Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
15. Jul 1996 - 16. Oct 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study (OECD No. 486) performed on analogue substance 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Karl Thomae GmbH, Biberach an der Riss, Germany.- Weight at study initiation: 254g (mean)- Housing: Makrolon cages, type III- Diet: Standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) was available ad libitum.- Water: drinking water from bottles was available ad libitum.ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24 °C- Humidity (%): 30-70%- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). During the time between treatment and perfusion the day/night rhythm was not followed.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle/solvent used: CMC (carboxymethyl cellulose).- Justification for choice of solvent/vehicle: Due to the Iimited solubility of the test substance in water, a 0.5% CMC formulation was selected as the vehicle.- Concentration of test material in vehicle: Test group 3 and 4: 5g/100ml; Test group 5 and 6: 10g/100ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: To achieve homogeneity of the test substance in the vehicle, the test substance formulation was stirred constantly during removal and administration.
Duration of treatment / exposure:
12 and 18h
Frequency of treatment:
The treatment consisted of a single oral administration.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:500 mg/kg Basis:nominal conc.dose group 3 and 4
Remarks:
Doses / Concentrations:1000 mg/kgBasis:nominal conc.dose group 5 and 6
No. of animals per sex per dose:
3
Control animals:
yes, historical
Positive control(s):
2-acetylaminofluorene;- Justification for choice of positive control(s): 2-acetylaminofluorene is a well-established UDS-inducing agent.- Route of administration: oral- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Isolated primary rat hepatocytes.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Culture conditions (seeding and attachment period):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 (WMF 1) to remove non-adherent celIs.LabelingThe medium (WMEI) was replaced by 2 ml labeling medium, and the cells were incubated with 5% CO2 at 37°C and > 90% humidity for 4 hours. After the labeling period, cells were washed with HBSS or WMEI (about 37°C); then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 :1, v/v) for at least 30 minutes, rinsed 2 - 4 times with aqua dest. and air-dried. The dried coverslips were mounted ceIl side up on glass slides using Corbit-Balsam and dried overnight.Autoradiography:The slides were coated with KODAK NTB-2 photographic emulsion (about 37°C) for about 5 - 10 seconds. After drying at room temperature in the dark (Iightproof boxes) for about 16 hours, the coated slides were started in the dark with a desiccant (in the presence of a drying agent) at -20°C for 2 -10 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15°C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5 -10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a second coverslip using Corbit-Balsam.METHOD OF ANALYSIS: Quantification of UDS/Microscopic evaluation:The quantification of UDS was performed microscopically generally using 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. For each celI, the following counts were performed with an automatic image analyzer (ARTEK):- the nuclear grain (NG) count (= number of silver grains overlying the nucleus)- the cytoplasmic grain (GG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).The following parameters were calculated:- the net nuclear grain (NNG) count of each cell (= nuclear grain count minuscytoplasmic grain count; NG - GG)- the mean nuclear grain (NG) count- the mean cytoplasmic grain (GG) count- the mean net nuclear grain (NNG) count- the percentage of cells in repair (cells showing net nuclear grain counts of > 0)- the percentage of cells in repair (cells showing net nuclear grain counts of > 5)Slides were coded before microscopic evaluation.
Evaluation criteria:
The test substance was considered positive if a dose-related increase was demonstrated in both of the following:- The mean number of net nuclear grain (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 very clear negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
(500 mg/kg and 1000 mg/kg)
Toxicity:
no effects
Remarks:
no cytotoxicity noted (trypan blue exclusion technique)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
After the single oral administration of the test substance preparation, all animals showed symptoms in form of:-piloerection-orange discoloration of for- and hindlimbs, tail and ears-yellow to brownish discoloration of the lymphatic organs, the adiposal tissue and the liver -anogenital region smeared with feces and test substanceThe urine of the animals was discolored orange/darkbrown.Furthermore, 5 out of 11 animals in the 1,000 mg/kg group died.

Any other information on results incl. tables

DNA repair activity

Perfusion 12 hours after treatment (mean of 3 animals ± standard deviation)

 Test Group  Vehicle control 500 mg/kg 1000 mg/kg positive control       
 NG counts 6.53± 1.84 7.06 ±0.70 8.19±1.89 18.11 ±4.44      
 CG counts 10.17±2.04 10.73±0.77 10.41 ±1.26 11.23 ±1.60      
 NNG counts -3.64 ±0.47 -3.67 ±0.62 -2.21 ±2.45 6.88 ±2.88      

% cells in repair

NGG > 0

9.33 ± 4.51 7.00 ± 4.36 20.00 ± 18.19 78.67 ±7.57      

% cells in repair

NGG > 5

0.00 ± 0.00   0.00 ± 0.00  5.67 ± 8.96  54.00 ±11.53      
               
               

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

Perfusion 18 hours after treatment (mean of 3 animals ± standard deviation)

 Test Group  Vehicle control  500 mg/kg  1000 mg/kg positive control       
 NG counts  6.35 ± 0.36  6.54 ± 0.54 7.48±0.88 20.43 ± 1.63      
 CG counts  11.08 ±0.30  9.90 ± 0.85 10.94±1.16 11.88 ±0.56       
 NNG counts  -4.73 ±0.65  -3.36 ± 0.59  -3.45 ±0.53 8.56 ±1.34       

 % cells in repair

NGG > 0

7.67 ± 4.04   15.00 ± 4.00  14.67 ±4.73  91.67 ±4.04      

 % cells in repair

NGG > 5

0.00 ± 0.00   0.33 ±0.58  0.67 ±0.58  65.67 ±9.50      
               
               

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

mean per group (mean of 3 animals)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negativeThe test substance did not cause a relevant increase in unscheduled DNA synthesis, as measured by an increase in net nuclear grain counts, and it was concluded that the test substance was negative in this in vivo assay using primary rat hepatocytes.