4-chloro-3,5-xylenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

genetic toxicity in vitro, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

88-04-0

Test material form:

solid: crystalline

Details on test material:

4-chloro-3,5-xylenol

Specific details on test material used for the study:

PCMX
cream coloured, crystalline solid
285/13847
27 September 2001
room temperature, in the dark

Method

Target gene:

Duplicate cultures of human lymphocytes

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

N/A

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated
for chromosome aberrations at up to three dose levels, together with vehicle and positive controls.
Four treatment conditions were used for the study, ie. in Experiment 1, 5 hours in the presence of
an induced rat liver homogenate metabolising system (S9), at a 1 % final concentration with cell
harvest after a 19-hour expression period and a 5-hour exposure in the absence of metabolic
activation (S9) with a 19-hour expression period. In Experiment 2, the 4-hour exposure with
addition of S9 was repeated (using a 2% final S9 concentration), whilst in the absence of
metabolic activation the exposure time was increased to 24 hours.

Vehicle / solvent:

ethanol

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: MEM culture medium.

Details on test system and experimental conditions:

After approximately 48 hours incubation at 37°C, 5% C02 in humidified air, the cultures were
transfered to tubes and centrifuged. Approximately 9 ml of the culture medium was removed,
reserved, and replaced with the required volume of MEM (including serum) and 0.1 ml of the
appropriate solution of vehicle control or test material was added to each culture. For the positive
control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 10% S9-mix (ie 1 %
final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary
Toxicity Test

Rationale for test conditions:

Preliminary
Toxicity Test

Evaluation criteria:

The mitotic index data are presented in Table 1. The test material showed evidence of toxicity in
all three exposure groups. A precipitate of the test material was observed in the parallel blood-free
cultures at the end of the exposure, at 1600 μg/ml, in all three exposure groups. Microscopic
assessment of the slides prepared from the treatment cultures showed that metaphase cells were
present up to 100 μg/ml in the 4(20)-hour treatment in the presence and absence of metabolic
activation (89). The maximum dose with metaphases present in the 24-hour continuous exposure
was 50 μg/ml.
Dose selection for Experiment 1 was based on toxicity.

Statistics:

Group Final concentration of PCMX {μg/ml)
S( 19)-hour without S9 o•, 12.s, 2s•, so•, 100•, 1so, 200, MMC o.4•
S( 19)-hour with S9 o•, 12.s , 2s•, so•, 100•, 1so, 200. CP 10•

Results and discussion

Test results

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range
expected for normal human lymphocytes.
All the positive control treatments gave statistically significant increases in the frequency of cells
with aberrations indicating the satisfactory performance of the test and of the activity of the
metabolising system.
The test material induced dose-related, statistically significant increases in the frequency of cells
with aberrations, in one of two separate experiments. The response was observed in the presence
of a 2% final concentration of S9 but not when I% S9 was used. In all exposure groups the dose
range included a dose level that induced approximately 50% mitotic inhibition.

Remarks on result:

other: A positive response was recorded

Any other information on results incl. tables

Table 1 Mitotic Index - Preliminary Toxicity Test

4-HOUR TREATMENT, 20-HOUR RECOVERY -S9

4-HOUR TREATMENT, 20-HOUR RECOVERY +S9

24-HOUR TREATMENT -S9 - CONCENTRATION

4(20)h WITHOUT S9 4(20)h WITH S9 24h WITHOUT S9

(μg/ml} MlTOTIC %OF MITOTIC %OF MITOTIC %OF

INDEX CONTROL INDEX CONTROL INDEX CONTROL

0 8.50 100 6.55 100 6.90 100

6.25 - - - - - -

12.5 - - - - 5.20 75

25 - - - - 3.20 46 -

50 6.25 74 5. 10 78 2. 15 31

100 2.65 31 2.95 45 0.00NM -

200 O.OONM - O.OONM - - -

400 - - - - - -

- 800 - - - - - - 1600 -P - -P - -P -

PCMX: CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES JN VITRO

Table 5 Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Number of Aberrations Total Number of Frequency of Aberrant

Treatment Group Replicate

Mitotic Number of Chromatid Chromosome Others Aberrations Cells(%)

Index(%) Cells Scored Gaps

Breaks Exchanges Breaks Exchanges x (+Gaps) (-Gaps) (+Gaps) (-Gaps)

A 11 .95 100 I 0 0 0 I 0 2 I 2 I

Vehic le Control B 8 15 100 0 0 0 0 0 0 0 0 0 0

Total 200 I 0 0 0 I 0 2 I 2 I

(100) (1.0) (0.5)

A 5.85 100 0 2 0 2 0 0 4 4 3 3

25 B 8.20 100 0 I 0 0 0 0 I I I I

μg/ml Total 200 0 3 0 2 0 0 5 5 4 4

(70) (2 .0) (2.0)

A 5.50 100 I - . I 0 I 0 0 3 2 3 2

50 B 4.95 100 4 I 0 0 0 0 5 I 5 I . -. - - .

μg/ml Total 200 5 2 0 I 0 0 8 3 8 3

(52) (4.0) (1.5)

A 6 45 100 0 3 0 0 0 0 3 3 3 J

100 B 6 70 100 0 0 0 0 0 0 0 0 0 0

μg/ml Total 200 0 3 0 0 0 0 3 J 3 3

(65) (1.5) (1.5)

A 0.00 TOXIC -- ------ - · - - -

150 B 0.00 TOXIC - - -- - -

μg/ml Total

(0)

Positive Control A 3.60 100 3 2 I 0 0 0 6 3 5 2

CP IO B 4.40 100 9 -- 8 6 2 0 0 25 16 18 13 - ·- ~ μg/ml Total 200 12 10 7 2 0 0 31 19 23 15•••

(40) ( 11.5) (7.5}

Applicant's summary and conclusion

Conclusions:

The test material induced a dose-related, statistically significant increase in the frequency of cells
with chromosome aberrations in the presence of a liver enzyme metabolising system in the second
experiment onJy. The use of a higher concentration of S9 in the second experiment (2% vs 1 %)
was therefore considered to be critical to the induction of chromosome aberrations. The test
material was therefore considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

The test material was therefore considered to be clastogenic to human lymphocytes in vitro.