Extract obtained from powder of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol

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

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals.

Author:

Hayashi, M. Kishi, M, Sofuni, T and Ishidate, M

Year:

1988

Bibliographic source:

Fd Chem Toxicol, 26, 487-500.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Propylene glycol was administered by single intraperitoneal injection at dose levels of 0, 2500, 5000, 10000 and 15000 mg/kg bw to groups of 6 male mice. Animals were killed 18 hr post-administration and femoral bone marrow cells were scored for the number of micronucleated polychromatic erythrocytes.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

Details on test material:

Source: Dow Chemical company, Freeport, Texas
Purity: 99%
Appearance: clear liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): propylene glycol
- Analytical purity: 99%
- Supplier: The Japan Food Additives Association, Tokyo

Test animals

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan
- Age at study initiation: 8 weeks
- Diet: food pellets CE-2 (Japan Clea, Tokyo), ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline;

Duration of treatment / exposure:

Single injection

Frequency of treatment:

Single injection

Post exposure period:

18 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw (total dose)

Doses / concentrations 4

Dose / conc.:

10 000 mg/kg bw (total dose)

Doses / concentrations 5

Dose / conc.:

15 000 mg/kg bw (total dose)

No. of animals per sex per dose:

6 males/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s):
- Route of administration:
- Doses / concentrations:

Examinations

Tissues and cell types examined:

Femoral bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on the pilot study using the multi-sampling at multi-dose levels method.

DETAILS OF SLIDE PREPARATION:
Mice were killed by cervical dislocation 18 hr after injection with propylene glycol. Femoral marrow cells were isolated, smeared onto clean glass slides, fixed with methanol and stained with Giemsa. The preparations were coded and analysed blind.

METHOD OF ANALYSIS:
One thousand polychromatic erythrocytes (PCEs) per mouse were examined using light microscopy (x100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of PCEs among the total erythrocytes was also evaluated by observation of 1000 erythrocytes on the same slide.

Evaluation criteria:

A positive result was recorded only when one or more treatment groups showed a statistically signiicant difference (p < 0.01) from the spontaneous level of micronucleated polychromatic erythrocytes and the trend test indicated a positive dose-response (p < 0.05)

Statistics:

The frequency of MNPCEs in each treatment was compared with the binomial distribution of historical control data. A result was considered positive if the increase in MNPCEs differed from the spontaneous data at P<0.01. Any dose-response relationship was tested using the Cochran-Armitage trend test, with P<0.05 indicating a positive result.

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:

Three of six mice from the top dose group died during the course of the study. There was no statistically significant increase or trend in MNPCE numbers following ip administration of propylene glycol at doses up to and including 15000 mg/kg. The percentage of PCEs in the top dose group appeared decreased relative to controls (31% versus 54%) suggesting that the test substance had reached the bone marrow. Significant, dose-related increases in MNPCEs were obtained with 5 of 39 chemicals included in these investigations indicating that the test system was capable of detecting a positive response

Any other information on results incl. tables

Table 1. Results of the micronucleous test using mouse bone marrow cells with propylene glycol.

Concentrationmg/kg bw	MNPCE (%)	PCE (%)	Mortality
0 (vehicle control)	0.12± 0.08	54.1± 5.9	0/6
2500	0.19± 0.04	47.4± 11.9	0/6
5000	0.17± 0.14	47.1 ± 8.9	0/6
10000	0.10 ± 0.13	38.2± 15.0	0/6
15000	0.24 ± 0.25	31.1± 11.1	3/6

MNPCE = micronucleated polychromatic erythrocytes; PCE = polychromatic erythrocytes. The valuyes are exressed as mean ± SD.

Applicant's summary and conclusion

Executive summary:

Propylene glycol produced no detectable increase in micronucleated polychromatic erythrocytes when administered by ip injection to mice at doses up to 15000 mg/kg.