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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:

- Physical state: solid, brown

- Stability under test conditions: the stability of the test substance under storage conditions throughout the study period was guaranteed until 2012 as indicated by the sponsor, and the sponsor holds this responsibility. The homogeneity of the test substance is guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Han rats, Crl:WI (Han), from Charles River Laboratories Germany GmbH
- Age at study initiation: 8 – 10 weeks
- Weight at study initiation: 233.7 g (± 8.80 g)
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type M III
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- 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: corn oil
- Concentration of test material in vehicle: 100 or 200 mg/ml, respectively for the low and high dose groups
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was suspended in corn oil. To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations, and the determination of the concentrations in the vehicle was carried out by means of UV/VIS spectrometer.
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
The livers were perfused 3 and 14 hours after the administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
4 (4 animals were treated per test group, but only three animals were prepared. In case of a failing preparation the reserve animals would have been prepared. Remaining reserve animals were killed by cervical dislocation after anesthetization with Narcoren on the same day.)
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; 2-acetylaminofluorene (2-AAF; 5 mg/ml in corn oil)
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Liver; hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: in a pretest for the determination of the acute oral toxicity, at 2000 mg/kg bw recommended as the highest dose according to the OECD Guideline, all animals (male and female) tolerated without any signs of toxicity. Due to missing differences in clinical observations between male and female animals, males was used in the main experiment as requested by the current OECD Guideline 474. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg bw was administered as further dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: The primary rat hepatocytes were isolated by means of in situ perfusion under cold conditions (e.g. beakers and centrifuge tubes on ice).
- Liver perfusion and preparation of the hepatocytes: 3 and 14 hours after treatment the animals were anesthetized, and, after removal of the livers and isolation of the hepatocytes, the cells were washed and mixed thoroughly with a pipette. The cell suspension was then filtered through sterile gauze and the volume was adjusted prior to centrifugation, after which the supernatant was discarded and the sediment resuspended to yield single cell suspensions.
- Determination of the cell viability: viability of the hepatocytes was determined by means of vital staining (trypan blue vital dyeexclusion method), and the yield was determined by cell counting in the counting chamber.
- Culture conditions (seeding and attachment period): the isolated hepatocytes of each animal (200000 viable hepatocytes per mL) were seeded into 4 - 6 wells (with coverslips) of a 6-well-plate for autoradiographic evaluation. 2 mL of this suspension (corresponding to 400000 viable cells) was seeded per well (1.9 cm2). Subsequently, the cells were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity

METHOD OF ANALYSIS:
- Preparation of test cultures; labeling for the UDS determination: after an attachment period (at leat 2 hours), 2 mL labeling solution containing 3H-thymidine was added to the adherent wells for an incubation period of 4 hours under the conditions specified above. The wells were then rinsed, incubated 12 hours with unlabeled thymidine solution, washed, the cells were fixed with ethanol/acetic acid (raatio 3:1) and rinsed 3 times. The coverslips were then taken out of the wells and air-dried. The dry coverslips were mounted cell-side-up on glass slides using Corbit-Balsam TM. After drying overnight, autoradiography was carried out.
- Autoradiography:
Following operations were carried out in the darkroom under red light: the slides were immersed in undiluted Kodak nuclear track emulsion NTB at about 37°C for about 5 – 10 seconds, dried at room temperature overnight and subsequently stored in the dark with a desiccant at about -20°C for at least 3 days. Thereafter, the slides were left at room temperature for at least 3 hours and then they were treated with Kodak D-19 developing solution at about 15°C for 3 - 5 minutes. Immediately afterward, the developing solution was rinsed with water. The rinsed slides were fixed in Kodak fixer for about 5 minutes. Subsequently, the slides were rinsed with running deionised water for about 5 – 10 minutes.

The slides were stained with hematoxylin-eosin and after having dried in the air, they were covered with a second coverslip using Corbit-Balsam TM. In general, only 3 out of 6 slides prepared per test group were initially developed autoradiographically. The remaining slides were kept as a reserve for second autoradiography, if required.

- Microscopic evaluation: at least 100 cells in good morphological condition were evaluated per animal using at least 2 slides. Three animals were evaluated per test group. The cells were selected at random. Care was taken that no cell was evaluated twice and cells were recorded from all 4 quadrants of the slides. The few number of heavily radiolabeled hepatocytes (proliferating cells) can be recognized easily and were excluded from evaluation.
The following parameters were determined per cell using an automatic image analyzer: (1) Nuclear grain count (NGC): number of silver grains overlying the nucleus; (2) Cytoplasmic grain count (CGC): number of silver grains in one adjacent cytoplasm area about the size of the nucleus
The following parameters were calculated based on the data of the evaluation: (1) Net nuclear grain count (NNGC) per cell: nuclear grain count minus cytoplasmic grain count (NGC - CGC); (2) Mean nuclear grain count (NGC); (3) Mean cytoplasmic grain count (CGC); (4) Mean net nuclear grain count (NNGC); and (5) Percentage of cells in repair: amount of cells showing a net nuclear grain count of ≥ 5.


