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Genetic toxicity: in vivo

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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:
guideline study

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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
- Physical state: homogeneous colourless-clear liquid
- Analytical purity: 50.5%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: ca. 50% glutaraldehyde in water
- Lot/batch No.: 50-4402
- Storage condition of test material: refrigerator (under N2 conditions)

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland GmbH
- Age at study initiation: about 10 to 12 weeks
- Weight at study initiation: about 256 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type III
- Diet (e.g. ad libitum): standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) , ad libitum
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: not specified
- Other: feed, water and bedding were analyzed for contaminants.

- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Administration / exposure

Route of administration:
oral: gavage
purified water
Details on exposure:
All test solutions were prepared once weekly before administration. The amount of test material or volume to be administered was related to the specific weight of the individual animals on the day of treatment.

In a pretest for the determination of the acute oral toxicity (male and female rats), deaths were observed down to a dose of 400 mg/kg body weight (1 out of 14 animals were found dead the day after test substance administration). The clinical signs observed were piloerection, squatting posture, gasping respiration and bloody snout and the general state of the animals was poor. However, there were no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the main experiment.
Therefore, a dose of 400 mg/kg body weight was selected as the maximal tolerated dose level (MTD) for the main assay, and 200 mg/kg body weight were administered as further dose; both dosages refer to the aqueous test substance and not to the active ingredient, i.e.
Glutaraldehyde (about 50%).

The test series consisted of following groups:
Control group 1 and 2: vehicle controls with animals receiving 10 mL water/kg bw.
Test groups 3 and 4: animals receiving 200 mg test substance/kg bw, application volume 10 mL/kg bw of a 2% test solution.
Test groups 5 and 6: animals receiving 400 mg test substance/kg bw, application volume 10 mL/kg bw of a 4% test solution.
All the animals were treated once and were examined for clinical signs of toxicity and body weight change.
The animals were sacrificed and their livers perfused 3 hours and 14 hours after treatment.
Duration of treatment / exposure:
Not applicable as single application by gavage
Frequency of treatment:
Single application
Post exposure period:
3 and 14 hrs after single dose administration
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
the test concentrations refer the test substance as such and not to the active ingredient
Dose / conc.:
400 mg/kg bw/day (nominal)
the test concentrations refer the test substance as such and not to the active ingredient
No. of animals per sex per dose:
3 animals were used per group.
Control animals:
Positive control(s):
As a positive control, 50 mg of 2-acetylaminofluorene/kg bw, suspended in corn oil, was administered to the animals once orally in a volume of 10 mL/kg body weight.
The stability of 2-AAF is well-defined under the selected culture conditions, since 2-AAF is a well-established UDS-inducing agent.


Tissues and cell types examined:
The hepatocytes were isolated from the liver of the treated animals 3 and 14 hours after treatment according to the procedure described by Butterworth BE et al., Mutat. Res. 189(2): 123-133, 1987). The main steps were as follows:
- Anesthetization of the animals with Metofane by inhalation;
- Liver perfusion with ethylene glycoI-bis(ß-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) solution followed by collagenase solution;
- Obtention of a cell suspension;
- Filtration, centrifugation steps with removal of supernatant and obtention of cell pellets;
- Resuspension of the cell pellets to yield single ceIl suspensions.

The hepatocytes were examined for cell viability as measured by the trypan blue exclusion technique, and for conspicuous changes in cell morphology.
DNA damage and repair was measured by incorporation of 3H-Thymidine using autoradiography technique. For this purpose about 400,000 viable cellson coverslips were seeded per well and 6 wells per animal were used for the UDS assay. For labeling, the initial medium was replaced by labeling medium, and the cells were incubated for 4 hours. The cells on the coverslips were then fixed, rinsed and air-dried before being mounted cell side up an glass slides using Corbit-Balsam.
Autoradiography was carried out according to the procedure of Butterworth et al. (see above), and for this purpose the slides were coated with KODAK NTB-2 photographic emulsion.
about 5 -10 seconds.
In fact, for microscopical evaluation and quantification of UDS, a total of 100 cells/animal was examined.
Following parameters were considered:
- the net nuclear grain (NNG) count/cell,
- the mean nuclear grain (NG) count,
- the mean cytoplasmic grain count (CG) count,
- the mean net nuclear grain count,
- the % of cells in repair (cells with NNG >= 0; 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.
Due to clear negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
The microscopical evaluation and quantification of UDS showed that the results obtained for the GA-treated animals were within the range of negative controls, as can be seen from the table included below.
Evident signs of toxicity were seen in animals treated with the test material at both test doses and included piloerection, squatting posture and bloody snout.
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
For all treated animals including those serving for positive control, the cell viability after 3 and 14 hours was within 94 - 102% of control.
The microscopical evaluation and quantification of UDS showed that for the negative controls, the frequencies of mean nuclear grain counts were within historical control range.
For the positive controls, the increase in unscheduled DNA synthesis was as expected.
The results obtained for the glutaraldehyde-treated animals were within the range of negative controls, as can be seen from following table.

Any other information on results incl. tables

Test group

Negative Control.

Protectol GDA 200 mg/kg bw

Protectol GDA 400 mg/kg bw

Positive control

Cells harvested after 3 hours following treatment

NG counts*

6.79 +/- 0.99

7.79 +/- 1.26

6.47 +/- 0.27

21.23 +/- 1.82

CG counts*

12.07 +/- 1.46

14.61 +/- 0.26

12.45 +/- 0.19

15.46 +/- 1.40

NNG counts**

- 5.28 +/- 0.65

- 6.82 +/- 1.02

-5.98 +/- 0.42

5.77 +/- 2.57

% cells in repair (NNG >= 0)

8.33 +/- 0.58

6.00 +/- 3.61

3.33 +/- 1.53

80.33 +/- 9.07

% cells in repair (NNG >= 5)

0.00 +/- 0.00

0.00 +/- 0.00

0.00 +/- 0.00

50.67 +/- 14.98

Cells harvested after 14 hours following treatment

NG counts*

7.88 +/- 2.31

7.66 +/- 0.43

6.48 +/- 0.64

19.82 +/- 2.38

CG counts*

13.01 +/- 2.70

13.67 +/- 1.17

11.57 +/- 1.36

13.63 +/- 1.62

NNG counts**

-5.13 +/- 0.61

-6.01 +/- 0.76

-5.09 +/- 0.72

6.20 +/- 1.04

% cells in repair (NNG >= 0)

8.33 +/- 0.58

6.33 +/- 4.51

6.33 +/- 2.08

80.00 +/- 3.61

% cells in repair (NNG >= 5)

0.00 +/- 0.00

0.00 +/- 0.00

0.00 +/- 0.00

52.33 +/- 4.73

NG, nuclear grains; CG, cytoplasmic grains, NNG, net nuclear grains; *, mean per group (3 animals) with standard deviation

Applicant's summary and conclusion