2,2,6,6-tetramethylpiperidinooxy

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:

1995-05-02 to 1995-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1993

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

EC Number:

218-760-9

EC Name:

4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

Cas Number:

2226-96-2

Molecular formula:

C9H18NO2•

IUPAC Name:

1-λ1-Oxidanyl-2,2,6,6-tetramethylpiperidin-4-ol

Test material form:

solid: flakes

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: young adults
- Body weight at study initiation: 29.9 ±6.0 g
- Housing: conventional, 5 mice/sex in Macrolon cages type III
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity:30 - 70 %
- Air changes: 15 per h
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1995-05-03 To: 1995-05-12 (toxicity test); 1995-05-15 To: 1995-05-22 (main study)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: physiol. saline
- Amount of vehicle: 10 mL/kg bw

Frequency of treatment:

single exposure

Post exposure period:

24 or 48 hrs

Doses / concentrations

Dose / conc.:

1 200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 100 mg/kg bw in 10 mL/kg bw

Examinations

Tissues and cell types examined:

femoral bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
MTD (limit dose: 2000 mg/kg bw): highest dosage causing signs of toxicity but no mortality within 48 hrs of test compound administration), determined in pre-test

DETAILS OF SLIDE PREPARATION:
After sacrifice, bone marrow was suspended in FCS and erythrocytes were purified in a cellulose chromatography column. The eluate was centrifuged and the pellet was re-suspended in FCS/EDTA. Two slides were prepared per animal and stained with May-Grünwald Giemsa.

METHOD OF ANALYSIS:
Fully automated scoring: counting micronuclei in a total of appr. 2000 PCE (i.e. 100000 PCE per treatment group). Based on 2000 PCE scored, the PCE/NCE index was calculated.

Evaluation criteria:

A test compound is considered an inducer of micronuclei in PCE, if at least one group of mice treated with the test compound reveals a statistically and biologically relevant increase in micronucleated PCE (as compared to the negative control group of the same sampling time).

Statistics:

Heterogeneity Chi square calculated first. If homogenous: Pearson's contingency 2x2 chi square test (DF = 1), including a Yates correction factor. If inhomogenous: transformation and t-test (one sided, two sample). The latter also for evaluation of PCE/NCE ratios.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 750, 1000, 1250, 1500, 1750 or 2000 mg/kg bw
- Solubility: soluble in vehicle up to limit concentration (2000 mg/kg bw)
- Clinical signs of toxicity in test animals: clinical symptoms (hunched posture, sedation, piloerrection and closed eyes), death at high dosages
- Evidence of cytotoxicity in tissue analyzed: not reported

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No
- Ratio of PCE/NCE: unaltered compared to controls

Applicant's summary and conclusion

Conclusions:

The genetic toxicity in vivo of 4-Hydroxy-TEMPO was tested in a GLP compliant OECD 474 guideline study (mouse micronucleus test). As a result, the test item was not genotoxic under the conditions tested.

Executive summary:

In an in vivo mouse micronucleus assay (95-0250-DGM), male and female NMRI mice were orally (gavage) exposed to a single dose of 4 -Hydroxy-TEMPO at a concentration of 1200 mg/kg bw (vehicle: saline solution). The maximum tolerated dose (MTD) was determined prior in a toxicity test. The post exposure period was 24 or 48 hrs. The following main signs of toxicity were observed: hunched posture, sedation, in males: staggering gait, convulsions. One male of the 48 h sampling group and one male of the satellite group died. No statistically or biologically significant increase in micronucleated polychromatic erythrocytes at the sampling times of 24 and 48 h in male and female animals. No alterations of the ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio) were detected. There was no indication of a clastogenic effect. The positive control induced the appropriate responses. The study satisfies the requirements of Test Guideline OECD 474 for the evaluation of in vivo mutagenicity (resp. clastogenicity: induction of micronuclei).