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Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian cell study: DNA damage and/or repair
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
(Revised Draft March 1996)
GLP compliance:
(Department of Toxicology, BASF AG)
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Test substance No. 96/125
Batch: 65-3997
Purity: 99.9%
Physical state: colorless crystals
Storage: refrigerator
Stability: verified by reanalysis throughout the study period
Homogeneity: guarantted on account of the high purity

Test animals

Details on test animals or test system and environmental conditions:
Designation: Wistar rats (Chbb : THOM ; SPF)
Source: Dr . Karl Thomae GmbH, Biberach an der Riss, Germany
Mean body weight at test initiation: ca. 258.1 g
Housing: individually in Makrolon cages, type III, in fully air-conditioned rooms
Environmental conditions: temperature 20 - 24°C, relative humidity 30 - 70%
Identification: by means of cage cards
Light/dark cycle: 12 hrs/12 hrs
Feed: standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
Drinking: drinking water from bottles, ad libitum
Bedding, feed and drinking analysis for contaminants: yes

Administration / exposure

Route of administration:
oral: gavage
Purified water, 10 ml/kg bw
Details on exposure:
Preliminary test for determination of test doses:
In an acute oral toxicity pretest, the animals survived a dosage of 2000 mg/kg bw (recommended as the highest dose according to the OECD Revised Draft Guideline) without showing any clinical signs. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present study; 1000 mg/kg, 500 mg/kg and 250 mg/kg bw administered as further doses.

Test groups:
The test was conducted with 12 groups of 3 animals each, i.e. 2 vehicle control groups, 2 positive control groups, and 4 x 2 test groups (see below). Within each set of two groups, the animals of one group were subjected to liver perfusion after 4 hours following treatment, whereas the animals of the second group were subjected to liver perfusion after 18 hours.

Dosage levels and application volumes:
Two test groups were given 250 mg test substance/kg bw, i.e. 10 ml/kg bw of a solution with a concentration of 2.5 g test substance/100 ml;
Two test groups were given 500 mg test substance/kg bw, i.e. 10 ml/kg bw of a solution with a concentration of 5.0 g test substance/100 ml;
Two test groups were given 1000 mg test substance/kg bw, i.e. 10 ml/kg bw of a solution with a concentration of 10.0 g test substance/100 ml;
Two test groups were given 2000 mg test substance/kg bw, i.e. 10 ml/kg bw of a solution with a concentration of 20.0 g test substance/100 ml.
Duration of treatment / exposure:
Following the single oral administration of the test substance, the liver was perfused after 4 and 18 hours post treatment.
Frequency of treatment:
Single gavage.
Post exposure period:
4 and 18 hours
Doses / concentrations
Doses / Concentrations:
250, 500, 1000, 2000 mg/kg bw

No. of animals per sex per dose:
Each test group consisted fo 3 animals.
Control animals:
Positive control(s):
- 2-acetylaminofluorene (2-AAF) was tested as positive substance at a dose of 50 mg/kg bw;
- Corn oil was used as solvent and the application volume was 10 ml/kg bw;
- Two groups of 3 animals each were treated with 2-AAF;
- For the first group, liver perfusion was done after 4 hours following treatment;
- For the second group, liver perfusion was done after 18 hours following treatment.


Tissues and cell types examined:
- Following perfusion of the liver of the anesthetized animals, the organ was removed and the hepatocytes were isolated. For isolation of primary rat hepatocytes, the method of Butterworth et al. was used (Mut. Res. 189: 113-121, 1987);
- The isolated cells were subjected to washing, centrifugation and resuspension steps, until obtaining single cell suspensions;
- Cell viability was checked by means of Trypan blue staining, and the cells were counted;
- The cells were seeded on coverslips which were placed in wells containing attachment medium; about 400,000 viable cells were seeded/well;
- Six wells/per animal were used for the UDS assay;
- After a 2 hour incubation (37 °C, 5% CO2, > 90% humidity), medium was renewed and non-adherent cells were removed;
- After a further incubation, the medium was replaced by fresh medium containing 3H-thymidine, and the cells were incubated again for 4 hours;
- The medium was renewed after the 4 hours of incubation, and the cells were incubated in unlabeled medium for another 14 hours;
- The cells were fixed (ethanol/acetic acid, 3:1) for at least 30 minutes, rinsed and air-dried. The coverslips were mounted cell side up on glass slides using Corbit-Balsam, and were dried overnight;
- DNA damage and repair was measured as expressed by the incorporation of 3H-thymidine, using autoradiography (Butterworth et al. (1987);
- The cells were examined for viability, morphological changes, and reduction in cell material following treatment;
- The UDS quantification was performed microscopically on 2 or 3 slides per test group; a total of 100 cells/animal was examined;
- Counting was performed by means of an automatic image analyzer (ARTEK); following parameters were considered:
- (1) Net nuclear grain (NNG) count/cell
- (2) Mean nuclear grain (NG) count
- (3) Mean cytoplasmic grain count (CG) count
- (4) Mean net nuclear grain count
- (5) Percentages of cells in repair (cells with NNG >= 0 and cells with NNG >= 5)

Evaluation criteria:
Acceptance criteria:
-Clearly negative results in the untreated and in the vehicle controls in the range of historical control data.
-Clearly positive results in the positive control group (>=30% of cells in repair, as mean of 3 animals) in the range of historical control data.
-Viability (trypan blue staining) of at least 70% in hepatocytes from negative control animals.

Evaluation criteria:

-Positive response: a positive response implicates a dose-related increase in mean number of NNG counts (> 0 at one of the test points) and in percentage of cells in repair (i.e. cells with NNG >= 5), which must be >= 20.

-Marginal response: a response is considered marginal when the dose-related increase in percentage of cells in repair is >= 5 outside the values of both the concurrent negative control and the historical control (>= 5 < 20), and when the dose-related increase in mean number of NNG counts is near to but without exceeding 0. In this case, an additional confirmatory test is needed.

-Negative response: a negative response implicates that both, the NNG counts and the percentage of cells in repair are within the range of negative control.
Because of clear negative findings, no statistical evaluation was needed.

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:
1,3,5-Trioxan up to doses of 2000 mg/kg bw did not lead to any clinical signs of toxicity.
The single administration of 50 mg/kg bw 2-AAF as positive control substance did not cause any evident signs of toxicity.
The single oral administration of the vehicle in a volume of 10 ml/kg bw was tolerated by all animals without any signs or symptoms.

1,3,5-Trioxan did not lead to an increase in the mean number of net nuclear grain counts from 500 mg/kg up to 2000 mg/kg bw. The values at any dose were nearly the range of the concurrent negative controls (vehicle controls) and the range of historical control data. Therefore, an evaluation of the lowest dose group of 250 mg/kg bw was omitted.
The induction of DNA repair by the positive control substance 2-AAF clearly demonstrated the sensitivity of the test method applied.

Any other information on results incl. tables

DNA Repair Activity 
Dose Percent cells in repair
4-Hour Harvest 18-Hour Harvest
0 8.67 7.33
250 Not examined Not examined
500 10.0 12.0
1000 8.67 11.7
2000 10.0 6.33
Positive Control 78.7 84.7

Applicant's summary and conclusion