Propyl 3,4,5-trihydroxybenzoate

Genetic toxicity: in vivo

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

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Principles of method if other than guideline:

- Principle of test: Comet assay in mice similar to OECD guideline 489
- Short description of test conditions & parameters analysed / observed: 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

Test material information

Constituent 1

Reference substance name:

Propyl 3,4,5-trihydroxybenzoate

EC Number:

204-498-2

EC Name:

Propyl 3,4,5-trihydroxybenzoate

Cas Number:

121-79-9

Molecular formula:

C10H12O5

IUPAC Name:

propyl 3,4,5-trihydroxybenzoate

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 or test system 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): 12 hours light/12 hours dark

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

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

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

No. of animals per sex per dose:

4 males

Control animals:

yes, concurrent vehicle

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:

CRITERIA FOR DOSE SELECTION: 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.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were sacrificed 3 or 24 hours after treatment.

DETAILS OF SLIDE PREPARATION: 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. 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) 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. 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.

METHOD OF ANALYSIS: 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope were examined and photographed. The length of the whole comet and the diameter of the head were measured for 50 nuclei per organ per animal.

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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

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:

In a ddY mouse comet assay conducted similar to OECD guideline 489, 4 male mice were once orally treated with propyl gallate at a dose of 2000 mg/kg bw. All mice were euthanized at 3 hours and 24 hours after exposure, at which time organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) 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.