N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

EC Number:

940-393-4

Cas Number:

1702355-94-9

Molecular formula:

C35 H66 N8

IUPAC Name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Test material form:

solid: bulk

Details on test material:

- State of aggregation: solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 27.63 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Based on preliminary solubility testing dimethyl sulfoxide (DMSO) was the most suitable
vehicle, whereas only a homogeneous suspension was obtained. Using the vehicles DMSO
and corn oil subsequently a homogeneous suspension of the test substance was obtained.
Therefore, a mixture of DMSO and corn oil (ratio 2:3) was selected as vehicle, which has
been demonstrated to be suitable in the mouse micronucleus test and for which historical
control data are available.

Details on exposure:

The substance to be administered per kg body weight was suspended in DMSO/corn oil.
To achieve homogeneity of the test substance in the vehicle, the test substance preparation
was stirred with an ultraturrax.
All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

24h and 48h

Frequency of treatment:

single

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control
20 mg cyclophosphamide (CPP)
10 mL positive control preparation
sacrifice after 24h

Examinations

Tissues and cell types examined:

preparation of the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: pre-test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single treatment, exposure for 24h and 48h

DETAILS OF SLIDE PREPARATION: stained with eosin and methylene blue for about 5 minutes, were stained with Giemsa solution for 15 min, rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam

METHOD OF ANALYSIS: 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored

OTHER:

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• A statistically significant and dose-related increase in the number of PCEs containing
micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle
control value and the range of the historical vehicle control data (see Appendix 4).
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant
increased above the concurrent vehicle control value and is within the range of the
historical vehicle control data

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN
(BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test
according to WILCOXON) was carried out to clarify the question whether there are
statistically significant differences between the untreated control group and the treated dose
groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative
frequencies of cells containing micronuclei of each animal were used as a criterion for the
rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

An inhibition of erythropoiesis induced by the treatment of mice with was detected at a dose of 2 000 mg/kg body weight at 48-hour sacrifice interval

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The administration of the test substance led to severe clinical signs of toxicity at the top dose
of 2 000 mg/kg body weight 48 hours after administration. Following clinical symptoms were
observed shortly before sacrifice: piloerection, hunched posture, reduced general condition,
eyelid closure, lacrimation and diarrhea.
The single statistical significant increase in the number of micronucleated polychromatic
erythrocytes after treatment with 2 000 mg/kg body weight at 48-hour sacrifice interval is
based on the low value of the respective vehicle control group, and therefore, it has to be
regarded as biologically irrelevant.
The ratio of PCE/NCE was clearly influenced at the highest administered dose of
2 000 mg/kg body weight at delayed 48-hour preparation interval as indication for target
organ toxicity. These data confirm the severe clinical observations in the treated animals
48 hours after test substance administration.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative