[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

publication

Title:

Safety Evaluation of a Peptide Product Derived From Sardine Protein Hydrolysates (Valtyron)

Author:

Katsuhiro Osajima et al.

Year:

2009

Bibliographic source:

International Journal of Toxicology Vol. 28 N. 5 2009 341-356

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Remarks:

This information was not provided in the publication

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Fish protein hydrolysate

IUPAC Name:

Fish protein hydrolysate

Details on test material:

Material obtained via enzymatic hydrolysis of sardine muscle

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

were acclimated to laboratory conditions for 11 days. The environmental conditions under which the mice were housed were specifically regulated. The temperature
and humidity ranges were 19C to 25C and 30% to 70%, respectively. The ventilation rate was 15 air changes per hour, and artificial light was provided for 12 h/d (ie, 12-hour light/dark cycle). The animals were housed in aluminum cages, with 4 animals per cage during the acclimation period and 5 animals per cage after group assignment. A standard commercial pellet diet (CE-2; Clea Japan, Inc., Tokyo, Japan) and water were provided ad libitum. At the start of the study period, the mice were 8 weeks old and body weights ranged from 32.7 to 38.9 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% (wt/vol) aqueous solutions of carboxymethyl cellulose sodium (0.5% CMC-Na), After preparation, the solvent solution was autoclaved
at 121C for 20 minutes and stored under refrigeration (2-6C) until use. In the case of the highest concentration of the sardine peptide product
tested (ie, 200mg/mL), a suspension was prepared by precipitation. Each sardine peptide test solution or suspension was prepared shortly before use, and
because the test article was a peptide mixture, sonification was not required.

Details on exposure:

the sardine peptide product was administered to the mice once daily at dose levels of 250, 500, 1000, or 2000 mg/kgBWby gavage using a stomach tube and a disposable syringe.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

at 24-hour intervals

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group received a single intraperitoneal injection of mitomycin C (MMC) at a dose
level of 2 mg/kg BW using a disposable syringe and needle

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

To prepare bone marrow smears, both ends of the femurs were cut and bone marrow fluid was washed out with about 1 mL of fetal bovine serum. Thereafter, the fluid was centrifuged at 1000 rpm (210 g) for 5 minutes and the supernatant discarded. A small amount of cell suspension was pipetted onto a lipid-free, clean glass slide, and a smear specimen was prepared using a cover-slip. Once the specimen was sufficiently dry, it was fixed with methanol for 5 minutes and immediately stained with 3% Giemsa solution. Following staining, the specimen was gently washed with a sodium–potassium phosphate buffer solution and subsequently was treated with a 0.004% (wt/vol) aqueous citric acid solution for approximately 3 seconds, washed with distilled water, and finally dried.

Evaluation criteria:

Coded specimens were examined using a light microscope at a final magnification rate of x600. For every 2000 polychromatic erythrocytes (PCEs) in each individual sample, the number of micronucleated polychromatic erythrocytes (MN-PCEs) was counted to determine the MN-PCE prevalence. In addition, as an index of growth suppression of bone marrow cells, the numbers of PCEs and normochromatic erythrocytes (NCEs) were scored in 500 cells to determine
the ratio of PCE to total erythrocytes (PCE/PCEþNCE).

Statistics:

The statistical significance of differences in the prevalence of MN-PCEs between the negative control group and the test groups or the positive control group was determined by the method of Kastenbaum and Bowman. A difference of P < .05 was considered statistically significant. To determine the statistical significance of differences between the groups in the rate of bone marrow cell proliferation, the Student’s t test was applied. A difference of P < .01 was considered statistically significant.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No animals died following administration of the sardine peptide product, and no changes in general appearance or behavior attributable to the administration of the sardine peptide product were observed. Body weights of the sardine peptide-treated animals were comparable to the negative control.

The prevalence of MN-PCE in bone marrow cells of mice administered the sardine peptide product at dose levels of 250, 500, 1000, and 2000 mg/kg BW was 0.08, 0.06, 0.07, and 0.08%, respectively. The values observed in the sardine peptide-treated mice did not differ significantly (P < .05) from the value determined in the negative control group (0.04%). The prevalence of MN-PCE observed in the positive control group (3.83%) was significantly higher (P < .05) than the corresponding negative control group value. Of all the erythrocytes examined, the PCE incidence was determined to be 46.88%, 44.76%, 40.52%, and 40.08% in animals administered the sardine peptide product at dose levels of
250, 500, 1000, and 2500 mg/kg BW, respectively. The reductions in the ratios of PCE to total erythrocytes (PCE/PCE+NCE) observed at the two highest dose levels (1000 and 2000 mg/kg BW) were statistically significant (P .01) compared with the ratio observed for the negative control group (49.96%). The PCE/PCE+NCE value in the positive control
group (37.72%) also was significantly lower than that of the negative control group. The prevalence of MN-PCE and the ratio of PCE to total erythrocytes in both the negative and the positive control groups were within the ranges of historical background data for the laboratory (mean + 3 standard deviation).

Applicant's summary and conclusion

Conclusions:

Examination of mice erythrocytes following administration of 2 successive gavage doses of the sardine peptide product at dose levels of up to 2000 mg/kg BW did not elevate the incidence of micronuclei compared with the solvent control.