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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent

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.
Controlsopen allclose all
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
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.