Registration Dossier

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start : 19 December 2001 Completed : 8 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: There are no deviations from the recommended guidelines.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - 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
Details on test material:
- Name of test material (as cited in study report): Phenyloxindole
- Physical state: Light beige powder
- Analytical purity: 99.80%
- Lot/batch No.: CHAD 9902K
- Expiration date of the lot/batch: 14 November 2002 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 indticed rat liver S9-mix
Test concentrations with justification for top dose:
Dose range finding test:
10, 33, 100, 333 and 1000 µg Phenyloxindole/ml culture medium with and without S9-mix.

First cytogenetic assay:
Without S9-mix : 33, 100, 333, 375 and 420 µg Phenyloxindole/ml culture medium (3 h exposure time, 24 h fixation time).
With S9-mix : 100, 150, 200, 250 and 300 µg Phenyloxindole/ml culture medium (3 h exposure time, 24 h fixation time). Part of this experiment was repeated with the following dose levels:
Experiment 1 A
With S9-mix : 200, 300 and 420 µg Phenyloxindole/ml culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:
Without S9-mix : 10, 15, 20, 25, 33, 42, 56, 75 and 100 µg Phenyloxindole/ml culture medium (24 h exposure time, 24 h fixation time)
10, 15, 18, 20, 22, 25, 28 and 33 µg Phenyloxindole/ml culture medium (48 h exposure time, 48 h fixation time)
With S9-mix: 200, 300, 375 and 420 µg Phenyloxindole/ml culture medium (3 h exposure time, 48 h fixation time)
Part of this second experiment was repeated with the following dose levels:
Experiment 2A:
Without S9-mix : l0, 20, 33, 56, 75, 100, 200, 300 and 420 µg Phenyloxindole/ml culture medium (48 h exposure time, 48 h fixation time).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period.

Migrated to IUCLID6: Without metabolic activation
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
15 µg/ml for a 3 h exposure period (24 h fixation time).

Migrated to IUCLID6: With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Whole blood treated with an anti-coagulant (heparin) obtained from healthy rnale subjects was cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals.

Within 4 h after blood collection, lymphocyte cultures were started. Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml F10 complete culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.

All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% C02 in air in the dark at 37 ± 1°C. The temperature, humidity and CO2-percentage were monitored throughout the experiment.

Lymphocyte cultures were cultured for 48 h and thereafter exposed to selected doses. Phenyloxindole was dissolved in dimethyl sulfoxide. Stock solutions above 42 mg/ml were treated with ultrasonic waves until Phenyloxindole had completely dissolved. Stock solutions of 42 mg/ml and lower were dissolved by vortexing only. Phenyloxindole concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted 1.0 % (v/v).

DURATION
- Exposure duration:
Range finding: 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
First cytogenetic assay: 3 h in the absence and presence of S9-mix.
Second cytogenetic assay: 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.

- Fixation time (start of exposure up to fixation or harvest of cells):
Range finding: 24 h fixation time (3 h and 24 h exposure ) and 48 h fixation time (48 h exposure).
First cytogenectic assay: 24 h fixation time.
Second cytogenetic assay: 48 h fixation time (3 h exposure ), 24 h fixation time (24 h exposure) and 48 h fixation time (48 h exposure).

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): for 10-30 min with 5% (v/v) Giemsa solution in tap water.

NUMBER OF REPLICATIONS: First and second assays: Lymphocyte cultures were exposed in duplicate.


NUMBER OF CELLS EVALUATED: At least 100 metaphase chromosome spreads per culture w ere examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.


DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or
greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.


Evaluation criteria:
A test substance w as considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P <0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P <0.05) increase in the number of cells with chromosome aberrations.
Statistics:
Chi-square test

Results and discussion

Test results
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding study: At a concentration of 1000 µg/ml Phenyloxindole precipitated in the culture medium. Therefore, a concentration of 1000 µg/ml was used as the highest concentration
First cytogenetic assay: a concentration of 420 µg/ml already precipitated in the culture medium. Therefore, a concentration of 420 µg/ml was used as the highest concentration.

RANGE-FINDING/SCREENING STUDIES:
Dose range finding study:
age 23, AGT = 16.8 h (Dec. 2001 )
First cytogenetic assay:
age 29, AGT = 17.6 h (Dec. 2001) (without S9-mix)
age 27, AGT = 17.3 h (Dec. 2001) (with S9-mix)
Second cytogenetic assay:
age 26, AGT = 16.6 h (Dec. 2001)
age 36, AGT = 16.6 h (Dec. 2001) (48 h without S9-mix)
(AGT= Average Generation Time of the cells).

Any other information on results incl. tables

The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range. At the 24 h continuous exposure time, in culture A of the DMSO-treated cultures 6 aberrant cells were observed when gaps were included, and 3 aberrant cells when gaps were excluded. Since the number of aberrant cells was just within our historical control data range, additional 100 cells were scored to confirm that the results of the blank were within the historical control data range. In the additional 100 cells, 4 aberrant cells were observed when gaps were included.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that Phenyloxindole is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

This study describes the effect of Phenyloxindole on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system. In the first cytogenetic assay, Phenyloxindole was tested up to 420 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. In the second cytogenetic assay, Phenyloxindole was tested up to 100 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 75 µg/ml for a 48 h continuous exposure time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of 1.8% (v/v) S9-fraction Phenyloxindole was tested up to 420 µg/ml for a 3 h exposure time with a 48 h fixation time. Positive control substances, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Phenyloxindole did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments. It is concluded that this test is valid and that Phenyloxindole is not clastogenic in human lymphocytes under the experimental conditions described in this report.