Bis(5-oxo-L-prolinato-N1,O2)zinc

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 December 2003 to 29 March 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

To investigate the potency of eliciting micronuclei in vivo of the test material, a micronucleus study was conducted using male ICR mice. Mice were intraperitoneally administered with the test material twice at 24-hour interval and the specimens were prepared at 24 hours after the last administration.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(5-oxo-L-prolinato-N1,O2)zinc

EC Number:

239-473-5

EC Name:

Bis(5-oxo-L-prolinato-N1,O2)zinc

Cas Number:

15454-75-8

Molecular formula:

C10H12N2O6Zn

IUPAC Name:

(3'aR)-3,3',6,6'-tetraoxo-3a,3'a,4,4',5,5',6,6'-octahydro-3H,3'H-1,1'-spirobi[pyrrolo[1,2-c]1-oxa-3-aza-2-zincacyclopentane]-1,1-bis(ylium)-2,2'-diuide

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystal

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks old
- Weight at study initiation: Body weight ranges at start of administration were 31.7 - 36.8 g, 32.8 - 37.7 g and 31.6 - 37.7 g in the preliminary, additional test for preliminary and main tests respectively.
- Housing: Six mice were housed in a cage (5 - 6 mice during the quarantine and acclimation period)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 12 days. Additional animals for the preliminary test were quarantined and acclimated for 1 day. General signs were observed every day and the animals were weighed on the next day of receipt and the end of the period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 °C (actual value 22 - 23 °C)
- Humidity (%): 40 - 70 % (actual value 57 - 66 %)
- Air changes (per hr): 10 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7:00 - 19:00)

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Adequate amount of the test material was weighed, suspended with agate mortar gradually adding olive oil to prepare 0.625 w/v% suspension, and then other suspensions (0.3125, 0.15625 and 0.078125 w/v%) were prepared by a serial dilution method. Each formulation was used for administering to the 62.5, 31.25, 15.625 and 7.8125 mg/kg group.
In the preliminary test, adequate amount of the test material was weighed, suspended with agate mortar gradually adding olive oil to prepare 20, 10 and 2.5 w/v% suspensions. Each formulation was used for administering to the 2 000, 1 000, 500 and 350 mg/kg group.
In the additional preliminary test, adequate amounts of the test material was weighed, suspended in agate mortar gradually adding olive oil to prepare 1.25 w/v% suspension and then other suspensions (0.625 and 0.3125 w/v%) were prepared by serial dilution method. Each formulation was used for administering to the 125, 62.5 and 31.25 mg/kg group.
In the main test, adequte amounts of the test material were weighed, suspended in agate mortar gradually adding olive oil to prepare 0.625 w/v% suspension and then other suspensions (0.3125, 0.15625 and 0.078125) were prepared by serial dilution. Each formulation was used for administering to the 62.5, 31.25, 15.625 and 7.8125 mg/kg group.

DOSING METHOD AND ROUTE
Intraperitoneal injection, which is widely used in the micronucleus test, was chosen as the route of administration. The number of doses was set at 24-h intervals. Disposable syringes and needles were used for administration. Dosing volume was calculated individually based on the determined body weight on the day of administration at a rate of 10 mL/kg.

Frequency of treatment:

The test material was administered twice at 24 h intervals.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

31.25 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

15.625 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

7.812 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 animals per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Mitomycin C
- Route of administration: Intraperitoneally administered once
- Concentration/ Dose: 2 mg/kg
- Justification for choice of positive control: Widely used in the micronucleus assay.
- Preparation: In both the preliminary and main test, one vial of mitomycin C (2 mg/vial) was dissolved in 5 mL water for injection on the day of use and then diluted 2-fold with physiological saline to prepare formulation for the positive control group (0.2 mg/mL). The formulation of the positive control was kept in well closed umber glass bottles and stored at room temperature until use.

Examinations

Tissues and cell types examined:

The potency of eliciting micronuclei and the frequency of total polychromatic erythrocytes of each article were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the preliminary test, according to information from the sponsor that the LD50 is > 2 000 mg/kg vial the oral route in rats, 2 000 mg/kg was selected as the highest dose. Other dose levels were set at 1 000, 500 and 350 mg/kg with the common ratio of 2.
Since all animals treated with the test material died by the next day of the first administration of the preliminary test an additional preliminary test was performed.
In the additional preliminary test, the highest dose of the test material was set at 125 mg/kg. Other dose levels were set at 62.5 and 31.25, with the common ratio of 2. All animals of the 125 mg/kg group died by the next day of the first administration and one animal of the 62.5 mg/kg group died after the second administration. In animals that survived, there were bradypnea, decrease in locomotor activity, abnormal fur, writhing etc. Bradypnea and writhing were also noted at the 31.25 mg/kg group. Tendency to suppress body weight changes were noted in the 31.25 mg/kg group.
In observation of the specimens, there was no tendency to depend on dose, nor time-point to prepare the specimens in the frequency of MNPCE and a total of PCE/RBC in the test material groups.
Based on the above results, the time to prepare the specimens was set at 24 h after the last administration the highest dose was set at 62.5 mg/kg and other dose levels were set at 31.25, 15.625 and 7.8125 mg/kg with a common ratio of 2. In addition, two groups with the negative control (olive oil) and the positive control group (MMC, 2 mg/kg) were set.

TREATMENT AND SAMPLING TIMES: Mice survived until the time to prepare the specimens were euthanized by cervical vertebra dislocation method.

