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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:
2003-01-14 to 2003-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21st July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Supplier: ATOFINA
- Chemical Name: Alpha-CUMYL PEROXYNEODECANOATE
- Batch number: 0210840414
- Description: colourless liquid
- Container: one plastic flask
- Storage conditions: at -20 °C and protected from light
- Purity: 75% in isododecane

The test item was dissolved in the vehicle at a concentration of 612.9 mg/mL for the first experiment and 306.45 mg/mL for the second experiment.
The preparations were made immediately before use.

Method

Species / strain
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Cells used: Human lymphocytes
- Modal number of chromosomes: 46
- Cell cycle time: 12 - 14
- Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.

MEDIA USED
RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account.
- Experiment I (with and without S9): 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM
- Experiment II (with S9): 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM
- Experiment II (without S9): 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol, batch no. V8M084248 M (Carlo Erba, 27106 Val de Reuil, France)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9: 3 µg / mL ( 3h) or 0.2 µg / mL (continuous experiment)
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 12.5, 25 or 50 µg / mL
Details on test system and experimental conditions:
Treatment
In two independent experiments, using duplicate cultures, the cells were tested, with and without S9 mix, with:
- at least five dose-levels of the test item,
- the vehicle control,
- the appropriate positive control.
To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.

In the first experiment, lymphocyte cultures were then exposed to the test or control items, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
• without S9 mix, cells were exposed continuously to the test or control items, until harvest,
• with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

Preparation of microscope slides
After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded, so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

Determination of cytotoxicity
The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a item induces mitotic inhibition. Mitotic index was determined without blind scoring. Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed. All metaphase analyses were performed blind. The following structural aberrations were recorded for each metaphase (c, d): gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication. The metaphase analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p= 0.05 was used as the lowest level of significance.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: pH was approximately 7.7 (7.4 for the vehicle control) and the osmolality equal to 313 mOsm/kg H2O (377 for the vehicle control).
- Other confounding effects: No emulsion was observed in the culture medium at the end of the treatment period.

Experiments without S9 mix:

Cytotoxicity:
Following the 3-hour treatment, a moderate to marked decrease in mitotic index (56-81% decrease) was noted at dose-levels ≥ 1.25 mM. Following the 20-hour treatment, a moderate decrease in mitotic index (41% decrease) was noted at 1.25 mM. Following the 44-hour treatment, a slight to marked decrease in mitotic index (31-70% decrease) was noted at dose-levels ≥ 0.63 mM.

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 0.31, 0.63 and 1.25 mM for the 3-hour and the 20-hour treatments, the latter inducing 56 and 41% decreases in mitotic index, respectively,
- 1.25 mM, for the 44-hour treatment, this dose-level inducing 70% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44-hour treatments.


Experiments with S9 mix:

Cytotoxicity:
A slight to marked decrease in mitotic index (29-78% decrease) was noted at dose-levels ≥ 0.63 mM at the 20-hour harvest time.
At the 44-hour harvest time, a moderate to strong decrease in mitotic index was noted at dose-levels ≥ 1.25 mM (59-89% decrease).

Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1.25, 2.5 and 5 mM, for the 20-hour harvest time in the first experiment, the latter inducing 59% decrease in mitotic index,
- 0.31, 0.63 and 1.25 mM, for the 20-hour harvest time in the second experiment, the latter inducing 68% decrease in mitotic index,
- 1.25 mM, for the 44-hour harvest time, this dose-level inducing 59% decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Any other information on results incl. tables

Table 1: Experiment 1 without S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 14M 100 0 0 1 0 0 0 0 0 5 3 1 2.0 1 1.5
23F 100 1 2 0 0 1 1 0 0 3 2
0.31 22M 100 1 2 1 0 2 2 0 0 9 7 6 4.0 4 3.0
6F 100 0 0 1 1 0 0 0 0 2 2
0.63 5M 100 0 2 0 0 0 0 0 0 6 2 2 3.0 0 1.0
15F 100 1 2 2 0 0 0 0 0 4 2
1.25 39M 100 2 3 1 0 1 1 0 0 10 5 6 5.0 3 2.5
12F 100 0 2 2 0 0 0 0 0 4 2
MMC 9M 50 0 4 18 11 3 0 0 0 89 78 21 52.0 19 48.0
***
3µg/mL 18F 50 0 7 28 14 4 0 0 0 31 29

Table 2: Second experiment without S9 mix: chromosome aberration (20 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 48M 100 0 2 2 0 0 0 0 0 5 3 4 2.5 1 1.5
57F 100 2 0 0 0 1 0 0 0 1 2
0.31 51M 100 0 1 0 0 0 0 0 0 3 1 1 1.5 4 0.5
75F 100 0 1 1 0 0 0 0 0 2 2
0.63 64M 100 1 1 1 0 0 0 0 0 6 1 2 3.0 0 0.5
44F 100 0 4 0 0 0 0 0 0 4 2
1.25 60M 100 0 0 0 0 0 0 0 0 3 2 0 1.5 3 1.0
71F 100 1 1 2 0 0 0 0 0 3 2
MMC 54M 50 0 3 5 4 1 0 0 0 30 23 10 24.0 19 21.0
***
3 µg/mL 49F 50 0 4 6 5 2 0 0 0 14 29

