Hydrocotyle asiatica, ext.

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocotyle asiatica, ext.

EC Number:

283-640-5

EC Name:

Hydrocotyle asiatica, ext.

Cas Number:

84696-21-9

Molecular formula:

not applicable

IUPAC Name:

Hydrocotyle asiatica, ext.

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

young healthy adult

Sex:

male/female

Details on test animals or test system and environmental conditions:

5 of each sex per dose group

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The test item was suspended in 0.9% NaCl

Duration of treatment / exposure:

Single application

Post exposure period:

Bone marrow cells of the treated animals were collected 24 h and 48 h after the single application.

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.:

400 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

5 of each sex per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes: CPA; Cyclophosphamide

Examinations

Tissues and cell types examined:

Bone marrow was obtained from the femurs

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Within the main experiment the chosen concentration of 2000 mg/kg bw led to severe signs of toxicity as well as lethality within the first 24 hours. Based on animal welfare aspects the remaining animals of the 2000 mg/kg bw dose group were euthanized. Thus, as a result of the observed severe toxicity and lethality the maximal tolerable dose were adjusted.
1MTD: 1000 mg/kg bw
0.4 MTD: 400 mg/kg bw
0.1 MTD: 100 mg/kg bw
The animals treated with doses of 0.1 MTD, 0.4 MTD and 1 MTD showed dose-dependent mild to moderate signs of systemic toxicity. The animals treated with 1 MTD (24 h and 48 h) showed moderate signs of systemic toxicity such as reduction of spontaneous activity, catalepsis, prone position, constricted abdomen, piloerection, bradykinesia, kyphosis, recumbency, opisthotonos, ataxia, half eyelid closure and eye closure (Table 4, 5). 24 hours (1 MTD, 24 h), respectively 48 hours (1 MTD, 48h) after the treatment, no signs of toxicity were observed any more. Animals treated with 400 mg/kg bw (0.4 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals (Table 4, 5), except bradykinesia, recumbency and eye closure. 24 hours after treatment no toxic symptoms were observed anymore. Animals treated with 0.1 MTD showed mild signs of toxicity. 24 hours after treatment no signs of toxicity were observed anymore for the 0.1 MTD dose group animals.
For all dose groups, including positive and negative controls, 2000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (24 h, 48 h) were within the range of the historical control data. The mean values noted for all dose groups, which were treated with the test item (24 h, 48 h), were within the historical negative control data range.
No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases/decreases (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.
Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a significant increase in micronucleus frequency. This demonstrates the validity of the assay.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Centella asiatica purified dry extract did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, Centella asiatica purified dry extract is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

This study was performed to investigate the potential of Centella asiatica purified dry extract to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, which is the endpoint of this test to assess genotoxicity.

The test item was suspended in 0.9% NaCl. The volume administered i.p. was 10 mL/kg bw. Bone marrow samples were collected for micronuclei analysis 24 h and 48 h after a single application of the test item.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

Therefore, Centella asiatica purified dry extract is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.