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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study, no test repetition

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
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): diisopropylnaphthalene isomer, KMC, RKKO 131006
- Physical state: clear liquid
- Analytical purity: 99%
- Isomers composition: no data
- Storage condition of test material: at room temperature in the dark

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Hams F12 (from Imperial) supplemented with 5% foetal calf serum (Gibco)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254 induced livers of male SD rats
Test concentrations with justification for top dose:
Pre-test to determine 50% reduction in the mitotic index
1.9, 3.8, 7.6, 15.2, 30.4, 60.8, and 121.5 µg/mL (highest concentration soluble in test medium)
Main experiment
without S-9 mix: 0.95, 4.75, and 9.5 µg/mL
with S-9 mix: 6, 30, 45, and 60 µg/mL (preliminary test 7.5, 37.5, 75 µg/ml; highest dose was too toxic.)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S-9: mitomycin C (Sigma London Chemical Company Ltd.) at 0.4 µg/mL; with S-9: cyclophosphamide (Sigma) at 20 µg/mL
Details on test system and experimental conditions:
Test was performed with duplicate cultures for positive controls and test substance and quadruplicate cultures for solvent controls

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 20 h in cultures without S-9 mix; 4 h in cultures with S-9 mix
- Expression time (cells in growth medium): 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 21 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.25 µg/mL); inhibitor was added 2 h before the end of the 21 h incubation period
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF CELLS EVALUATED: 100 metaphases per culture (200 or 400 total)

DETERMINATION OF CYTOTOXICITY
- Method: reduction of mitotic index (preliminary independent experiment)

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: ≥15 µg/ml (without S-9); ≥ 60 µg/ml (with S-9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES
The mitotic index was determined for 7 graduate test substance concentrations (without and with S-9 mix each). From the data an EC50 was estimated (reduction of mitotic index by 50%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test sustance concentration of ≥ 15 µg/mL and ≥ 60 µg/mL resulted in a reduction of the mitotic index by 50% and more (without and with S-9 mix, respectively).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The first main test (with S-9 mix, maximum concentration 75 µg/mL) was dismissed and not continued for chromosomal analysis. The reason was a reduction in the mitotic index at 75 µg/mL, higher than expected from pre-testing. Therefore, a second main trial was initiated with a somewhat reduced upper concentration (60 µg/mL). Cytotoxic screening was relinquished for the second main test because cytotoxicity had been demonstrated in prior screening.

 

No significant increase in chromosomal damage was seen in cultures treated with diisopropylnaphthalene at any dose level in either the presence or absence of metabolic activation.

 

Positive controls caused large, statistically highly significant increases in chromosomal damage, thus demonstrating the sensitivity of the test system and the efficiency of the S-9 mix.

 

In this in-vitro cytogenetic test diisopropylnaphthalene did not show any evidence of clastogenic activity.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In an in-vitro mammalian chromosome aberration test, diisopropylnaphthalene did not demonstrate any increase in chromosomal damage at any dose level either without or with metabolic activation. There was no evidence of any clastogenic activity under the conditions of the test used.