darkness.
- Storage condition of test material: Moisture protected, light protected
- Solubility: Solubility in different solvent is as follows:
0.06 weight% in water (pH 7.2)
1.3 weight% in acetone/water 1:1 (pH 7.4)
> 10 weight% in DMSO

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source: Dr. S. Brendler-Schwaab, Bayer AG, D-42096, Wuppertal
- Type and identity of culture media: Earle's Minimal Essential Medium containing 10 % v/v FCS and 100 Units /100 µg/mL PenStrep
- Properly maintained: Yes, large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank of Cosmital, allowing the repeated use ofthe same cell culture batch in experiments.
- Periodically checked for Mycoplasma contamination: Yes, the cells were regularly checked for the absence of mycoplasma contamination.
- Propagation of cells: Thawed stock cultures were propagated at 37°C in 75 cm2 plastic flasks. The cells were cultivated in culture medium at 37°C in a humidified atmosphere containing 5% CO2.

Additional strain / cell type characteristics:

other: Cell lines have a modal chromosome number of 22 and a population doubling time of 10-14 hours.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 413, 826 and 1652 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.
- Concentration of vehicle in the culture medium: 1% v/v

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Remarks:

(with metabolic activation)

Positive control substance:

benzo(a)pyrene

Controls 2

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium (Earle’s Minimal Essential Medium containing 2 % v/v FCS and 100 Units /100 µg/mL PenStrep)

DETAILS OF PROCEDURE AND DURATION
- Seeding: Cells were seeded 18 hours before the experiment (4 x 10(5) cells/mL) in 6-well-plates.
- Exposure duration: Immediately before the experiment, cell monolayers were washed with PBS and incubated with the test material dissolved in DMSO. Before incubating the cells with the test material, 200 µL of media were pipetted off and replaced by 200 µL of S9-mix. The cells were exposed to test material/positive control/vehicle control for 3 hours in the absence and in the presence of metabolic activation.
- Cell harvest: Incubation was followed by removal of the treatment medium, rinsing of the cells 1x with PBS and harvesting of cells using a Trypsin/EDTA solution. The effect of Trypsin was stopped by addition of culture medium and cells were separated by repeated pipetting. An aliquot of the cell suspension was diluted 10-fold with pre-warmed (37˚C) LMA (low melting agarose) solution (0.7 % w/v).
- Preparation of slides/Lysis for Comet Assay: 75 µL of the LMA-cell suspension was dropped on frosted slides pre-coated with NMA (normal melting agarose) and covered with a cover slip. Each slide was transferred to a cooled plate for solidification and the slide was placed in freshly prepared lysis solution for at least 1 hour at 4 °C in the dark.
- Electrophoresis: Before electrophoresis, a washing step was performed by equilibrating the slides for 20 min with electrophoresis buffer. The electrophoresis was performed for 30 min at 25 V, corresponding to 1.1 V/cm, at 300 mA. Electrophoresis was followed by 20 min of neutralisation in the neutralization buffer (TRIS-Base (48.5 g/L), dissolved in water, pH adjusted to 7.5 with Hcl).
- Staining: The DNA of the cells was stained with the fluorescence dye SYBR Gold (0.1 µg/mL SYBR Gold in 50 mM TRIS/HCI-Buffer (pH 7.4)) for 15 min immediately before evaluation of the slides.
All steps were performed under avoidance of direct light.

NUMBER OF REPLICATIONS PER CONCENTRATION: 2

NUMBER OF CELLS EVALUATED: 100 cells/concentration level

EVALUATION OF COMETS:
Evaluation of comets was performed with an Olympus fluorescence microscope with a 20x objective (200 fold magnification) and suitable fluorescence filters, using image analysis software (Komet 5, Kinetic Imaging, a subsidiary of Andor Technology, Belfast BT12 7AL, UK). % Tail DNA was used as assessment parameter. Tail length and olive tail moment were also recorded.
% Tail DNA = 100 – [Head optical intensity /(Head Optical Intensity + Tail Optical Intensity)] x 100

DETERMINATION OF CYTOTOXICITY
- Method: Relative cell density and cell viability
After removal of an aliquot for slide preparation, 3 mL of PBS were added to the remaining cell suspension in the wells of the culture plate and thoroughly mixed before an aliquot of the cell suspension was mixed with the same volume of trypan blue. Relative cell density and cell viability were determined using a Neubauer chamber and calculated with the following formulae:
Relative cell density = [Number of viable cells/number of viable cells in the vehicle control] x 100
Cell viability: [Number of viable cells/(number of viable cells + number of dead cells)] x 100

Evaluation criteria:

