Registration Dossier

Diss Factsheets

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:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carried out using scientifically valid protocols equivalent to relevant guidelines; adapted for this endpoint according to REACH Annex XI section 1.1.2

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
not applicable
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
not applicable
Principles of method if other than guideline:
According to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo) indicates DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents. The test is usually based on the incorporation of 3H-TdR into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle.
Test substances are generally administered as a single treatment administered orally by gavage. The number of animals should be at least 3 analysable animals per group. Where a significant historical database has been accumulated, only 1 or 2 animals are required for the concurrent negative and positive control groups. At least two dose levels are used, the highest dose as defined as the dose producing signs of toxicity. The highest dose may also be defined as a dose that produces some indication of toxicity in the liver (e.g. pyknotic nuclei).
A limit test may be performed if no adverse effects are observed < 2000 mg/kg unless human data/exposure indicate a need for a higher limit dose level.
Liver cells are prepared from treated animals normally 12-16 hours after dosing. An additional earlier sampling time (normally 2-4 hours post-treatment) is generally necessary unless there is a clear positive response at 12-16 hours. However, alternative sampling times may be used when justified on the basis of toxicokinetic data.
- Freshly isolated mammalian liver cells are incubated usually with medium containing 3HTdR for an appropriate length of time, e.g. 3 - 8 hours are then developed and slides developed and analysed for morphology and signs of overt cytotoxicity.
- Slides should be coded before grain counting. Normally 100 cells are scored from each animal from at least two slides; the scoring of less than 100 cells/animal should be justified. Grain counts are not scored for S-phase nuclei, but the proportion of S-phase cells may be recorded.
- The amount of 3H-TdR incorporation in the nuclei and the cytoplasm of morphologically normal cells, as evidenced by the deposition of silver grains, should be determined by suitable methods.
- Grain counts are determined over the nuclei (nuclear grains, NG) and nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, CG). CG counts are measured by either taking the most heavily labelled area of cytoplasm, or by taking an average of two to three random cytoplasmic grain counts adjacent to the nucleus. Other counting methods (e.g. whole cell counting) may be used if justified.
Examples of positive and negative response criteria are given in OECD Guideline 486. A positive result from the UDS test with mammalian liver cells in vivo indicates that a substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A negative result indicates that, under the test conditions, the test substance does not induce DNA damage that is detectable by this test.
This is an accurate outline of the procedures used in this, study no. 961122 Ogorek.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate
EC Number:
603-837-5
Cas Number:
134605-64-4
Molecular formula:
C20H18ClF3N2O6
IUPAC Name:
1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Substance type: Tan solid powder.
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Tif: RAJ (SPF)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 150 - 350 g
- Assigned to test groups randomly: Yes.
- Fasting period before study: 12 hrs
- Housing: Individually in makrolon cages before and after treatment
- Diet (e.g. ad libitum): Pelleted, certified standard diet administered ad libitum up to 12 hours before dosing.
- Water (e.g. ad libitum): Tap water ad libitum, drinking water quality of recorded specifications
- Acclimation period: Four days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-75%
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30/09/1996 To: 14/10/1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Mixture of Carboxymethylcellulose (0.5% [w/v] in bidistilled water) and Tween 80 (0.1% [v/v] in bidistilled water); 20 ml/kg
- Justification for choice of solvent/vehicle: A solubility test was not performed. From an earlier in vivo study with this test compound,
a mixture of Carboxymethylcellulose (0.5% [w/v] in H20) and Tween 80 (0.1% [v/v] in H20), was known to be a suitable vehicle for preparation of applicable suspensions of the test substance up to a top dose level of 5000 mg/kg body weight.
- Concentration of test material in vehicle: 62.5 and 250 μg/ml respectively.
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A solubility test was not performed. A mixture of Carboxymethylcellulose (0.5% [w/v] in H20) and Tween 80 (0.1% [v/v] in H20), was known to be a suitable vehicle for preparation of applicable suspensions of the test substance up to a top dose level of 5000 mg/kg body weight from a previous reported study. Further information on the preparation of the dose solutions was not reported other than information on dose levels from a preliminary test.

Animals were treated with the selected doses of the test substance, with the vehicle alone or with the positive control by oral application. The treatment regimen was designed in accordance with published standard protocols. For determining UDS treatment, a period of 16 hours was selected for the groups treated with the test substance and with the vehicle alone. With the positive control substance a time point resulting in an optimal UDS response is usually selected. For DMN this time is known to be about 2 hours.
Duration of treatment / exposure:
16 hours main study group and negative control; 2 hours for positive control.
Frequency of treatment:
Single treatment oral dose
Post exposure period:
Not applicable. Primary hepatocytes were freshly isolated by in situ-collagenase perfusion.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1250 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
4 male per group
Control animals:
yes, concurrent vehicle
other: positive control received the promutagen dimethylnitrosamine (DMN, 10 mg/kg, 10 ml/kg) dissolved in water.
Positive control(s):
dimethylnitrosamine
- Justification for choice of positive control(s): Not reported.
- Route of administration: Oral gavage
- Doses / concentrations: 10mg/kg dissolved in water (10 ml/kg)

Examinations

Tissues and cell types examined:
Primary hepatocytes freshly isolated from rats previously treated with the test substance
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Prior to the study test the maximum tolerated dose (MTD) of the test substance to be applied was determined in a tolerability test. The MTD is defined as the highest dose which does not induce severe toxicity or lethality. In two consecutive steps each one male rat was dosed with 5000 mg/kg. Body weight and signs of toxicity were recorded hourly for the first few hours after application and once the following two days.
The rat survived and showed piloerection on the day of treatment. In the second step of the tolerability test (confirmatory experiment) one male rat was dosed with 5000 mg/kg. Again, the animal survived and piloerection was noted after application. Based on these results the doses of 1250, 2500 and 5000 mg/kg were chosen for the DNA-rapair assay.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
For determining UDS treatment, a period of 16 hours was selected for the groups treated with the test substance and with the vehicle alone. With the positive control substance a time point resulting in an optimal UDS response is usually selected. For DMN this time is known to be about 2 hours.
Primary hepatocytes were freshly isolated by in situ-collagenase perfusion.

DETAILS OF SLIDE PREPARATION:
The procedure was as follows: the liver was perfused in situ through the portal vein. The liver was then carefully excised and placed into a dish containing the formulated wash solution. After opening the Glisson's capsule, the cells were dispersed by gently shaking of the liver in the solution. The cells were then filtered (mesh width of 61 μm) and washed once with the same wash solution (sedimentation rate of 50 g for 3 minutes at 2°C). Finally, the cells were suspended in Williams' medium E and analysed for viability by Trypan blue exclusion. Freshly isolated male rat hepatocytes were cultured. Cells were allowed to attach to the cover-slips during an attachment period of 1.5-2 hours. Unattached cells were then removed by washing with BSS and the cultures were refed with culture medium.
The UDS assay was initiated by adding 10 μCi/ml 3H-thymidine (6-3H-thymidine, specific activity 27-28 Curies/mMol). After 4 hours' incubation the cells were washed twice with BSS and further incubated overnight in renewed medium containing
0.25 mMol/1 cold thymidine. In the morning, the cells were washed twice with BSS and fixed with ethanol/acetic acid, 3/1, v/v. The cover-slips were mounted on microscope slides and prepared for autoradiography. Four slides from each animal were autoradiographed; the remainder were kept in reserve. Preparation and development of autoradiographs was performed in a dark room at 20 °C. Autoradiographic emulsion (diluted with two vol. H2O) was used as film material. Preparing the emulsion and coating the slides was performed in red light conditions. The air-dried slides were exposed in light- and airproof boxes containing desiccants at 4 °C for five days. Autoradiographs were developed, rinsed in acetic acid (1%) and fixed in Hypam solution (diluted 1: 10 in water). The autoradiographs were stained in hematoxylin solution, rinsed in tap water and counter-stained in eosin.

METHOD OF ANALYSIS:
Prior to scoring, the slides were coded. Silver grains over the nuclei and cytoplasm of the hepatocytes were counted using an electronic counter (DOMINO) attached to a microscope (ZEISS) at a magnification of 2000x. Two slides (50 cells/slide) from each animal of the treatment groups and of the respective positive and the negative controls were scored. The number of silver grains over the nuclei (nuclear grain counts) were counted and the mean values with the standard deviations were calculated. The results of the nuclear grain counts were tabulated.
The incorporation of radioactive material in the cytoplasm was determined by counting the silver grains in three cytoplasmic regions adjacent to the nucleus, each with an area equivalent to that of the nucleus (cytoplasmic grain counts). The results were tabulated.
The net nuclear grain counts were calculated by subtracting the average grain count over the cytoplasm from the total over the nuclei. The results were tabulated.
To make sure that not only effects on the majority of the cells within the treated hepatocyte population were recognised but also effects on particularly sensitive subpopulations of the hepatocyte culture, the percentage of cells in repair (percentage of cells with a net nuclear grain count value of 2.0 or more) was calculated for each slide and the results were tabulated.

Cells which were in the DNA-synthesis phase show more than 120 silver grains/nucleus. These cells were excluded from the determination of the silver grain/nucleus count.

