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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 January 05,2010 to March 18,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well described GLP compliant study conducted to recognised international test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisobutyl hexahydrophthalate
EC Number:
275-069-5
EC Name:
Diisobutyl hexahydrophthalate
Cas Number:
70969-58-3
Molecular formula:
C16H28O4
IUPAC Name:
bis(2-methylpropyl) cyclohexane-1,2-dicarboxylate
Details on test material:
- Name of test material : DIBE (diisobutylhexahydrophthalate)
- Physical state: Liquid
- Lot/batch No.: T60211/349
- Expiration date of the lot/batch: 2012-12-15
- Storage condition of test material: room temperature, protected from light

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First test:
In the presence and absence of S9 mix 1.86, 4.65, 11.6, 29.1, 72.7, 182, 454, 1140 and 2840 µg/mL.
Second test:
In the absence of S9 mix: 0.389, 0.777, 1.55, 3.11, 6.22, 12.4, 24.9, 49.8, 99.5 and 199 µg/mL.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Final concentration: 0.5 µg/mL (3 hour treatment) 0.3 µg/mL (21 hour continuous treatment)

Migrated to IUCLID6: without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Final concentration: 18 µg/mL (3 hour treatment)

Migrated to IUCLID6: with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h
- Fixation time:
Three hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.2 µg/mL. After 3 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 10 minutes at 1000rpm. The cell pellets were treated with a hypotonic solution and after a period of incubation the suspensions were again centrifuged and the cell pellets fixed by addition of cold fixative (methanol : acetic acid) followed by 2 further washes in fresh fixative.

SELECTION AGENT (mutation assays):. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 46 centromeres were analysed. Polyploid and endoreduplicated cells were noted when seen.

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED:1000cells in each plate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the negative control range of the laboratory.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
The numbers of cells bearing aberrations in the control and treated cultures were compared using Fisher's exact test. This comparison was performed both including and excluding gaps from the aberration counts. The values obtained at all the treatment dose-levels combined were compared with the control values. Since multiple dose-levels were compared with the negative controls, the problem of Type I error (chance 'positive' results) arose. Accordingly, significance levels for each treatment-level were also presented after application of Bonferroni's correction.
Solvent controls were used as the reference point for comparison in the statistical evaluation and the evaluation of the results.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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: None
- Effects of osmolality: None
- Evaporation from medium: No
- Water solubility: Limited
- Precipitation: Addition of substance solution in DMSO to test medium resulted in observable rpecipitation at concentrations above 1420 microgram/mL
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES: Wide concentration range examined a part of main experiments

COMPARISON WITH HISTORICAL CONTROL DATA: Within test laboratory's acceptance limits

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For both experiments, the mitotic index (MI) was scored for each of the treatment series. For the first main experiment, dose levels of 2840, 1140, 454, 182, 72.7, 29.1, 11.6, 4.65 and 1.86 g/mL were used in the absence and presence of S9 metabolism. Following treatment in the absence of S9 metabolism, severe toxicity was observed at exposure levels of 2840, 1140, 454 and 182 µg/mL, where few metaphases were recovered. Moderate toxicity was observed at 72.4 µg/mL, reducing the mitotic index to 41% of the control. No remarkable toxicity was observed over the remaining dose range. In the presence of S9 metabolism, severe toxicity was observed at the highest exposure level (2840 µg/mL), where few metaphases were recovered. Marked toxicity was observed at 1140 µg/mL, where the mitotic index was reduced to 36% of the concurrent negative control. Moderate toxicity was observed at 454 and 182 µg/mL, where the relative mitotic indexes were 51% and 73%, respectively. No toxicity was observed over the remaining dose range. For the second main experiment, following the continuous treatment in the absence of S9 metabolism, severe toxicity was observed at the highest exposure levels selected for treatment (199, 99.5 and 49.8 µg/mL), where no cells or few metaphases were recovered. Marked toxicity was observed at 24.9 µg/mL, where the mitotic index was reduced to 36% of the concurrent negative control. Moderate to slight toxicity was observed at 12.4, 6.22 and 3.11 µg/mL, (relative mitotic index from 63% to 81%). No relevant toxicity was observed at the three lower levels of 1.55, 0.777 and 0.389 µg/mL. One hundred metaphase spreads were scored for chromosomal aberrations from each culture with the exception of cultures treated with the positive control Mitomycin-C from the second main experiment, where due to the high incidence of cells bearing aberrations (excluding gaps), scoring was terminated at 50 metaphases. No increase in the incidence of cells bearing aberrations, including or excluding gaps, over the concurrent negative control value was observed in any experiment following treatment with the tested substance. In the first main experiment, the presence of one cell bearing more than five aberrations and one endoreduplicated cell was observed at the intermediate dose level selected for scoring in the absence of S9 metabolism. This observation was not considered related to treatment but probably due to a chance event. Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that teh tested substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

Diisobutylhexahydrophthalate (DIBE) has been assayed for the ability to cause chromosomal damage in cultured human lymphocytes followingin vitrotreatment in the absence and presence of S9 metabolic activation. Methods used were in accordance with OECD/EU test methods. The substance does not induce chromosomal aberrations in human lymphocytes.