- A test material was considered positive in this assay if it induces either a substantial increase (doubling of the % Tail DNA if compared to the control) and/or a dose-related increase in % Tail DNA.
- If, in case of an increase, the historical negative control range (3.93 - 6.57 %, mean value 4.8 % ±0.7 % SD in the absence of metabolic activation; 4.50 - 5.10 %, mean value 4.8 % ± 0.2 % SD in the presence of metabolic activation) is not exceeded at, at least one of the concentrations, the effect was not regarded as biologically relevant and needs to be checked for reproducibility in a second experiment.
- A test material was considered to be negative in the Comet assay if there was no biologically relevant or dose-related increase in the mean of % tail DNA.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(slight cytotoxicity was observed)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed by the unaided eye, neither at the beginning nor at the end of the incubation period.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, negative control values were compared to the historical control data (% Tail DNA (i) in absence of metabolic activation: 3.93 – 6.57%, mean value 4.8±0.7 %; (ii) in presence of metabolic activation: 4.50 - 5.10 %, mean value 4.8 ± 0.2%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell viability in the highest concentration of the test material was decreased to 93.7 % and 92.3 % in the absence and in the presence of metabolic activation, respectively.
- The relative cell density at the highest concentration of the test item was decreased to 87.6 % and 89.8 % in the absence and in the presence of metabolic activation, respectively.
For details refer to ‘Table 1’ under ‘Any other information on results incl. tables’ section.

RESULTS OF DNA MIGRATION:
None of the tested concentrations of test material did induce a biologically relevant or distinct dose-related increase of % Tail DNA. The mean values were well within the historical range of testing laboratory.

RESULTS OF CONTROL:
Mean % tail DNA values of the vehicle control cells were within the normal range of testing laboratory. The positive controls induced a substantial increase in % tail DNA values, both in the absence and in the presence of metabolic activation.
Therefore, the assay was regarded as valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Cytotoxicity results (study # 69269)

Concentration (µg/mL)	Absence of metabolic activation		Presence of metabolic activation
	Viability	Relative cell viability	Viability	Relative cell viability
0	95.4	100.0	94.0	100.0
413	93.0	100.0	93.2	95.7
826	92.7	88.2	94.0	91.4
1652	93.7	87.6	92.3	89.8
Positive control	93.9	82.3	90.2	79.0

Table 2: Summary of results of in vitroComet assay with 4-acetylamino-2-hydroxytoluene (study # 69269)

Concentration (µg/mL)	% Tail DNA ± SD
	Absence of metabolic activation	Presence of metabolic activation
0	4.50 ± 0.66	4.50 ± 0.45
413	5.13 ± 1.17	4.81 ± 0.40
826	3.94 ± 0.23	4.88 ± 0.85
1652	5.87 ± 0.54	4.65 ± 0.46
Positive control	14.79 ± 0.23	14.79 ± 0.23

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative (with and without metabolic activation)

Under the test conditions reported, 4-acetylamino-2-hydroxytoluene did not induce biologically relevant DNA damage in Chinese hamster V79 cells in vitro, as measured by the comet assay.

Executive summary:

4-Acetylamino-2-hydroxytoluene was investigated for the induction of DNA strand breaks in Chinese hamster V79 cell in the in vitro alkaline single cell gel electrophoresis assay.

The cells were exposed to the following test concentrations for 3 hours in the absence and in the presence of metabolic activation:

0 (DMSO, solvent), 413, 826 and 1652 µg/mL

The highest concentration applied in this experiment was 1652 µg/mL, corresponding to approximately 10 mM, which is the maximum recommended concentration.

Benzo(a)pyrene and methylmethanesulfonate served as positive controls for the experiments with and without metabolic activation respectively. The cytotoxicity of test substance was determined by measuringrelative cell density and cell viability. The genotoxicity potential of test substance was determined by detection of a significantly elevated level of DNA damage indicated by DNA strand breaks.DNA migrated out (appearing as a comet with head and tail) from the nucleus in single cell gel electrophoresis was used as a measure of DNA strand breakage.The percentage of DNA fragments in the tail (% tail DNA) was assessed for genotoxicity potential of the test material.

% Tail DNA = 100 – [Head optical intensity /(Head Optical Intensity + Tail Optical Intensity)] x 100

The test material showed a slight cytotoxic effect, reducing the cell viability at the highest concentration of the test material to 93.7 % and 92.3 % in the absence and in the presence of metabolic activation, respectively. The relative cell density at the highest concentration of the test material was decreased to 87.6 % and 89.8 % in the absence and in the presence of metabolic activation, respectively.

None of the tested concentrations of 4-Acetylamino-2-hydroxytoluene induced a biologically relevant or distinct dose related increase in DNA damage of the cells in the presence and absence of metabolic activation.Furthermore, the mean % tail DNA values of the test material were well within the historical range of negative control.

Appropriate reference mutagens used as positive controls led to a distinct increase of DNA damage, indicating that the experiments were sensitive and valid.

Based on above, 4-acetylamino-2-hydroxytoluene did not induce biologically relevant DNA damage in Chinese hamster V79 cells in vitro, as measured by the comet assay.