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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
publication
Title:
The comet assay with 8 mouse organs: results with 39 currently used food additives.
Author:
Sasaki Y.F. et al.
Year:
2002
Bibliographic source:
Mutation Research 519, 103-119.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
only 4 animals per dose group, only one dose, no in vivo positive control, only 50 cells scored per organ per animal
Principles of method if other than guideline:
Mice were exposed to the limit dose of 2000 mg/kg and eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) were removed and were afterwards processed to be analysed in a Comet assay.
GLP compliance:
not specified
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kanto Chemical Co. Inc., Tokyo, Japan

Test animals

Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Co., Shizuoka, Japan
- Age at study initiation: 7 weeks of age and used after 1 week of acclimatization.
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: no
- Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Photoperiod (hrs dark / hrs light): 12h light-dark

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Details on exposure:
4 male mice per group were administered orally 2000 mg/kg, and untreated mice served as controls. The oral dose of 2000 mg/kg was determined via preliminary simple acute toxicity experiment on 4-5 animals. No death was observed at 2000 mg/kg, so the LD50 was defined at >2000 mg/kg.
Duration of treatment / exposure:
Once orally
Frequency of treatment:
once
Post exposure period:
not specified
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Justification of dose: LD50 was determined using simple acute toxicity "experiments on four–five animals." No death was observed at 2000 mg/kg, so the LD50 was defined as >2000 mg/kg. Whether 2000 mg/kg was nominal or the actual dose received was not specified.
No. of animals per sex per dose:
2000 mg/kg: 4 males
Control animals:
yes, concurrent no treatment
Positive control(s):
not specified

Examinations

Tissues and cell types examined:
Eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow).
Details of tissue and slide preparation:
Eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) were removed and were afterwards processed to be analyzed in a Comet assay. The length of the whole comet and the diameter of the head were measured for 50 nuclei per organ per animal. Migration was calculated as the difference between length and diameter for each 50 nuclei.

The liver, kidney, lung, and brain were minced, suspended in 4 ml chilled homogenizing solution (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, and then homogenized gently using a Potter–Elvehjem type homogenizer at 500–800 rpm, in ice [9]. The glandular stomach, colon, and urinary bladder were opened and rinsed with physiological saline; the mucosa was scraped into 4 ml chilled homogenizing buffer and homogenized gently using a Potter–Elvehjem type homogenizer at 500–800 rpm, in ice. To obtain nuclei, the homogenate was centrifuged at 700×g for 10 min at 0 ◦C, and the precipitate was re-suspended in chilled homogenizing buffer at 1 g organ weight/ml. of the covering slide. Finally, 75 l of agarose GP-42 was quickly layered on again. Slides prepared from nuclei isolated by homogenization were placed in a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) [9] and kept at 0 ◦C in the dark for about 1 night, then in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 10 min in the dark at 0 ◦C [9]. Electrophoresis was conducted at 0 ◦C in the dark for 15 min at 25 V (0.96 V/cm) and approximately 250 mA. The slides were neutralized and then stained with 50 l of 20 g/ml ethidium bromide (Wako Pure Chemical Industries Ltd.) [9]. We examined and photographed 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope.
Evaluation criteria:
not specified
Statistics:
Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P value less than 0.05 was considered statistically significant.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
not examined
Negative controls valid:
not specified
Positive controls valid:
not examined
Remarks on result:
other: no in vivo positive control included in the experiment
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Propyl gallate did not increase DNA damage in any of the organs studied. Additionally, no death, morbidity, or clinical signs were observed.

Applicant's summary and conclusion

Conclusions:
This experimental study in a small group of male mice shows that a single oral 2000 mg/kg exposure to Propyl gallate did not increase DNA damage in any of the organs studied at 3 hours and 24 hours after the exposure.
Executive summary:

The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of Propyl gallate to induce genotoxicity in murine organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) 3 and 24 h after treatment. In the present study, 4 male ddY mice were exposed once orally to 2000 mg/kg of Propyl gallate. Untreated mice served as negative controls. All mice were euthanized at 3 hours and 24 hours after exposure, at which time the either organs were removed and processed to be analyzed in a Comet assay. The length of the whole comet and the diameter of the head were measured for 50 nuclei per organ per animal. Migration was calculated as the difference between length and diameter for each 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared. The results of this study indicate that Propyl gallate did not increase DNA damage in any of the organs studies at 3 hours and 24 hours after exposure.