2,5-xylenol

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

Study period:

18 May 2004 to 20 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,3-xylenol

EC Number:

208-395-3

EC Name:

2,3-xylenol

Cas Number:

526-75-0

Molecular formula:

C8H10O

IUPAC Name:

2,3-dimethylphenol

Constituent 2

Reference substance name:

2,4-xylenol

EC Number:

203-321-6

EC Name:

2,4-xylenol

Cas Number:

105-67-9

Molecular formula:

C8H10O

IUPAC Name:

2,4-dimethylphenol

Constituent 3

Reference substance name:

2,5-xylenol

EC Number:

202-461-5

EC Name:

2,5-xylenol

Cas Number:

95-87-4

Molecular formula:

C8H10O

IUPAC Name:

2,5-dimethylphenol

Constituent 4

Reference substance name:

2,6-xylenol

EC Number:

209-400-1

EC Name:

2,6-xylenol

Cas Number:

576-26-1

Molecular formula:

C8H10O

IUPAC Name:

2,6-dimethylphenol

Constituent 5

Reference substance name:

3,4-xylenol

EC Number:

202-439-5

EC Name:

3,4-xylenol

Cas Number:

95-65-8

Molecular formula:

C8H10O

IUPAC Name:

3,4-dimethylphenol

Constituent 6

Reference substance name:

3,5-xylenol

EC Number:

203-606-5

EC Name:

3,5-xylenol

Cas Number:

108-68-9

Molecular formula:

C8H10O

IUPAC Name:

3,5-dimethylphenol

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, working cell stocks were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: not stated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S9

Test concentrations with justification for top dose:

In the absence of S9 activation: 37.5, 75, 150, 300, 600, 800, 1000 and 1200 µg/mL (4 hour treatment) and 12.5, 25, 50, 100, 200, 300, 400, 500 and 600 µg/mL (20 hour treatment).
In the presence of S9 activation: 37.5, 75, 150, 300, 600, 800, 1000 and 1200 µg/mL (4 hour treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selected following a solubility test using water and DMSO to determine the highest soluble stock concentration up to 500 mg/mL.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

10 and 20 µg/mL in water for the treatments without S9 activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

1 and 2 mg/mL in water for the treatments with S9 activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (2 hours prior to the scheduled harvest)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): a minimum of 200 metaphase spreads per treatment

DETERMINATION OF CYTOTOXICITY
- Method: 4 h treatment: viability, cell counts, 20 h: mitotic index; cloning efficiency; relative total growth; other:

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Positive for the induction of structural and numerical chromosome aberrations.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 h / -S9: 47% at 550 µg/mL (the highest dose evaluated for aberrations); mitotic index: 53% reduction at 550 µg/mL relative to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

A repeat CHO assay was performed due to lack of sufficient scorable cells in the highest doses and a lack of at least 50% reduction in mitotic index at the lower doses in the first assay.

The toxicity of mixed xylenols to CHO cells treated for 4 hours in the absence of S9 activation was 47% at 550 µg/mL (the highest dose evaluated for aberrations). The mitotic index showed a 53% reduction at 550 µg/mL relative to the solvent control. The % of cells with structural aberrations was significantly increased (p<0.05, Fisher's exact test) at 300 and 500 µg/mL. The % of structurally damaged cells in the MMC positive control group was statistically significant at 22%.

Treatment in the presence of S9 activation showed 54% toxicity at 550 µg/mL. The mitotic index at 550 µg/mL showed a 63% reduction relative to the solvent control.

The % of cells with structural aberrations was significantly increased (p<0.05, p<0.01 Fisher's exact test) at 400 and 500 µg/mL, respectively. The Cochran-Armitage test was also positive for a dose response (p<0.05). The % of structurally damaged cells in the CP positive control group was statistically significant at 17.5%.

Table 1: Summary of results from the repeat chromosome aberration assay with mixed xylenols

Treatment (µg/mL)	Treatment time (h)	Mean mitotic index	Cells scored		Aberrations per cell (mean ± SD)	Cells with aberrations
			Numerical	Structural		Numerical	Structural
Without S9 activation
DMSO	4	11.2	200	200	0.000 ± 0.000	3.0	0.0
Mixed xylenols
300	4	12.6	200	200	0.035 ± 0.210	11.0**	3.0*
400	4	11.2	200	200	0.045 ± 0.322	10.0**	2.0
550	4	5.3	200	200	0.040 ± 0.262	7.5*	3.0*
MMC, 0.2	4	4.7	200	200	0.300 ± 0.704	3.0	22.0**
With S9 activation
DMSO	4	13.8	200	200	0.015 ± 0.158	7.0	1.0
Mixed xylenols
300	4	14.0	200	200	0.015 ± 0.122	8.5	1.5
400	4	12.6	200	200	0.070 ± 0.395	8.5	4.0*
550	4	5.1	200	200	0.185 ± 0.635	6.0	11.5**
CP, 10	4	6.1	200	200	0.255 ± 0.680	5.0	17.5**

SD = standard deviation
Cells were harvested 20 hours after the initiation of the treatments.
Severely damaged cells were counted as 10 aberrations.
* = Significant at p< 0.05 using Fisher’s exact test.
** = Significant at p< 0.01 using Fisher’s exact test.

Applicant's summary and conclusion

Conclusions:

The results of the assay indicate that under the conditions of the study, mixed xylenols caused a positive response in chromosome aberrations in both the presence and absence of metabolic activation.