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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
human lymphoblastoid cells (TK6)
Details on mammalian cell lines (if applicable):
N/A
Additional strain 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:
ethanol
Controls
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: MEM culture medium.
Details on test system and 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:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: A positive response was recorded
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.

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.