1,4-dihydroxy-2,2,6,6-tetramethyl piperidinium-2-hydroxy-1,2,3-propanetricarboxylate

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:

1999-03-24 to 1999-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21,1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-370-5

EC Name:

-

Cas Number:

220410-74-2

Molecular formula:

C34H67N3O13

IUPAC Name:

tris(1,4-dihydroxy-2,2,6,6-tetramethylpiperidin-1-ium) 2-hydroxypropane-1,2,3-tricarboxylate

Test material form:

solid: flakes

Details on test material:

- Identification: TKA 45021
- Appearance: light-red flakes
- Analytical purity: 93.64%
- Lot/batch No.: pax 2068 / rd 100
- Test Item arrived at Test Facility: 1999-03-29
- Storage conditions: ambient temperature

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Canada, St. Constant, Canada (Dose Limit and Dose Range Finding); and Raleigh, North Carolina, USA (Micronucleus Study)
- Housing: in sanitary polycarbonate cages separated by gender, with up to five animals per cage during acclimation, and full dose group after randomization
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: 28.5-35.0 g (males only in main study)
- Diet: PMI® Feeds, Inc. Certified Rodent Diet # 5002 (pellets) ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 64 to 79°F (17,8 °C to 26,1 °C)
- Humidity: 30 to 70%
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12-hour light / 12-hour dark cycle

IN-LIFE DATES: From: 1999-04-01 To:1999-04-29

Administration / exposure

Route of administration:

intravenous

Vehicle:

- Vehicle/solvent(s) used: sterile water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to dosing, the top stock of the test item was prepared by adding the appropriate volume of sterile water for injection, to a preweighed quantity of the test article. The remaining stocks were prepared by dilution from the top stock.

Duration of treatment / exposure:

not applicable (single i.v. treatment)

Frequency of treatment:

once

Post exposure period:

Micronucleus assay: 24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
50.0, 100, and 200 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

6 males (a replacement dose group of 8 animals was included at the top dose level in anticipation of mortality)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: p.o.
- Doses / concentrations: 80.0 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes (polychromatic erythrocytes, PCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Observed toxicity in Dose Range Finding assay

SAMPLING TIME: Bone marrow was sampled 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow was removed from the femurs of the first five surviving animals in a dose group. Following centrifugation to pellet the tissue, the supenatant was removed by aspiration and portions of the pellet were spread on slides, air dried and stained in May-Grünwald solution followed by Giemsa.

METHOD OF ANALYSIS:
The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal.
The frequency of PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 200 erythrocytes on the slide.

Evaluation criteria:

The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test item that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

The criteria for the identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.

Statistics:

ANOVA (Winer, 1971) was applied when the variances were homogeneous, ranked proportions were used for heterogeneous variances. If the ANOVA was statistically significant (p <0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was performed.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 150, 200, 300 and 500 mg/kg bw
- Clinical signs of toxicity in test animals: at 200 mg/kg bw and above
500 mg/kg bw: all animals died (6/6)
300 mg/kg bw - fatalities: 2/3 males, 1/3 females
200 mg/kg bw - fatalities: 1/2 males, 0/2 females
lower concentrations: no deaths, no clinical signs
- Evidence of cytotoxicity in tissue analyzed: no


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PCE/NCE: no statistically significant decrease
- Appropriateness of dose levels and route: the high dose produced mortality in two animals of the 24 h harvest group
- Statistical evaluation: Yes