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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23 January to 6 March 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study undertaken at GLP accredited laboratory to internationally accepted guidelines. The restriction is due to the use of the read across approach: the test was performed not with S-205 but with Black 400, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile .

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
ODB-2
IUPAC Name:
ODB-2
Constituent 2
Reference substance name:
Black 400
IUPAC Name:
Black 400
Constituent 3
Reference substance name:
89331-94-2
EC Number:
618-263-0
Cas Number:
89331-94-2
IUPAC Name:
89331-94-2
Details on test material:
- Name of test material (as cited in study report): ODB-2
- Physical state: White powder
- Analytical purity: 99% minimum
- Lot/batch No.: 8A-24433-10
- Expiry date of the lot/batch: October 1991
- Storage condition of test material: Room temperature in the dark
- Molecular formula (if other than submission substance): C35 H36 N2 O3
- Molecular weight (if other than submission substance): 532.7
- Smiles notation (if other than submission substance): CCCCN(CCCC)c6ccc5c(Oc2cc(C)c(Nc1ccccc1)cc2C54OC(=O)c3ccccc34)c6
- InChI: InChI=1/C35H36N2O3/c1-4-6-19-37(20-7-5-2)26-17-18-29-33(22-26)39-32-21-24(3)31(36-25-13-9-8-10-14-25)23-30(32)35(29)28-16-12-11-15-27(28)34(38)40-35/h8-18,21-23,36H,4-7,19-20H2,1-3H3

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.5, 1, 5, 10, 15, 30, 40, 50 µg / ml

Mutation tests: -S-9 mix
Test 1 1, 2.5, 5, 10, 15, 30, 50 µg / ml
Test 2 1, 2.5, 5, 10, 15, 30, 50 µg / ml

Mutation tests: +S-9 mix
Test 1 0.1, 0.5, 1, 2.5, 5, 10, 15 µg / ml
Test 2 0.1, 0.5, 1, 2.5, 5, 10, 15 µg / ml
Vehicle / solvent:
- solvent used: DMSO;
- Justification for choice of solvent: commonly used and accepted.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Used in the absence of S-9 mix
Positive controls:
yes
Positive control substance:
other: 20-methylcholanthrene
Remarks:
Used in presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:as a solution

DURATION
- Preincubation period:
- Exposure duration: 3 hours @ 37°C
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours

SELECTION AGENT (mutation assays):4 pg trifluorothymidine TFT/mI

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 600 cells plated in cloning medium for estimation of viability (200 cells/90 mm petri dish) and a total of 3 x 1E+06 cells in selective medium for quantitation of mutation (1E+06 cells/90 mm petri dish)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and cloning efficiency

Evaluation criteria:
The criteria which must be satisfied before a positive response may be claimed are:

1. The induction of at least a two-fold increase in mutant frequency relative to the concurrent control by the test agent.
2. The demonstration of a statistically significant response.
3. Evidence of a dose-related response.
4. The response must be reproducible.
5. The data are acceptable according to the criteria detailed in Arlett et ai. (1989), in 'Statistical Evaluation of Mutagenicity Test Data', pp. 66 - 101, Cambridge University Press.
Statistics:
The general method of statistical analysis was by analysis of variance of the mutant frequencies

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary test on ODB-2 treatment with 0.5 - 50 µg/ml in the presence and absence of S-9 mix resulted in relative growth in suspension of 106 - 2% and 108 - 17% respectively. The concentrations used in the main test were based on this data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In the absence of S-9 mix, treatment with 1 - 50 µg ODB-2/ml in tests 1 and 2 resulted in relative growth in suspension of 111 - 39% and 99 - 35% respectively. Cultures treated with 1, 15,30 and 50 µg/ml in test 1 and test 2 were assessed for viability. The resulting cell survival levels were 91 - 31% in test 1 and 93 - 26% in Test 2 relative to the controls.

In the presence of S-9 mix, treatment with 0.1 - 15µg ODB-2/ml in tests 1 and 2 resulted in relative growth in suspension of 110 - 31% and 112 - 5% respectively. Cultures treated with 0.5,5, 10 and 15 µg/ml in test 1 and 0.5, 1, 2.5 and 5 µg/ml in test 2 were assessed for viability. The resulting cell survival levels were 86 - 31% in test 1 and 95 - 23% in test 2 relative to the controls.

ADDITIONAL INFORMATION ON GENOTOXICITY:

In the absence of S-9 mix, cultures treated with 1, 15,30 and 50 µg/ml in test 1 and test 2 were assessed for induced mutation.Statistically
significant increases in mutant frequency were observed in both tests in the absence of S-9 mix. In test 2 the response was also dose-related. .Although this data does not fulfil all the criteria for a positive response (1. The induction of at least a two-fold increase in mutant frequency relative to the concurrent control by the test agent), the detection of a dose-related increase may be indicative of mutagenic potential.

In the presence of S-9 mix, cultures treated with 0.5,5, 10 and 15 µg/ml in test 1 and 0.5, 1, 2.5 and 5 µg/ml in test 2 were assessed for induced mutation.Statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be non-biologically significant.
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous without metabolic activation

The evidence for mutagenic potential of ODB-2 in the absence of S-9 mix is equivocal in this in vitro test system. There is no evidence for mutagenic potential in the presence of S-9 mix using this in vitro test system.
Executive summary:

S-205 and ODB-2 (Black 400), which is tested for its genotoxicity in a mouse lymphoma TK locus assay, are both colour precursor for heat sensitive record sheets. These substances have been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile. Due to the fact that S-205 and ODB-2 have nearly the same chemical structure, the same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. Therefore, a read-across from S-205 to data obtained with ODB-2 is scientifically justified.

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