1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: 6-12 weeks
- Weight at study initiation: 37-45 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: individually in type II cages; bedding of soft wood granules, type BK8/15 (J. Rettenmaier & Soehne, Fuellstoff-Fabriken, 73494 Ellwangen-Holzmuehle) was used
- Diet (e.g. ad libitum): fixed-formula feed 3883 (10 mm cubes), produced according to specification by Provimi Kliba SA, CH-4303 Kaiseraugst, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22.5
- Humidity (%): 45-54
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous Cremophor emulsion, resulting suspension was set to pH 6 to 8 using 0.02 N aqueous NaOH. Cyclophosphamide was dissolved in physiological saline solution.
- Justification for choice of solvent/vehicle: suitability for intraperitoneal application
- Concentration of test material in vehicle: 1, 2, 4 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg intraperitoneal
- Lot/batch no. (if required): Fluka, batch 444193/1 52304077

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a microdismembrator for 3 minutes, and formed white turbid suspensions. The suspensions were set to pH 6 to 8 using 0.02 N aqueous NaOH. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally. Cyclophosphamide was dissolved in physiological saline solution and administered in the same way.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

2 injections 24 hours apart

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in the form of Endoxan 100 mg injection vials of dry substance (Baxter Oncology GmbH), batch 4N120
- Justification for choice of positive control(s): proven cytostatic agent and known clastogen with bifunctional alkylation action.
- Route of administration: intraperitoneal (only 1 injection)
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow cells (erythrocytes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the range-finder study with doses of 20, 40, 100 and 1000 mg/kg, each applied to groups of 3 males and 3 females; 40 mg/kg AE 1170437 technical were chosen as MTD for males.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals received 2 intraperitoneal injections 24 hours apart, 24 hours after the last application the animals were sacrificed and the bone marrow cells were isolated.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared according to the method of Schmid (Mutat Res 31: 9-15; 1975. DFG; Kommission fuer Mutagenitaetsfragen; Mitteilung III, 53-61). At least one intact femur was prepared from each sacrificed animal. The bone marrow cells were flushed out from the femur with fetal calf serum. After centrifugation the supernatant was discarded and a homogeneous cell suspension prepared from the pellet. One drop of this suspension was then placed on a well-cleaned slide and spread out with a suitable object. The slides were dried overnight and stained automatically on the following day with an Ames Hema-Tek Slide Stainer from the Miles Company. Unspecific background staining was subsequently removed with methanol, then the slides were rinsed with deionised water and left to dry. Thereafter the slides were fixed with xylene, embedded in covering agent and covered with a covering glass. Slides were not evaluated until the covering agent had dried.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.

Evaluation criteria:

A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible. In both cases, normally a second test will be performed.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Statistics:

The AE 1170437 technical group(s) with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing for up to 3 hours after the second treatment

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Groups consisting each of three males and three females received two intraperitoneal injections (animals of the 1000 mg/kg group died prior to second treatment) separated by 24 hours.

- Dose range: 20, 40, 100, 200, 1000 mg/kg bw AE 1170437 technical

- Solubility: AE 1170437 technical was suspended in 0.5% aqueous Cremophor (Fluka, batch 444193/1 52304077) emulsion, using a microdismembrator for 3 minutes, and formed white turbid suspensions. The suspension was set to pH 6 to 8 using 0.02 N aqueous NaOH. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.

- Clinical signs of toxicity in test animals: In males the following symptoms were recorded up to their death or for up to at least 3 hours after the second application, starting at 20 mg/kg: apathy, running in circles, roughened fur, lateral and sternal recumbency, spasm, palmospasm, twitching, difficulty in breathing, and slitted eyes. In addition, 3 of 3 males died in the 1000 mg/kg group, 2 of 3 males died in the 200 mg/kg group and 1 of 3 males died in the 100 mg/kg group. In females, except twitching, the same symptoms plus periodically stretching of body were recorded. In addition, 3 of 3 females died in the 1000 mg/kg group and 2 of 3 females died in the 200 mg/kg group as well as in the 100 mg/kg group.
- Rationale for exposure: Based on these findings, 40 mg/kg AE 1170437 technical were chosen as MTD for males. Due to the results of the dose range finder it is concluded, that there are no substantial differences between sexes in toxicity. Therefore, no females were used. Intraperitoneal application is one of the common routes recommended by the guideline.
- Harvest times: 24 hours after last treatment
- High dose with and without activation: activation not required, in vivo study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with AE 1170437 technical, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.4/2000 (1s=1.1) in the negative control, and 3.0/2000 (1s=1.9), 3.0/2000
(1s=2.2) and 3.0/2000 (1s=2.6) in the AE 1170437 technical groups (10, 20 and 40 mg/kg bw, respectively).

- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to normochromatic erythrocytes in males was not altered by the treatment with AE 1170437 technical, being 2000: 3247 (1s=362) in the negative control, 2000: 3741 (1s=976) in the 10 mg/kg group, 2000: 3465 (1s=436) in the 20 mg/kg group and 2000: 4219 (1s=1050) in the 40 mg/kg group. No relevant variations were thus noted for males.

- Appropriateness of dose levels and route: After two intraperitoneal administrations of 10, 20 and 40 mg/kg AE 1170437 technical, treated males showed the following compound-related symptoms for up to 3 hours after the second treatment: apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing. These symptoms demonstrate relevant systemic exposure of males to AE 1170437 technical. Thereafter, their external appearance and physical activity remained unaffected. There was no substance-induced mortality. Except for loss of weight in one male, no symptoms were recorded for the control groups. No animals died in these groups.

- Statistical evaluation: There was no biologically significant variation between the negative control and AE 1170437 technical groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The results with AE 1170437 gave no indications of clastogenic effects for male mice after two intraperitoneal treatments with doses up to and including 40 mg/kg bw.

Any other information on results incl. tables

Summary of Results of the Micronucleus Test with AE 1170437 technical:

Experimental groups	Number of evaluated PCE	Number of NCE/2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
negative	10000	3247 ± 362	1.2 ± 0.6	2.4 ± 1.1
2 x 10 mg/kg	10000	3741 ± 976	1.4 ± 0.9	3.0 ± 1.9
2 x 20 mg/kg	10000	3465 ± 436	1.2 ± 0.4	3.0 ± 2.2
2 x 40 mg/kg	10000	4219 ± 1050	0.7 ± 0.7	3.0 ± 2.6
Cyclophosphamide 20 mg/kg	10000	4036 ± 714	1.1 ± 0.8	29.0 ± 9.6*

*P<0.01 in non-parametric Wilcoxon ranking test

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MNNCE: micronucleated normochromatic erythrocytes

MNPCE: micronucleated polychromatic erythrocytes

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 29.0/2000 (1s=9.6), which represents biologically relevant increases in comparison to the negative control. A biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control was not possible since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [2000: 4036 (1s=714), as against 2000: 3247 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Conclusion:

There was no indication of a clastogenic effect of intraperitoneally administered AE 1170437 technical in the micronucleus test on the male mouse, i.e. in a somatic test system in vivo.