2,4-dichloro-3,5-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:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: U.S. FDA Good Laboratory Practice regulations (21CFR58) , the U.S. EPA GLPs (40CFR792 and 40CFR160) , and the OECD guidelines

Deviations:

no

GLP compliance:

yes

Type of assay:

other: chromosome aberrations in CHO cells

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-3,5-xylenol

EC Number:

201-793-8

EC Name:

4-chloro-3,5-xylenol

Cas Number:

88-04-0

Molecular formula:

C8H9ClO

IUPAC Name:

Phenol, 4-chloro-3,5-dimethyl-

Test material form:

solid: crystalline

Details on test material:

Sponsor's identification
Description
Batch number
Date received
Storage conditions
PCMX
cream coloured, crystalline solid
285/13847
27 September 2001
room temperature, in the dark

Specific details on test material used for the study:

The test article, Ottasept chloroxylenol, was received by Microbiological Associates, Inc. on 11—30—87 and was assigned the code number T5800. The test article was characterized by the Sponsor as a white crystalline solid which should be stored in a cool, dry place with an expiration date of 5 years.

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Mammalian Cells: Chinese hamster ovary (CHO—KI) cells (repository number CCL 61, American Type Culture Collection, Rockville, MD)

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

not applicable

Metabolic activation system:

n/a

Test concentrations with justification for top dose:

The S—9 was prepared according to established procedures . Adult male Sprague—Daw1ey rats, 200—300 gm, were induced by a single intraperitoneal injection of Aroclor 1254 at a dose of 500 mg/kg body weight five days prior to sacrifice. The animals were sacrificed and the livers aseptically removed. The excised tissue was rinsed three times in cold sterile O. 15 M KCI and then homogenized in a Polytron Tissuemizer at a concentration of 1:3 (w/ v) in O . 15 M KCI. The supernatant fraction (S—9) was collected following centrifugation at 9000 x g for 10 minutes at O c portioned into aliquots for daily use, and stored frozen at s—70 0 c until used. Each bulk preparation of S—9 was assayed for its ability to metabolize 2—aminoanthracene and 7, 12 dimethyl benz (a) — anthracene to forms mutagenic to S. typhimurium TAIOO

Vehicle / solvent:

Irmediately prior to use, the S—9 was mixed with the cofactor pool to contain 15 ul S—9, 1.4 mg NADP and 2.7 mg isocitric acid per ml growth medium with 2 . serum.
The toxicity test was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of test article effect on mitotic indices and cell cycle delay.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: The solvent vehicle for the test article was used as the solvent control at the same concentration as that found in the test article—treated groups

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Remarks:

Growth medium was used in the untreated control.

Details on test system and experimental conditions:

The toxicity test was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of test article effect on mitotic indices and cell cycle delay. CHO cells were seeded for each treatment condition at approximately 5 x 10 5 cells/ 25 cm 2 flask and were incubated at 37+1 O c in a humidified atmosphere of C02 in air for 16—24 hours. Treatment was carried out by refeeding the flasks with 5 ml complete medium for the non—activation study or with 5 ml S—9 reaction mixture for the activation study to which was added 50 ul dosing solution of test article in solvent or solvent alone. The cells were treated for six hours in the non—activated system; two hours after initiation of treatment, 50 ul of 1mM BrdU was added to each flask and incubation continued as required. In the activation system, the cells were treated for two hours after which the treatment medium was removed, the cells washed with PBS , refed with 5 ml complete medium containing 0.01 mM BrdU and returned to the incubator for a total of 24.5 hours from BrdU treatment. Extension of the incubation time by O .5 hours was documented in the raw data with a protocol deviation report. Two hours prior to harvest by trypsinization, Colcemid was added to each flask at a final concentration of O .1 ug/ml. Metaphase preparations were prepared and stained for sister chromatid differentiation using a modified fluorescence plus giemsa technique (Perry and Wolf, 1974) . Slides were evaluated for the percentage of first, second and third—division metaphase cells for estimation of the test article effect on cell cycle kinetics. The mitotic index was determined for each treatment condition as the percentage of mitotic cells in a population of 500 cells scored.

Rationale for test conditions:

For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 10 5 cells/ 25 cm 2 flask and were incubated at 37+1 O c in a humidified atmosphere of 5+1% C02 in air for 16—24 hours. Treatment was carried out by refeeding duplicate flasks with 5 ml complete medium for the non—activation study or with 5 ml S—9 reaction mixture for the activated study to which was added 50 ul of dosing solution of test or control article in solvent or solvent alone. An untreated control consisting of cells in complete medium was also included.

Evaluation criteria:

The toxic effects of treatment are expressed as mitotic inhibition relative to the solvent—treated control and are presented for the toxicity and aberration study. The number and types of aberrations found are presented for each treatment group . The percentage of structurally damaged cells (percent aberrant cells) in the total population of cells examined was calculated for each group. The frequency of structural aberrations per cell (mean aberrations per cell) also was calculated and reported for each group. Chromatid and chromosorne gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell .

Statistics:

Statistical analysis of the frequency of structural aberrations per cell was performed using the Student's t test. The t test was used to compare pairwise the number of aberrations per cell of each treatment group with that of the solvent control. Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The positive and negative controls fulfilled the requirements for a valid test.
statistically significant increases in mean chromosome aberrations per cell were observed at 75 and 150 ug/ml in both the nonactivated and S—9 activated test systems . Statistically significant increases in percent aberrant cells were observed at 75 ug/ml in the nonactivated test system and at 75 and 150 ug/ml in the S—9 activated test system. No dose response was observed in either the nonactivated or S —9 activated test system. Under the conditions of the assay described in this report Ottasept chloroxylenol was concluded to be equivocal in the nonactivated test system and suspect in the S—9 activated CHO cytogenetics test system.

Executive summary:

The positive and negative controls fulfilled the requirements for a valid test.

statistically significant increases in mean chromosome aberrations per cell were observed at 75 and 150 ug/ml in both the nonactivated and S—9 activated test systems . Statistically significant increases in percent aberrant cells were observed at 75 ug/ml in the nonactivated test system and at 75 and 150 ug/ml in the S—9 activated test system. No dose response was observed in either the nonactivated or S —9 activated test system. Under the conditions of the assay described in this report Ottasept chloroxylenol was concluded to be equivocal in the nonactivated test system and suspect in the S—9 activated CHO cytogenetics test system.