Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Cytotest Cell Research GmbH & Co. KG
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HGPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: HAM's F12 (Seromed, D-1000 Berlin, FRG) supplemented with 10 % fetal calf serum (FCS; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
Experiment 1: 30, 100, 200, 300, 400, 800 µg/ml (without S9 mix); 1, 5, 20, 100, 1000, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 2: 80, 300, 600, 800, 1000, 1200 µg/ml (without S9 mix); 342, 1000, 1692, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 3: 1, 5, 10, 20, 30, 50 µg/ml (with S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: substance is not soluble in water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 600 µg/ml = 4.8 mM dissolved in medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation Migrated to IUCLID6: 3.85 μg/ml = 15.0 μM dissolved in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 or 16 days

Colony staining with 10% methylene blue in 0.01 % KOH solution


NUMBER OF REPLICATIONS: in duplicate per experimental point


NUMBER OF CELLS EVALUATED: 10^6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced plating efficiency observed in some plates with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRETEST:
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test article was observed and compared to the controls. Toxicity of the test article was evidenced by a reduction in plating efficiency (PE). The plating efficiency of the CHO cells was reduced after treatment with 1000 µg/ml (without metabolic activation). With metabolic activation toxicity was observed after treatment with 2000 µg/ml and between 40 µg/ml and 300 µg/ml. Therefore, the first experiment was performed with six (without metabolic activation) and eight concentrations (with metabolic activation) ranging from 1 to 3420 µg/ml.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results:

Experiment µg/ml S-9 mix mean number cells/flask factor mean number mutant colonies/flask mean number mutant colonies/10^6 cells
seeded found
1 negative control - 542 280,5 0,52 2.8 ± 0.8 13,8
600, EMS - 530 186 0,35 61.0 ± 6.6 542,9
30 - nc
100 - 618 298,5 0,48 1.4 ± 1.1 7
200 - 608 362 0,6 2.4 ± 2.1 9,4
300 - nc
400 - 612 459 0,75 5.2 ± 2.9 17,8
800 - 610 423,5 0,69 3.8 ± 1.3 13,4
negative control + 498 289,5 0,58 2.6 ± 1.1 12,1
solvent control + 498 260,5 0,52 0.2 ± 0.4 0,8
3850, DMBA + 519 123 0,24 109.4 ± 14.2 1215,6
1 + nc
5 + nc
20 + nc
100 + 508 221 0,44 4.6 ± 2.9 28,6
1000 + 525 233,5 0,44 1.2 ± 1.1 7,6
2000 + nc
2700 + 454 273 0,6 0.8 ± 0.8 3,1
3420 + 492 264 0,54 2.6 ± 1.7 13,5
2 negative control - 519 282,5 0,54 3.2 ± 2.9 13,8
600, EMS - 506 148,5 0,29 142.6 ± 3.5 1343,5
80 - 529 253,5 0,48 1.4 ± 0.9 6,7
300 - 504 232 0,46 1.4 ± 1.7 7,5
600 - nc
800 - 522 294 0,56 0.2 ± 0.4 0,8
1000 - 507 282 0,56 0.4 ± 0.5 1,7
1200 - nc
negative control + 506 267 0,53 2.0 ± 0.7 8,4
solvent control + 514 258 0,5 1.8 ± 1.1 8
3850, DMBA + 500 159 0,32 153.0 ± 14.8 1048,5
342 + 511 236,5 0,46 1.2 ± 1.3 5,8
1000 + nc
1692 + 525 211 0,4 0.2 ± 0.4 1,3
2000 + nc
2700 + 515 224,5 0,44 0.2 ± 0.4 1
3420 + 503 251,5 0,5 2.0 ± 2.0 8,8
3 negative control + 530 341 0,64 1.2 ± 0.8 4,5
solvent control + 506 314 0,62 0.4 ± 0.5 1,7
3850, DMBA + 504 298 0,59 77.2 ± 2.9 320,7
1000 + 531 290,5 0,55 0.4 ± 0.5 2,1
5000 + nc
10000 + 525 320 0,61 1.6 ± 1.5 6,2
20000 + 504 260,5 0,52 0.2 ± 0.4 0,9
30000 + 532 313 0,59 0.6 ± 0.5 2,4
50000 + 519 264 0,51 0.4 ± 0.5 1,9

nc: culture not continued

Applicant's summary and conclusion

Conclusions:
The analogue substance was tested for gene mutation in mammalian cells follwoing OECD 476. The tested substance under the reported experimental conditions did not induce gene mutation at the HGRPT locus in CHO cells.