Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted following OECD/EU guideline and in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
Only two animals were used for evaluation of the 2-4 hour treated groups due to no cells being present on the slides. Clear negative results mean that the missing animal would provide limited additional data, study integrity is not affected
GLP compliance:
yes (incl. certificate)
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test item 206474/B

Identification K1600 black dye
Appearance Black powder
Batch G-173
Purity/Composition 100%(Limit:>98.5% On Dry Basis)
Test item storage At room temperature
Stable under storage conditions until 09 November 2016

Test animals

Species:
rat
Strain:
Wistar
Sex:
male

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single dose
Post exposure period:
12-16 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle

Examinations

Tissues and cell types examined:
DNA repair in male rat hepatocytes.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Dose range finding study

In the dose range finding test three males were dosed with 2000 mg K1600 black dye per kilogram body weight. In two animals the hairless body parts were dark coloured within 1 hour after dosing. The next day this staining was disappeared. The other animal showed no treatment related clinical signs or mortality after dosing. Black faeces was observed in the bedding of the cages. The study duration was two days.

 

On the second day a pilot perfusion was performed with two animals to identify any adverse effects on the liver at this dose level that could interfere with a good performance of the main experiment.

No macroscopic effects in the liver were observed and the viability of the liver cells was 69 and 79%, indicating that hepatotoxicity would not adversely influence the performance of thein vivorat hepatocyte-repair assay.

 

In vivo rat hepatocyte DNA-repair assay

Based on the results of the dose range finding study and the pilot perfusion dose levels of 2000 and 1000 mg/kg body weight were selected as appropriate doses for thein vivorat hepatocyte-repair assay.

 

Three male rats were used per sampling time in each treatment group, except for the vehicle and positive control group in whichtworatswereused per sampling time.

The body weights recorded immediately prior to dosing are presented inTable2(APPENDIX1).

 

Due to unknown reasons the slides of the test item treated animals (12-16 hours sampling time) could not be used for microscopic evaluation. Therefore this part of the experiment was repeated.

  Mortality / signs of toxicity

The animals of the negative and positive control groups showed no treatment related clinical signs or mortality. In addition the animals dosed with 1000 mg K1600 black dye/kg body weight (2 - 4 hours sampling time) showed no treatment related clinical signs.

 

Hairless body parts of animals treated with 2000 mg K1600 black dye/kg body weight were dark coloured within 1.5 and 12 hours after dosing (2 - 4 hours and 12 - 16 hours treatment time, respectively). All test item treated animals of the 12 - 16 hours treatment time had black faeces.

 

 Viability of the hepatocytes

At the 2 - 4 hour sampling time,the viability of the hepatocytes, used for the evaluation ofDNArepair inducing ability of K1600 black dye was at least 76% indicating no direct liver toxicity. A mean viability of 89% was found for the vehicle control culture.

 

At the 12 - 16 hour sampling time, the viability of the hepatocytes, used for the evaluation ofrepair inducing ability of K1600 black dye was at least 53% indicating no direct liver toxicity. A mean viability of 86% was found for the vehicle control culture. In the repeat experiment the viability of the hepatocytes, used for the evaluation ofrepair inducing ability of K1600 black dye was at least 81% indicating no direct liver toxicity. A mean viability of 96% was found for the vehicle control culture.

 

1.2.3.    ResultsDNArepair assay

In all slides used for grain counts sufficient cells of normal morphology to permit a meaningful assessment of unscheduled-synthesis were present. Preparations showed no or slight overt cytotoxicity (e.g. pyknosis£50%).

 

As a result of oral dosing with K1600 black dye the NNG per coverslip and per animal, as well as the group average revealed no positive response in this assay at any of the dose levels.

 

The percentage of cells in repair (repair taken as NNG≥5), both per individual animal and for the group average, revealed no increase at any dose.

 

1.2.4.    Acceptability

The NNG in the solvent-treated control cultures was within the historical control data range

 

Oral dosing of a male rat with dimethylnitrosamine () resulted in a NNG of 45.5 with 100% of the cells in repair (NNG>5). Oral dosing of a male rat with 2-acetylaminofluorene (2-AAF) resulted in a net nuclear grain count (NNG) of 34.5 and 31.7 with 100% of the cells in repair (NNG≥5).

 

In the scored coverslips the mean background of a single area of the same size as the corresponding nuclear area, located outside the cytoplasm, was always equal to or less than 13 grains, indicating that the autoradiographic procedure functioned adequately.

 

It can be concluded that the test system was functioning correctly.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
When treated orally with K1600 black dye at doses up to 2000 mg/kg body weight (maximum required dose) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2 - 4 or 12 - 16 hours after dosing, respectively.

In conclusion, K1600 black dye was not genotoxic in the DNA repair assay using hepatocytes obtained from male rats following in vivo exposure to the test item under the conditions described in this report.
Executive summary:

Evaluation ofrepair inducing ability of K1600 black dye in male rat hepatocytes (in vivorat hepatocyterepair assay).

 

This report describes therepair inducing ability of K1600 black dye in male Wistar rat hepatocytes, measured as unscheduledsynthesis ().

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch G-173 of K1600 black dye was a black powder with a purity of 100% (Limit:>98.5% On Dry Basis). The test item was suspended in physiological saline.

 

In the dose range finding study male animals were dosed by oral gavage with 2000 mg K1600 black dye per kg body weight. In two animals the hairless body parts were dark coloured within 1 hour after dosing. The next day this staining was disappeared. The other animal showed no treatment related clinical signs or mortality after dosing. Black faeces was observed in the bedding of the cages. The study duration was two days. On the second day a pilot perfusion was performed with two animals dosed with 2000 mg/kg body weight. The viability of the hepatocytes was within the normal range (69% and 79%).

 

Two groups of 5 male Wistar rats received a single oral dose of 2000 or 1000 mg K1600 black dye/kg body weight for both sampling times (2 - 4 and 12 - 16 hours). Hairless body parts of animals treated with 2000 mg/kg body weight were dark coloured. The animals treated with 1000 mg/kg body weight showed no treatment related clinical signs after dosing. All test item treated animals of the 12 - 16 hours treatment time had black faeces.

 

Corresponding vehicle treated groups served as negative controls. Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (, 10 mg/kg body weight) or
2-acetylaminofluorene (2-AAF, 50 mg/kg body weight) were harvested 2 - 4 or 12 - 16 hours after dosing respectively. No treatment related clinical signs or mortality were noted in control animals. Two slides per animal and one to two animals for each control group were examined.

 

As a result of oral dosing with K1600 black dye the net nuclear grain count (NNG) per slide and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG≥5), both per individual animal and for the group average, revealed no significant increase at any dose.

 

The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system functioned properly.

 

It is concluded that K1600 black dye is not genotoxic in the-repair assay using hepatocytes obtained from male rat liver followingin vivoexposure for 2 - 4 or 12 - 16 hours to K1600 black dye up to concentrations of 2000 mg/kg body weight under the conditions described in this report.