OTHER:
- After the administration of the test substance up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Cytotoxicity test: in addition to the determination of viability using the trypan blue vital dye-exclusion method, the hepatocytes were checked microscopically for morphological changes or reduction of the cell material.
Evaluation criteria:
- Acceptance criteria: the in vivo UDS test is considered valid if the following criteria are met: (1) viability (trypan blue vital dye-exclusion method) of at least 70% in liver cells from the vehicle control animals. (2) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 300 cells per test group. (3) The mean net nuclear grain count (NNGC) of the negative controls (vehicle controls) has to be below zero and within the range of the historical negative control data. (4) The mean NNGC of the positive control group has to be distinctly increased compared to the concurrent negative control group. The values should be clearly above zero and they should be within the range of the historical positive control data or above. In addition, the percentage of cells in repair should be distinctly increased compared with the concurrent negative control group.
- Assessment criteria: a test substance is considered positive if an increase is demonstrated in both of the following: (1) the mean NNGC must exceed zero at one of the dose groups. (2) The mean NNGC clearly exceeds the value of the concurrent negative control group at one of the dose groups. Statistical significance may give further evidence for a positive evaluation. However, both biological relevance and statistical significance should be considered together. A dose-related increase of the percentage of cells in repair (NNGC ≥ 5) with values of ≥ 20%, and a dose-related increase in the mean number of NNGC of about zero is considered to be an indication for a marginal response which needs to be confirmed / clarified in a further experiment.
A test substance is considered negative if the following criteria are met: (1) in all dose groups, the mean NNGC are close to the values of the concurrent negative control group and within the range of the historical negative control data.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
no relevant increase in the mean number of net nuclear grain counts (NNGC) was observed either 3 hours or 14 hours after single oral administration of the test substance.
Toxicity:
no effects
Remarks:
the test substance led to discoloration of feces at the 3 and 14 hours sacrifice interval
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- In both parts of the study the mean net nuclear grain counts per animal of the test substance treated dose groups (-4.30 to -3.09) were close to the respective vehicle control values (-4.62 to -3.55) and within the historical negative control data range (-1.96 to -7.92).

- The rate of cells in repair (NNGC ≥ 5) per animal was in the range from 0% to 1%. The values were within the range of the respective vehicle control values (0% to 1%) and within the historical negative control data range (0% to 1%).

- Besides at 3-hour and 14-hour sampling time the positive control substance 2-AAF (50 mg/kg body weight) induced clearly increased DNA repair activity indicated by distinctly increased mean net nuclear grain counts per animal (0.87 to 9.96) and distinctly increased rates of cells in repair (26% to 74%). The values of both parameters were in the expected range of the historical positive control data except for one animal at 3-hour sampling time. Although the values obtained for that animal were slightly below the historical control data range the criteria of acceptance for positive controls were fulfilled. The mean net nuclear grain count (0.87) was below zero and both the mean net nuclear grain count and the rate of cells in repair (26%) were distinctly increased compared to the respective vehicle control group.

- Cytotoxicity: the cell viability indicated by trypan blue exclusion was not influenced by test substance treatment. The values varied between 70.1% and 90.0%. Thus, the cell preparation was adequate and the test substance treatment did not influence this quality parameter. The number of primary hepatocytes were not reduced and there were no changes in cell morphology after test substance treatment in any case.

Any other information on results incl. tables

Table 1: Summary table – DANN repair activity

Test group (mg/kg bw)

Sampling time (hours)

Number of animals

Net nuclear grain count ± standard deviation

Cell in repair (%)

Vehicle control

3

3

-4.16 ± 0.53

0

Test substance (1000)

3

3

-3.95 ± 0.31

0

Test substance (2000)

3

3

-3.71 ± 0.39

1

Positive control (2-AAF, 50)

3

3

4.26 ± 4.67

45

Vehicle control

14

3

-4.39 ± 0.20

0

Test substance (1000)

14

3

-3.75 ± 0.21

1

Test substance (2000)

14

3

-3.50 ± 0.35

1

Positive control (2-AAF, 50)

14

3

7.80 ± 1.88

62

 

Applicant's summary and conclusion