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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Reactive Yellow 138:1 Na/K

Test animals

Details on test animals or test system and environmental conditions:
- Source: ICI Barriered Animal Breeding Unit (BABU), Alderley Park, Macclesfield.
- Age at study initiation: 6-8 weeks
- Assigned to test groups randomly: the animals were allocated according to the order in which they were removed from the stock cage
- Housing: 5/cage
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period:

- Temperature (°C): 21 ± 2
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
The vehicle for the test agent and negative control substance was deionised water

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For dosage,the test substance was dispersed in deionised water by homogenisation. Concentrations prepared for UDS assay were 125 and 200mg/mi. All dosing preparations were administered at a volume of 10 mL/kg bw.

2-AAF was prepared as a 2.5mg/mi suspension in 0.5% hydroxypropylmethyl cellulose in 0.1% aqueous PS80 (HPMC) by high speed homogenisation. DMH.2HCl was dissolved at 3 mg/mL in deionised water by hand shaking. All dose solutions were used within a day of preparation.

Duration of treatment / exposure:
Preparation of hepatocytes took place 2 or 16 hours after dosage.
Frequency of treatment:
Single dose

Post exposure period:
2 and 16 h

Doses / concentrationsopen allclose all
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
preliminary study: 5 (treatment group only)
5 males/dose in treatment groups
2 males/group in vehicle and positive control groups

Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control substances were 2-acetylaminofluorene (2-AAP) for 16 hour treatments and dimethylhydrazine dihydrochloride (DMH.2HCl) for 2 hour treatments.


Tissues and cell types examined:

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a preliminary study, in which the acute MTD for the test substance was >2000 mg/kg

DETAILS OF SLIDE PREPARATION: Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic coverslips.
Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After approximately 4 hours incubation at 37°C within a 5% CO2/95% (v/v) air atmosphere, the medium was removed, the cells washed three times and the cultures incubated overnight with medium containing an excess of unlabelled thymidine.
Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 days at 4°C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS. Slides were scored under code.
Coded hepatocyte preparations were examined for the induction of UDS using a microscope-mounted image analyser linked to a computer for data analysis

METHOD OF ANALYSIS: The number of silver grains over the nucleus [N] was determined. Then an equivalent area of cytoplasm tangential to the nucleus and with the highest apparent number of silver grains was scored [C]. The difference between these two values [N-C] was the net nuclear grain count. Appendix C shows the total number of cells scored for each animal.
Normally 60 cells were scored from each animal using two slides. The third slide was not normally read, unless a total of 60 cells could not be obtained from the first two slides examined.
Evaluation criteria:
Criteria for a positive response:
- A mean animal net nuclear grain count [N-C] value of greater than zero represents a biologically significant departure from normal.
- The radiolabelling of the nucleus exceeds that of the cytoplasm, indicating of a real net synthesis of nuclear DNA
Criteria for a negative response:
- The mean net nuclear grain count of all test substance treated animals is less than 0.

Results and discussion

Test results
Key result
no effects
orange discolouration of urine and internal organs served as proof for exposure
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:

Applicant's summary and conclusion

Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw

Executive summary:

 The test substance was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay incorporating an autoradiographic technique. Male rats were given a single oral dose by gavage at 1250 or 2000 mg/kg body weight. The highest test treatment, 2000 mg/kg, was the limit dose for this assay. Two sampling times were used, 2 hours and 16 hours following administration of the chemical. Two independent experiments were carried out at each time point, validated by concurrent control treatments of rats with the vehicle and with the carcinogens 2-acetylaminofluorene (2-AAF) or dimethylhydrazine dihydrochloride (DMH.2HCl).

Hepatocytes from treated rats were assessed for the induction of UDS at both dose levels, 1250 and 2000 mg/kg bw. Examination of the mean net nuclear grain count and percentage of cells in repair showed that the test substance did not induce DNA repair, as measured by UDS, at either dose level or time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg, the test substance did not induce DNA repair in the liver of the rat in vivo.