Reaction mass of m-cresol and 2-chloro-m-cresol and 6-chloro-m-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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP compliant study which followed OECD Guideline 482 without deviations, tested with the source substance CAS 59-50-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Chlorocresol

EC Number:

200-431-6

EC Name:

Chlorocresol

Cas Number:

59-50-7

Molecular formula:

C7H7ClO

IUPAC Name:

4-chloro-3-methylphenol

Details on test material:

- Name of test material (as cited in study report): Preventol CMK
- Physical state: White crystals
- Analytical purity: 99.9%
- Lot/batch No.: 792/1987
- Stability under test conditions: Not reported

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

hepatocytes: Rat primary hepatocytes from an adult male F344 rat (150-300 g bw). A single animal was used for each trial.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with

Metabolic activation system:

intrinsic

Test concentrations with justification for top dose:

Trial 02: 0.25, 0.5, 2.5, 7.5, 10, 20 µg/mL
Trial 03: 2.53, 5.06, 7.58, 10.1, 15.2, 20.2, 30.3 µg/mL
Trial 01 was discarded due to excessive cytotoxicity.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance was not soluble in medium and was dissolved in DMSO at a concentration of 502 mg/mL forming a clear, colourless solution. With DMSO as vehicle, the test substance was soluble in the medium at concentrations up to 2010 µg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Migrated to IUCLID6: 0.1µg/mL (vehicle DMSO)

Details on test system and experimental conditions:

Freshly prepared rat hepatocytes were exposed to 6 (trial 02) and 7 (trial 03) concentrations of test substance ranging from 0.25 µg/mL to 30.3 µg/mL in the presence of 5 µCi/mL ³H-thymidine for 18-19 hours in medium with reduced serum content (1% FCS).
The cells were fixed, washed and air-dried prior to coating in the dark with photographic emulsion. The coated slides were stored at 4°C for 4-10 days and then developed. The slides were then rinsed and stained with haematoxylin/eosin.
Grain counting was done manually using a microscope with 1500x magnification and oil immersion. Each slide was examined for 3H-incorporation (silver grain formation) by counting 50 cells per slide, normally with 3 slides per dose group. The cytotoxicity was assessed using (trypan blue exclusion). Trial 01 was discarded due to excessive cytotoxicity.

Evaluation criteria:

The assay was deemed acceptable if the following criteria were met:
1. The viability of the hepatocytes must exceed 50%.
2. The viability of the monolayer cell cultures used for the UDS assay must be 70% or greater.
3. The number of viable cells in the vehicle control cultures should remain reasonably stable throughout the experiment; the number of viable cells in the vehicle control cultures must be ≥ 50% at the beginning of the treatment period.
4. For each of the 50 cells on each slide, the number of nuclear grains is scored, as well as numbers of three cytoplasmic grain counts from nuclear-sized areas adjacent to each nucleus.
5. The average net nuclear grain counts (NG) in the negative control cultures should range between -5 to +1. No more than 10% of the cells should contain ≥ 6 grains, or 1% of the cells ≥ 20 grains.
6. For the positive control 2-AAF (0.1 µg/mL), one might expect mean values of 13 NG with 83% and 21% of the nuclei having more than 6 and 20 grains, respectively.
7. For the conditions described, if a chemical yields ≥ 6 NG in the population average or ≥ 6 NG in ≥ 10% of the cells or ≥ 20 NG in ≥ 2% of the cells, the response is considered positive.
8. Grain count data obtained for a given treatment are acceptable as part of the evaluation if obtained from at least two replicate cultures and at least fifty cells per culture.
9. A minimum of 6 dose levels will be analysed for NG. Repeat trials need only augment the number of analysed dose levels in the first trial to achieve a total of 6 different dose levels.
10. The highest analysed dose must give rise to cytotoxicity or result in insolubility of the test material or reach a maximum concentration of 5 mg/mL.

Results and discussion

Test results

Species / strain:

hepatocytes: Rat primary hepatocytes from an adult male F344 rat (150-300 g bw). A single animal was used for each trial.

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The survival rate of hepatocytes was 43% at 20 µg/mL in trial 02.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

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: Results of trial 2 of the UDS assay in primary rat hepatocytes*

Concentration [µg/mL]	Net grains^a per nucleus	Avg % nuclei with ≥ 6 grains	Avg % nuclei with ≥ 20 grains	Survival^dat 21.5 h [%]
Vehicle control	-2.63	0.7	0.0	100.0
0.250	-2.69	3.3	0.0	n.d.
0.500	-2.05	2.7	0.0	100.3
2.50	-1.40	2.0	0.0	95.2
7.50	-1.32	1.4b	0.0b	77.9
10.0	-1.83	0.7	0.0	67.0
20.0	-1.62b	0.7b	0.0b	42.8
Pos. control	21.77b	98.6b	57.3b	99.8
*1^stassay was discarded due to excessive cytotoxicity^aAverage of net nuclear grain counts on triplicate coverslips (150 total cells) ^bAverage of net nuclear grain counts on duplicate coverslips (150 total cells for UDS) ^cMeans of percentages of cells with 5 or more net nuclear grains on triplicate coverslips ^dNumber of viable cells relative to vehicle controls n.d.: not determined

Table 2: Results of trial 3 of the UDS assay in primary rat hepatocytes

Concentration [µg/mL]	Net grains^a per nucleus	Avg % nuclei with ≥ 6 grains	Avg % nuclei with ≥ 20 grains	Survival^dat 20 h [%]
Vehicle control	-0.03	6.0	0.0	100.0
2.53	-0.43	3.3	0.0	100.6
5.06	-0.31	0.7	0.0	98.8
7.58	-0.62	0.7	0.0	98.2
10.1	-0.21	2.0	0.0	99.1
15.2	-0.62^b	0.0b	0.0b	98.7
20.2	0.14	0.7	0.0	87.7
30.3	n.d.	n.d.	n.d.	53.0
Pos. control	21.69	99.3	52.0	99.4
^aAverage of net nuclear grain counts on triplicate coverslips (150 total cells) ^bAverage of net nuclear grain counts on duplicate coverslips (100 total cells for UDS) ^cMeans of percentages of cells with 5 or more net nuclear grains on triplicate coverslips ^dNumber of viable cells relative to vehicle controls n.d.: not determined

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative