N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

Genetic toxicity: in vivo

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:

November 2001 - May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; documentation sufficient for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

For a short period of time the relative humidity was higher than 70 % (hoghest value 74 %). This deviation has no influence on the integrity and validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

yes

Remarks:

For a short period of time the relative humidity was higher than 70 % (hoghest value 74 %). This deviation has no influence on the integrity and validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

This study performed in the testing facility of RCC Cytotest Cell Research was conducted in compliance with Good Laboratory Practice Regulations."

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Name of test material (as cited in study report): CA 2343 A (Intermediate of CGA 293343)
- Physical state: solid
- Colour: colourless to light beige
- Molecular weight: 160.1
- Analytical purity: 96.7 %
- Solubility ion water: 16 g/l (25 °C)
- Batch No.: P.503005
- Expiration/Reanalysis date: September 2004
- Stability (of the test material itself): stable
- Stability in solvent: stable
- Storage condition of test material: 0 - 5 °C, light protected, humidity protected

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 6 - 10 weeks
- Quarantine: minimum 5 days
- Weight at study initiation: mean value 218.1 g (SD +/- 12.9 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 4 hours prior to pre-experiments, overnight prior to main experiment 2 hour treatment, 6 hours before 16 hours treatment
- Housing: single; Makrolon cages Type II, with wire mesh top, bedding: granulated soft wood bedding
- Diet: pelleted standard diet
- Water ad libitum: tap water



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 30 - 74
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: CMC (carboxymethyl cellulose; FLUKA) 0.5 % (w/v) in deionised water
- Amount of vehicle (oral administration): 10 ml/kg b.w.

Details on exposure:

see below

Duration of treatment / exposure:

single dose application

Frequency of treatment:

once via oral gavage

Post exposure period:

2 and 16 hours after treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2000 mg/kg b.w.
Basis:
nominal conc.
pre-experiment I: test material suspended in deionized water, 20 ml/kg;

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1500 mg/kg b.w.
Basis:
nominal conc.
pre-experiment II and III: test item was suspended in CMC (0.5 % v/v), 10 ml/kg

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg b.w.
Basis:
nominal conc.
pre-experiment II and III: test item was suspended in CMC (0.5 % v/v), 10 ml/kg

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1250 mg/kg b.w. (maximum dose)
Basis:
nominal conc.
main experiment; test item was suspended in CMC (0.5 % w/v)

Doses / concentrations 5

Remarks:

Doses / Concentrations:
312.5 mg/kg b.w. (minimum dose)
Basis:
nominal conc.
main experiment; test item was suspended in CMC (0.5 % w/v)

No. of animals per sex per dose:

4 animals

Control animals:

yes, concurrent vehicle

other: positive control

Positive control(s):

2 HOURS PREPARATION INTERVAL:
-Name: DMH (Fluka), > 99 % purity
- dissolved in 0.9 % NaCl solution
- dosing: 40 mg/kg b.w.
- route and frequency of administration: orally, once
- Volume administered: 10 ml/kg b.w.

16 HOURS PREPARATION INTERVAL:
-Name: 2-AAF (Fluka), > 99 % purity
- dissolved in dimethylsulfoxide/polyethylene glycol 400 (1+9)
- dosing: 100 mg/kg b.w.
- route and frequency of administration: orally, once
- Volume administered: 10 ml/kg b.w.

Examinations

Tissues and cell types examined:

liver hepatocytes

Details of tissue and slide preparation:

ISOLATION OF THE PRIMARY HEPATOCYTES:
- rats were anaesthetized with Na-thiopental
- liver was perfused through the vena portae with Hank’s balanced salt solution, supplemented collagenase (0.05 % (w/v)) adjusted to pH 7.4 and maintained at 37 °C
- isolated hepatocytes were washed twice with HBSS
- crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension
- determination of quality of the performed perfusion with trypan blue dye exclusion method for cell viability
- determination of number of cells


CULTURE CONDITION:
- washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with: Hepes (2.38 mg/ml), Penicillin (100 units/ml), Streptomycin (0.10 mg/ml), L-Glutamine (0.29 mg/ml), Insulin (0.50 µg/ml), fetal calf serum (100 µl/ml)
- complete medium was adjusted to pH 7.6
- at least three cultures were established from each animal
- aliquots of 2.5 ml with freshly isolated cell hepatocytes in complete culture medium were added to 35 mm six-well dishes containing one 25 mm rounf plastic coverslip per well coated with gelatine
- attachment period: approx. 1.h in a 95 % air / 5 % CO2 humidified incubator at 37 °C; thereafter culture medium was discarded and cell layer was rinsed once with PBS to remove non-adherent cells
- 3H-TdR in 2-0 ml culture medium was added to the cultures
- after labelling time of 4 hours, cells were washed twice with WME supplemeted with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine
- Cultures were incubated overnight using the same medium
- To prepare autoradiography the medium was replaced by a hypotonic solution of 1 (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection
- Cells were fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol and air-dried.


AUTORADIOGRAPHIC PROCESSING:
- cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 photographic emulsion in the dark
- coated slides were stored in light-proof boxes in the presence of an drying agent for 14 days at 4 °C
- photographic solution was then developed with KODAK Dektol Developer at room temperature, fixed in TETENAL and stained with hematoxylin/eosin

QUANTIFICATION OF UDS:
- microscopic evaluation with NIKON microscopes with oil immersion objectives
- cells for scoring were randomly selected according to a fixed scheme
- counting of the number of silver grains in the nuclear area, and of the number of grains of one nuclear-sized cytoplasm area adjacent to the nucleus
- evaluation of at least two slides per animal and 50 cells per slide
- heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting
- three animals per group were evaluated as described above

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.
A test item producing net grains not gr5eater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Test results 1

Sex:

male

Toxicity:

yes

Remarks:

pre-experiment I (2000 mg/kg b.w., formulated in deionised water, administered volume: 20 ml/kg b.w.)

Test results 2

Sex:

male

Toxicity:

yes

Remarks:

pre-experiment II (1000 mg/kg b.w., formulated in 0.5 % CMC, administered volume: 10 ml/kg b.w.)

Test results 3

Sex:

male

Toxicity:

yes

Remarks:

pre-experiment III (1500 mg/kg b.w., formulated in 0.5 % CMC, administered volume: 10 ml/kg b.w.)

Test results 4

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection, apathy, eyelid closure

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRE-EXPERIMENTS FOR TOXICITY:
- Dose range: 1000, 1500 amd 2000 mg/kg b.w.
- Clinical signs of toxicity in test animals (see tables below):
* 1000 mg/kg b.w.: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Apathy
* 1500 mg/kg b.w.: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Apathy, Bloody nose
* 2000 mg/kg b.w.: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Apathy, Bloody nose, death
- Rationale for exposure: determination of the maximum tolerable dose



RESULTS OF DEFINITIVE STUDY
- Toxic reactions (312.5 and 1250 mg/kg b.w.): piloerection, apathy, eyelid closure
- the viability of the hepatocytes was not substantially affected by the in vivo treatment with the test iten at any of the treatment periods or dose groups
- the interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes were in the range of the historical laboratory control
- no dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle controls
- neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 2 hours or 16 hours; therefore, the net grain values obtained after treatment with the test item were consistently negative
- no substantial shift to higher values was obtained in the percentage distribution of the nuclear grain counts
- positive controls revealed distinct increases in the number of nuclear and net grain counts

Any other information on results incl. tables

Pre-experiment I, 2000 mg/kg b.w. (number of dosed animals: 2)

Toxic reactions	Hours post-treatment
	1 h	2-4 h	5h	20-22h	24h
Reduction of spontaneous activity	2	2	--	--	--
Abdominal position	--	2	--	--	--
Eyelid closure	2	2	--	--	--
Apathy	2	2	--	--	--
Bloody nose	--	2	--	--	--
death	--	--	2	--	--

 

Pre-experiment II, 1000 mg/kg b.w. (number of dosed animals: 2)

Toxic reactions	Hours post-treatment
	1 h	2-4 h	5h	20-22h	24h
Reduction of spontaneous activity	2	2	2	2	2
Abdominal position	--	--	1	--	--
Eyelid closure	--	2	2	1	--
Apathy	1	2	2	2	2

 

Pre-experiment III, 1500 mg/kg b.w. (number of dosed animals: 2)

Toxic reactions	Hours post-treatment
	1 h	2-4 h	5h	20-22h	24h
Reduction of spontaneous activity	2	2	--	--	--
Abdominal position	--	2	--	--	--
Eyelid closure	2	2	--	--	--
Apathy	2	2	--	--	--
Bloody nose	--	2	--	--	--

On the basis of the data from the three pre-experiments the dose of 1250 mg/kg b.w. was selected as high dose (maximum dose) for the USD test in agreement with the sponsor monitor.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, under the experimental conditions reported, CA 2343 A (Intermediate of CGA 293343) did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
Therefore, CA 2343 A (Intermediate of CGA 293343) is considered to be non-effective in this in vivo / in vitro USD test system.

Executive summary:

The test item CA 2343 A (Intermediate of CGA 293343) was assessed in the in vivo / in vitro USD assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats.

The test item was formulated in 0.5 % (w/v) carboxymethylcellulose (CMC). Therefore 0.5 % CMC was used as vehicle control. The volume administered orally was 10 ml/kg body weight. After a treatment period of 2 and 16 hours, the animals were anaesthetized and sacrificed by liver perfusion. Primary hepatocytes cultures were established and exposed for 4 hours to³H-TdR (methyl-³H-thymidine), which is incorporated into the DNA if UDS occurs.

The test item was tested at the following dose levels:

2 and 16 hours preparation intervals: 312.5 and 1250 mg/kg b.w..

The highest dose was estimated by pre-experiments to be suitable, showing signs of toxicity. For each experiment group inclusive the controls, hepatocytes from three treated animals were assessed for the occurrence of UDS.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item.

No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle controls.

Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.) were used as positive controls. Treatment with the positive control substances revealed distinct increases in the number of nuclear and net grain counts.