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Toxicological information

Genetic toxicity: in vivo

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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: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Methyl-2-butenal, CAS: 107-86-8; Substance-No. 00/0680-1
- Analytical purity: 97%
- Physical state: Colorless to yellowish liquid
- Lot/batch No.: B 3290 from 28.08.2000
- Storage condition of test material: Room temperature (N2 conditions)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: mean weight of about 254 g
- Assigned to test groups randomly: randomization plan prepared with an appropriate computer program
- Housing: individually in Makrolon cages, type III, for the duration of about one week (until treatment) in fully air-conditioned rooms
- Diet: Standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: In comparison to other commonly used vehicles (e .g. DMSO, corn oil etc.) water is the most suitable (formation of an emulsion). Therefore, water was selected as vehicle.
- Concentration of test material in vehicle: 3.5 g/100 ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.

The UDS test measures DNA repair synthesis after excision and removal of a stretch of DNA containing the region of the damage induced. Repair is detected by the incorporation of tritrium-labeled thymidine (3H-thymidine) into the DNA that was not in the normal phase of the DNA synthesis (S-phase) and is determined by autoradiography. Cells undergoing repair are identified by an increase in the number of silver grains overlying the nuclei.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single application
Post exposure period:
none, animals were immediately killed after 3 and 14 hrs, respectively
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 350, 700 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
3 (males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substances: 2-acetylaminofluorene
- Justification for choice of positive control(s): 2-AAF is a well-established UDS-inducing agent
- Route of administration: oral
- Doses / concentrations: 50 mg kg bw (10 ml/kg bw)

Examinations

Tissues and cell types examined:
rat hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, deaths were observed down to a dose of 800 mg/kg body weight. 700 mg/kg were survived by all animals, but led to strong clinical signs such as abdominal position, squatting posture, piloerection, tremor, salivation, gasping respiration, eye discharge and eyelid closure ; the general state of the animals was poor. Therefore, a dose of 700 mg/kg body weight was selected as the highest dose in the present study; 350 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
The isolated hepatocytes were seeded on coverslips on 1 .9 cm2 well containing 2 ml of attachment medium (WMEC).
• about 400,000 viable cells were seeded per well.
• 6 wells per animal were used for the UDS assay.
After an attachment period of about 2 hours with 5% CO2 at 37°C and >= 90% humidity, the medium (WMEC) was replaced by fresh medium (WMEI) to remove non-adherent cells.
Evaluation criteria:
The quantification of UDS was performed microscopically using 2 or 3 slides per test group. 25 - 50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal.

A test substance is considered positive if a dose-related increase is demonstrated in both of the following :
• The mean number of NNG counts, which must exceed zero at one of the test points .
• The percentage of cells in repair (NNG >= 5) when >= 20 .
A dose-related increase in % cells in repair >= 5 outside the values of both the concurrent negative control and the historical control data base (>= 5 < 20) and a dose-related increase in the mean number of NNG counts near to but without exceeding zero is considered to be an indication for a marginal response which needs to be confirmed / clarified in a further experiment.
A test article producing both NNG counts and % cells in repair in the range of the negative control data is considered to be negative in the in vitro UDS assay.
Statistics:
Due to clear negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0-800 mg/kg bw
- Clinical signs of toxicity in test animals: abdominal position, squatting posture, piloerection, tremor, salivation, gasping respiration, eye discharge, eyelid closure; the general state of the animals was poor

The single oral administration of the test substance in a dose of 700 mg/kg body weights led to evident signs of toxicity; 350 mg/kg body weight were tolerated without any signs is or symptoms.
Hepatocytes were harvested 3 and 14 hours after administration of the test substance.
As a negative control, male rats were administered merely the vehicle, purified water, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range.
The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis.
On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes.

Any other information on results incl. tables

Test groups

Vehicle control

350 mg/kg

700 mg/kg

positive control

NG counts Mean ± SD**

4.88 ± 1.33

5.04 ± 0.89

4.70 ± 0.1 5

15.89 ± 3.01

CG counts

Mean ± SD**

9.35 ± 1.69

9.17 ± 1.45

8.67 ± 0.93

10 .05 ± 1.96

NNG counts

Mean ± SD**

-4.48 ± 0.37

-4.13 ± 0.70

-3.97 ± 0.78

5.84 ± 1.1 5

% cells in repair NNG ≥ 0

Mean ± SD**

9.67 ± 1.53

8.67 ± 4 .04

9.00 ± 3.6 1

86.67 ± 5.5 1

% cells in repair NNG ≥ 5

Mean ± SD**

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

51.33 ± 2.52

 DNA repair activity - perfusion 3 hours after treatment        

Test groups

Vehicle control

350 mg/kg

700 mg/kg

positive control

NG counts Mean ± SD**

4.78 ± 0.74

4.33 ± 0.44

5.24 ± 0.32

16.63 ± 2.24

CG counts

Mean ± SD**

8.92 ± 0.54

8.20 ± 0.76

9.36 ± 0.97

9.58 ± 1.05

NNG counts

Mean ± SD**

-4.14 ± 0.24

-3.87 ± 0.68

-4.12 ± 0.70

7.05 ± 1.25

% cells in repair NNG ≥ 0

Mean ± SD**

9.00 ± 3.61

8.33 ± 4.93

8.67 ± 0.58

88.00 ± 2.65

% cells in repair NNG ≥ 5

Mean ± SD**

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

57.67 ± 2.89

 DNA repair activity - perfusion 14 hours after treatment        

NG: nuclear grains

CG: cytoplasmic grains

NNG: net nuclear grains

** = mean per group (mean of 3 animals )

SD = standard deviation

Clinical examination:

The single oral administration of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms .

The oral administration of the test substance in a dose of 700 mg/kg led to strong clinical signs. 350 mg/kg were tolerated without any signs or symptoms.

The single administration of the positive control substance 2-AAF in a dose of 50 mg/kg body weight did not cause any evident signs of toxicity.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions of this assay, the test article 3-Methyl-2-butenal is considered to be negative in the in vivo UDS assay using rat hepatocytes.