Table 3: Experiment 2 without S9 mix: chromosome aberration (44 -hour treatment, 44 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 84M 100 0 2 0 0 2 0 0 0 4 2 4 2.0 2 1.0
97F 100 1 0 0 0 0 0 0 0 0 0
Jan 25 81M 52 0 1 2 0 0 0 0 0 15 11 3 5.5 2 3.5
87F 148 3 3 2 0 7 0 0 0 8 5

Table 4: Experiment 1 with S9 mix: chromosome aberration (3 -hour treatment, 20 -hour harvest)

Doses mM Slide Nb. Nb. of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
G Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 32M 100 0 1 1 0 0 0 0 0 5 3 2 2.5 1 1.5
8F 100 0 1 0 0 2 0 0 0 3 2
1.25 2M 100 1 4 0 0 1 0 0 0 9 2 5 4.5 1 1.0
33F 100 1 3 0 0 0 1 0 0 4 1
2.5 38; 100 2 3 1 0 1 1 0 0 13 9 6 5.5 3 3.5
26F 100 1 1 3 0 3 0 0 0 5 4
5 16M 100 0 7 0 0 0 0 0 0 10 1 7 5.0 0 0.5
4F 100 3 2 0 0 1 0 0 0 3 1
CPA 27M 50 0 6 26 5 1 0 0 0 91 78 22 52.0 21 48.0
***
50 µg/mL 17F 50 0 7 35 6 4 0 1 0 30 27

Table 5: Experiment 2 with S9 mix: chromosome aberration (4 -hour treatment 20 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 43M 100 0 1 0 0 0 0 0 5 4 1 2.5 1 2.0
53F 100 0 2 0 1 0 0 0 4 3
0.31 52M 100 0 0 0 0 0 0 0 3 1 0 1.5 0 0.5
69F 100 2 1 0 0 0 0 0 3 1
0.63 66M 100 1 1 0 0 0 0 0 5 4 1 2.0 1 1.5
47F 100 0 1 0 1 1 0 0 3 2
Jan 25 56M 100 0 1 0 0 0 0 0 6 4 3 3.0 1 2.0
72F 100 0 1 0 1 1 0 0 3 3
CPA 73M 50 0 10 3 3 0 0 0 38 31 16 28.0 13 23.0
***
12.5 µg/mL 45F 50 0 12 2 1 0 0 0 12 10

Table 6: Experiment 2 with S9 mix: chomosome aberration (3 -hour treatment 44 -hour harvest)

Doses mM Slide Nb. Nb. Of cells scored NA Structural chromosome aberrations
(type and number)
Cells with structural chromosome aberrations
Chromatid Chromosome MA PU Total
+G
Total
-G
Nb.
+G
% Nb.
-G
%
D Exch D Exch
0 77M 100 0 1 1 0 0 0 0 3 1 2 1.5 1 0.5
91F 100 0 0 0 0 0 0 0 1 0
1.25 83M 100 0 2 1 0 0 0 0 5 2 3 2.5 1 1.0
94F 100 1 1 1 0 0 0 0 2 1

NA: numerical aberrations, G: gap, D: deletion, Exch: exchange, MA: multiple aberrations, PU: pulverization.

M: male

F: female

0: vehicle control (DMSO)

MMC: mitomycin C

Statistical analysis: Chi-Quadrat test ***: p < 0.001 (performed only for cells with structural aberrations excluding gaps)

Nb.: number

(1): expressed as active

Applicant's summary and conclusion

Conclusions:
In this study, the test substance did not induce chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, the test item is not considered to be clastogenic in this test system.
Executive summary:

In an in-vitro mammalian chromosome aberration test conducted in accordance with OECD 473, the potential of the test item (75% in isododecane) to induce chromosome aberrations in cultured human lymphocytes was evaluated in the presence and absence of a liver metabolizing system (S9). In the first experiment, lymphocytes were exposed to 0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 mM with and without S9 mix for 3 hours, then rinsed. Cells were harvested 20 hours after beginning of treatment. For the second experiment, the cells were treated with 0.16, 0.31, 0.63, 1.25, 2.5 and 5 mM with S9 mix for 3 hours, then rinsed and with 0.04, 0.08, 0.16, 0.31, 0.63 and 1.25 mM without S9 mix until harvest. Cells were harvested 20 and 44 hours after the beginning of treatment. For all tested treatment groups, no dose-response relationship could be observed. The positive controls did induce the appropriate response. There was no significant increase in the frequency of cells with structural chromosomal aberrations in both experiments and at both harvest times. Based on these results, the test item did not induce chromosome aberrations in cultured human lymphocytes.