Hydrocotyle asiatica, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

july 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without metabolic activation: 10, 25, 50, 100, 250, 450 and 600 microg/mL.
With metabolic activation: 100, 250, 450, 600, 650, 700 and 800 microg/mL

Vehicle / solvent:

DMSO 1% (v/v)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Evaluation criteria:

Acceptability of the Assay
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT [8]:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range
50 - 170 mutants per 106 cells
- The cell number of the negative/solvent controls should undergo 8 - 32 fold increase during a 2 day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 106 cells
- The RTG must be greater than 10%.

Statistics:

Evaluation of Results
The test item is considered mutagenic if the following criteria:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: entella asiatica dry ext. is considered to be mutagenic in the in vitro mammalian cell gene mutation assay

Any other information on results incl. tables

Main Experiment - Toxicity Data, without metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	363000	916000	1520000	13.9	104.1	105.0	109.3
C2		380000	985000	1250000	12.3	92.1	100.0	92.0
S1	0	376000	873000	1370000	12.0	100.0	100.0	100.0
S2		400000	1020000	1450000	14.8
2	10	392000	944000	1250000	11.8	88.2	105.0	92.6
3	25	364000	886000	1370000	12.1	90.8	89.6	81.3
4	50	385000	898000	1490000	13.4	100.0	116.4	116.5
5	100	369000	987000	1470000	14.5	108.5	93.9	101.8
6	250	336000	705000	1450000	10.2	76.4	93.9	71.7
7	450	336000	332000	836000	2.8	20.8	100.0	20.7
8	600	322000	276000	465000	1.4	10.4	123.0	12.8
EMS	300	358000	925000	1400000	13.0	96.8	73.0	70.6
MMS	10	362000	839000	1350000	11.3	84.7	70.9	60.0

Main Experiment - Mutagenicity Data, without metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	82	74	104.6	11	19	21	21	18.0	99.9	/
C2		76	77	99.6	20	17	20	19	19.0	110.8	/
S1	0	76	78	101.2	19	15	16	10	15.0	84.3	/
S2		72	80	98.0	16	19	19	9	15.8	92.0	/
2	10	80	76	104.6	19	27	16	15	19.3	107.9	19.7
3	25	74	72	89.3	14	22	5	15	14.0	89.8	1.6
4	50	84	78	116.0	11	19	14	13	14.3	69.5	-18.6
5	100	72	77	93.5	18	24	21	20	20.8	130.4	42.3
6	250	72	77	93.5	23	25	22	16	21.5	136.1	47.9
7	450	71	82	99.6	25	15	22	19	20.3	119.5	31.3
8	600	85	80	122.6	39	12	17	18	21.5	107.8	19.6
EMS	300	58	74	72.7	67	68	72	60	66.8	824.7	736.5
MMS	10	67	63	70.6	61	55	69	63	62.0	742.4	654.3

 Main Experiment - Toxicity Data, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	265000	965000	1430000	13.8	91.4	115.0	105.1
C2		327000	1030000	1450000	14.9	99.0	106.3	105.2
S1	0	330000	985000	1430000	14.1	100.0	100.0	100.0
S2		362000	1110000	1450000	16.1
5	100	359000	1070000	1430000	15.3	101.4	107.9	109.4
6	250	403000	1020000	1410000	14.4	95.3	111.4	106.1
7	450	352000	611000	1430000	8.7	57.9	116.8	67.6
8	600	347000	467000	1200000	5.6	37.1	107.9	40.1
9	650	312000	355000	917000	3.3	21.6	127.0	27.4
10	700	333000	380000	888000	3.4	22.4	136.3	30.5
11	800	316000	294000	677000	2.0	13.5	115.0	15.5
B[a]P	2.5	384000	843000	1430000	12.1	79.9	82.7	66.1

 

Main Experiment - Mutagenicity Data, with metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	77	76	99.6	18	18	20	13	17.3	99.7	/
C2		73	75	92.1	18	23	18	18	19.3	121.7	/
S1	0	67	78	88.0	19	18	19	20	19.0	125.4	/
S2		69	74	85.4	21	16	20	21	19.5	133.2	/
5	100	73	76	93.5	17	20	15	15	16.8	102.7	-26.6
6	250	76	75	96.5	24	23	19	21	21.8	133.3	4.0
7	450	77	77	101.2	26	22	31	17	24.0	143.3	14.0
8	600	78	71	93.5	26	26	30	25	26.8	174.8	45.5
9	650	76	83	110.1	33	27	29	34	30.8	175.8	46.5
10	700	80	83	118.1	35	35	37	38	36.3	200.8	71.5
11	800	77	76	99.6	44	43	31	44	40.5	277.3	148.0
B[a]P	2.5	71	60	71.7	53	59	52	64	57.0	634.1	504.8

C:        Negative control

S:        Solvent control (1% DMSO; v/v)

e:        Number of cultures with cell growth.

f:         Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

g:       Mutant frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h:        Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

EMS:   Ethylmethanesulfonate

MMS:     Methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

Mutagenicity:
In the experiment without metabolic activation the mutant frequencies induced by the test item did not show any biologically relevant increase. A statistical analysis displayed that some of the mutant frequencies were significantly increased over those of the solvent controls. However, the GEF was not exceeded by the induced mutant frequency at any concentration. Therefore any differences observed in mutant frequency between the treated and concurrent control groups were concluded upon as not biologically relevant.

In the experiment with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 was exceeded at the concentration of 800 µg/mL (148.0 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies for the concentrations 600, 650, 700 and 800 µg/mL were significantly increased over those of the solvent controls. Moreover, a dose-response relationship was observed.

Clastogenicity:
The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.
In the main experiment with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the three highest dose groups, these concentrations of the test items were considered as potential clastogenic

Conclusion
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Centella asiatica dry ext. is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.