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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The test item did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice. In addition, the test item was not mutagenic in the Ames test in the absence and presence of metabolic activation. Weak positive results were observed in a chromosom abarration assay without metabolic activation. These findings were judged not relevant due to the negative results of the in vivo micronucleus assay.

Link to relevant study records

Reference

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.

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

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

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

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

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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames Test

A study was conducted according to OECD TG 471 for mutagenic activity in the Salmonella-Escherichia coli Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay. The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strains TAIOO and WP2MvrA and ten doses of test article ranging from 5000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TAIOO, TAl535, and TAl537 and Escherichia coli tester strain WP2MVA-A. The assay was conducted with five doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5000, 3330, 1000, 333, and 100 µg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test item did not cause a positive increase in the mean number of revertants per plate with any of the tested strains either in the presence or absence of microsomal enzymes prepared from phenobarbital/5,6-benzoflavone-induced rat liver (S9).

Chromosom abarration test

The objective of this in vitro assay was to evaluate the ability of the test item to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation according to OECD TG 473. The top concentration of the test item was 5000 µg/ml. The test item was dissolved in cell culture grade water at a concentration of 500 mg/mL. Concentrations of 33.9,48.4, 69.1, 98.7, 141, 202, 288,412, 588, 840, 1200,1720, 2450, 3500, and 5000 µg/mL were tested with and without metabolic activation. Cultures treated with concentrations of 1720, 2450, 3500, and 5000 µg/mL with and without metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed, except for a weak increase in cells with chromosomal aberrations at 5000 µg/mL in the nonactivation assay. The test item was considered negative for inducing chromosome aberrations in CHO cells with metabolic activation, but weakly clastogenic under nonactivation conditions. These weakly positive findings were judged not relevant as in vivo data (MN test) do not confirm these findings.

Micronucleus Assay

The objective of this study according to OECD TG 474 was to evaluate the test item for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-l®(ICR) BR mouse bone marrow.

Based on the results of the dose range finding study, the maximum tolerated dose was estimated to be 200 mg/kg bw. In the micronucleus assay, the test article was dissolved in sterile water for injection and dosed by intravenous injection to six males per dose level at each harvest time point. The animals were dosed at 50.0,100, and 200 mg/kg bw. Five animals dosed with the test article at 50.0 and 100 mg/kg bw dose levels and five animals dosed with the positive control article were euthanized approximately 24 hours after dosing for extraction of the bone marrow. Five animals dosed with the test article at the 200 mg/kg bw dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal. The test item, induced signs of clinical toxicity in the 200 mg/kg animals. There was no statistically significant decrease in the PCE:NCE ratio, demonstrating that the test article was not cytotoxic to the bone marrow. The test item did not induce a statistically significant increase in the frequency of micronucleated PCEs and is considered negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

 

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant in vivo study.

Justification for classification or non-classification

Based on the results of the two in vitro studies and the in vivo study and according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), the test item is not classified for genetic toxicity.