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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 19,2012 to August 01,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complaince with international guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Results obtained in a repeated experiment
Deviations:
yes
Remarks:
Results obtained in a repeated experiment
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Results obtained in a repeated experiment
Deviations:
yes
Remarks:
Results obtained in a repeated experiment
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material:PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1
- Substance type: liquid
- Physical state: Amber-yellowish/brown undefined liquid
- Analytical purity:no data
- Lot/batch No.: 0512/S
- Expiration date of the lot/batch: 26 January 2014
- Storage condition of test material: Room temperature

Method

Target gene:
no data
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes ,regular intervals.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment No. S9 Treatment time(hours) Harvest time (hours) Dose level(µg/mL)
1 ± 3 20 625, 313 ,156 ,78.1 and 39.1
2 - 20 20 80.0, 40.0 and 20.0

The dose levels of 0.30 and 0.10 µg/mL were selected for the scoring of cultures treated with Mitomycin-C (first and second experiment, respectively). The dose level of 15.0 µg/mL was selected for the scoring of cultures treated with Cyclophosphamide.
Vehicle / solvent:
distilled water, culture medium, DMSO, ethanol, acetone.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The medium used for the growth of the cells has the following composition:
Ham's F.10 499.0 ml
Streptomycin sulphate 50,000 IU/ml
Penicillin G 50,000 IU/ml 1.0 ml
Newborn Calf Serum 88.2 ml

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):Each 25 sq.cm. flask will be seeded with 300,000 cells in supplemented Ham's F10. These cells should be in exponential growth phase at the time of treatment.



NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED:1 x 10^6 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index,

Evaluation criteria:
In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:
(i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control.
(ii) The increases must exceed the historical control values.
(iii) The increases are reproduced in both replicate cultures.
The evaluation is based on the set of results, which excludes gaps. A more detailed explanation of the criteria for evaluation of the results is given in the Study Protocol.
Statistics:
For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps. Following treatment in the absence of S9 metabolism, a statistically significant increase in the incidence of cells bearing structural aberrations including and excluding gaps was observed in the first main experiment, at the intermediate dose level selected for scoring. No other statistically significant increases were observed in any experiment, at any dose level, in the presence or absence of S9 metabolic activation. A statistically significant increase of endoreduplicated cells was observed at the highest dose level after treatment in the presence of S9 metabolic activation

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: and osmolality: Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism. These results are not presented in this report but are retained in the study file and archived as indicated in the Study Protocol.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Deviations from Study Protocol

Results presented for the first experiment in the presence of S9 metabolic activation were obtained in a repeated experiment. In the original one, a low cellular growth was observed, probably due to the toxicity of S9 metabolic fraction. Data concerning the previous experiment are not presented in the report but will be archived as indicated in the Study Protocol.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce structural chromosomal aberrations in Chinese hamster ovary cells in vitro.However, treatment with the test item in the presence of S9 metabolic activation induced a biologically relevant increase in the number of cells with endoreduplicated chromosomes, indicating that the test item may have the potential to inhibit cell cycle progression. Statistically significant increases in aberrant cells, compared with the relevant control values, were seen in cultures treated with the positive controls Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

On the basis of these results, it is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.
It is also concluded that the test item induces endoreduplication in the presence of S9 metabolic activation.
Executive summary:

The test item PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of S9 metabolic activation. Two main experiments for chromosomal damage were performed.

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 20 hours corresponding to approximately 1.5 cell cycle length was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using the same harvest time. A continuous treatment until harvest at 20 hours was used.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO). For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol and on the basis of the solubility of the test item. Dose levels of 1250, 625, 313, 156, 78.1, 39.1,19.5 and 9.77 μg/mL were used both in the absence and presence of S9 metabolism.

The dose range used in the second experiment was modified to take into account the results observed in the previous experiment.

Dose levels of 160, 80.0, 40.0, 20.0, 10.0, 5.00, 2.50 and 1.25 μg/mL were used. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of population doubling or relative cell count. Based on the obtained results, the following dose levels were selected for scoring:

Experiment No.

 

S9

Treatment time

(hours)

Harvest time (hours)

Dose level

(ug/mL)

1

+

-

3

20

625,313 and 156

156, 78.1 and 39.1

2

-

20

20

80.0, 40.0 and 20.0

One hundred metaphase spreads were scored for chromosomal aberrations from each culture, with the exception of one culture treated with Mitomycin-C from the first main experiment, where, due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 50 metaphases. Following treatment with test item, no statistically significant increase in the number of cells bearing structural aberrations, including or excluding gaps, was observed at any dose level or treatment time in the absence or presence of S9 metabolic activation. For the first main experiment, a statistically significant increase of endoreduplicated cells over the concurrent negative control was observed at the highest dose level selected for scoring (625 μg/mL) in the presence of S9 metabolism. Few endoreplicated cells were also observed at the intermediate dose level selected for scoring (313 μg/mL).

Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatment with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system. It is concluded that PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 does not induce structural chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions. However, treatment with the test item induces an increase in the number of cells with endoreduplicated chromosomes only in the presence of metabolic activation and at the highest dose level selected for scoring (625 μg/mL), indicating that the test item may have the potential to inhibit cell cycle progression.