OTHER:
To confirm that the animals were actually exposed to the intended test concentrations and to confirm the stability of the test substance in the vehicle used, determination of the concentration of the substance in suspension was performed by the analytical unit. This determination was performed with a low dose and a high dose sample of the first and last segment of the DNA-repair test.
Evaluation criteria:
Criteria for a positive response
The test substance is generally considered to be active in the DNA repair test if one of the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains in relation to their respective vehicle control value show an increase at any dose level and the mean net nuclear value is 2.0 or higher.
2. The percentage of cells in repair show an obvious shift to higher values at any dose as compared to their respective vehicle control value.

Criteria for a negative response
The test substance is generally considered to be inactive in the DNA repair test if the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains as well as the percentage of cells in repair, do not significantly differ from the respective vehicle control value at any dose level.

Exceptions
At the limits of the criteria for a positive or for a negative response, or if the criteria for a positive response are only partially fulfilled, the Study Director decides by experience about the interpretation of the results.
Statistics:
Mean values and the standard deviations calculated. Comparison with historical laboratory mean control data from in vivo/in vitro DNA repair data

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no mortalities; piloerection 2-5 hrs after treatment in limit dose group only
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1250, 2500 and 5000 mg/kg
- Solubility: Not applicable.
- Clinical signs of toxicity in test animals: piloerection 2-5 hrs after treatment in limit dose group (5000 mg/kg) only

Any other information on results incl. tables

Table 1. Group mean net grain count values

Dose / mg/kg

Net nuclear grain count (NG)

Net grain count of cells in repair

Percent of cells in repair (NG >= 2)

 

Mean

SD

Mean

SD

Mean

SD

0 Negative Control (Vehicle only)

0

0.2

2.6

0.2

8.8

3.6

10 DMN Positive Control

12.5

1.6

12.5

1.6

100.0

0.0

5000

0.1

0.2

3.0

0.3

11.8

3.4

2500

0.0

0.2

2.8

0.1

11.0

4.1

1250

0.1

0.2

2.6

0.2

10.5

1.7

Hepatocyte preparations of good quality were obtained with all animals from all dosage groups. Viability after perfusion was determined to be between 83 and 95%.

 

Evaluation of the mean nuclear counts of silver grains in the vehicle controls and after treatment with test substance revealed no marked differences (Table 1). In consideration of the silver grain number over the cytoplasm, the mean net nuclear grain counts were calculated and there was no marked deviation between the treatment groups and the vehicle controls. Also the percentage of cells in repair (net nuclear grain count ~ 2.0) revealed no marked differences in comparison with the vehicle controls. By contrast, the positive control DMN (15 mg/kg) yielded a marked increase in the mean nuclear grain counts and the mean net nuclear grain counts in comparison with the negative. The percentage of cells in repair was also clearly increased.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.
Executive summary:

The test substance was investigated for DNA-damaging effects on rat hepatocytes after application in vivo and further investigation of unscheduled DNA synthesis (UDS) in vitro. The assay is designed to measure unscheduled DNA synthesis (UDS, DNA-repair synthesis) in freshly isolated rat liver cells (hepatocytes) as a consequence of DNA-damage induced after in vivo treatment of the animals with the test substance. The advantage of this test system is, that not only metabolites formed in the liver itself, but also those formed in the various other organ and cell systems may have an effect in the hepatocytes. Hepatocytes are isolated by perfusion technique, and unscheduled DNA-synthesis caused by the test substance or its metabolites is detected by reference to the incorporation of 3H-TdR into DNA during in vitro culture. DNA-repair is determined by scoring autoradiographs (counts of silver grains over the nuclei; nuclei with< 120 grains are analysed and counts of silver grains over nuclear-sized cytoplasmic areas). There is ample evidence that occurrence of unscheduled DNA-synthesis is closely correlated with mutagenic or carcinogenic properties of a chemical substance, or both. The substance was suspended in a mixture of carboxymethylcellulose (0.5% [w/v] in bidistilled water) and Tween 80 (0.1% [v/v] in bidistilled water and administered orally at single doses of 1250, 2500 and 5000 mg/kg. Hepatocytes from animals of the treatmentment groups and negative control groups were isolated 16 hours after administration of the test substance. Hepatocytes from animals of the positive control group (dimethylnitrosamine, 15 mg/kg) were isolated 2 hours after application.

In the UDS assay, comparison of the mean nuclear and the mean net nuclear counts of silver grains in the vehicle controls and in the hepatocytes of rats treated with the various doses of the substance revealed no marked deviations. Also the percentage of cells in repair after treatment with the test substance was not significantly shifted when compared with the animals of the vehicle control.

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.