DETAILS OF SLIDE PREPARATION: To collect bone marrow cells, right femur was washed with a little volume of foetal calf serum (about 1.0 mL/femur). The bone marrow cell suspension was centrifuged at 1 000 rpm for 5 minutes and the supernatant was discarded. The bone marrow cells in the tube were well stirred with a Pasteur pipette to get homogeneous cell suspension. A drop of the suspension was dripped on a slide glass and the smear was prepared using a cover glass. The specimens were prepared in duplicate. The specimens were dried at room temperature, fixed with methanol for 5 minutes and stained with 3 % Giemsa solution in Sorensen-phosphate buffer (1/15 M, pH 6.8) for 30 minutes. The stained specimens were washed with Sorensen-phosphate buffer, dipped in 0.004 % citric acid solution for several second, washed with distilled water, and then dried at room temperature.

METHOD OF ANALYSIS: One of the specimens prepared 2 slides were observed by blind test method (the specimens were identified by quarantine No.) with a microscope equipped ocular lenses (x 10) and an oil immersion objective lens (x 100). To evaluate the depression of proliferation of erythroblast of the test article, a ratio of total polychromatic erythrocytes count to total erythrocytes (PCE / RBC) was calculated by counting 1000 erythrocytes in each animal. A ratio of micronucleated polychromatic erythrocytes (MNPCE) was calculated by counting 2 000 total polychromatic erythrocytes (total PCE) in each animal.

Evaluation criteria:

Acceptance criteria were as follows:
1) The frequency of MNPCE in the negative control group in the main test was lower than the upper limit of frequency in the background data collected from the negative control groups in the facility (0.11 + 0.09 %, mean + 3 x standard deviation).
2) The frequency of NPCE in the positive control group was higher than the upper limit of frequncy in the background data.

Statistics:

Numbers of MNPCE were statistically analysed by binominal test (one-sided 5 %) according to a table of Kastenbaum and Bowman (Mutation Res., 9: 527-549, I 970) using SAS Release 6.1 2 (SAS Institute Inc.). To evaluate depression of proliferation of the bone marrow cells, the ratio of total polychromatic erythrocytes to total erythrocytes were analyzed by Bartlett's test for homogeneity of variances (significant level, one-sided: 5 and 1 %). Because the variances were homogeneous, Dunnett's test was perfomed to compare each of the treated group means with the mean of the negative control group (significant level, two-sided, 5 and 1 %). The Statistical analysis was performed using StatLight® (Yukms Corp.).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Death occurred in 1 animal (1/6) of the 62.5 mg/kg group on the next day of the first administration. Bradypnea (6/6), decrease in locomotor activity (6/6), writhing (2/6), abnormal fur (5/6) and loose stool (2/6) were noted in the same group. No abnormalities were noted in other groups. Depression of body weight change was noted in 62.5 mg/kg. No marked changes were noted in other groups in body weight changes.

In the positive control group, which received MMC (2 mg/kg) intraperitoneally once, the frequency of micronucleated polychromatic erythrocytes (MNPCE) at 24 hours after administration was 4.41 ± 1.06 %, which was much higher than that of the negative control group (0.13 ± 0.04 %). Number of MNPCE (529) was judged as positive by binominal test according to the table of Kastenbaum and Bowman. The frequency of MNPCE of the group was higher than the upper limit of the frequency in the background data of the negative control. On the other hand, the frequency of MNPCE in the negative control group was lower than the upper limit of the frequency in the background data. Since the results met the acceptance criteria, the study was considered to be carried out properly.

The frequency of MNPCE was evaluated 24 hours after the last administration of the test material of each dosage (administration was perfomed twice at 24-hour interval). The frequency of 62.5, 31.25, 15.625 and 7.8125 mg/kg groups were 0.20 ± 0.06, 0.13 ± 0.07, 0.13 ± 0.06 and 0.12 ± 0.05 %, respectively and they were lower than the upper limit of the frequency in the background data. These results were judged as negative by binominal test according to the table of Kastenbaum and Bowman.

There was slight depression of proliferation in the ratio of total polychromatic erythrocytes to total erythrocytes in the 62.5 mg/kg group by comparing to the negative control group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative
Under the conditions of this study, the results illustrate that the test material has no potency of eliciting micronuclei in vivo when intraperitoneally administrated to male ICR mice at 7.8125, 15.625, 31.25 and 62.5 mg/kg twice at 24-hour interval.

Executive summary:

A micronucleus study was conducted using male ICR mice to investigate the potency of eliciting micronuclei in vivo by the test material.

Mice were intraperitoneally administered with 62.5, 31.25, 15.625, 7.8125 or 0 mg/kg (negative control) of the test material twice at 24 hour intervals and the specimens were prepared at 24 h after the last administration. The animals in the positive control group were intraperitoneally administered with 2 mg/kg Mitomycin C once and the specimens were prepared at 24 h after administration. The potency of eliciting micronuclei and the frequency of total polychromatic erythrocytes of each article were examined.

In the 62.5 mg/kg group death occurred in 1 animal (1/6). In the surviving animals, decreased body weights were noted. Bradypnea, decrease in locomotor activity and abnormal fur were also noted in general signs. The frequency of micronucleated polychromatic erythrocytes were lower than upper limit of the frequency in the background data collected from the negative control group and was judged negative in statistical analysis. In the positive control group the frequency was higher than the upper limit of the frequency in the background data and was judged as positive in statistical analysis.

In the 62.5 mg/kg group there were statistically significant differences in the ratio of total polychromatic erythrocytes to total erythrocytes from the negative control group and slight depression of proliferation of erythrocytes were recognised. However, this change was considered to be associated with the toxic changes attributed to administration of massive doses since occasion of death and decrease of bodyweight and other toxic changes in the surviving animals was noted in the same group.

The results suggest that the test material has no potency of eliciting micronuclei in vivo when administered intraperitoneally to male ICR mice under the conditions of this study.