Hydrocotyle asiatica, ext.

Endpoint summary

Description of key information

In vitro studies are available for the evaluation of skin irritation/corrosion potential and of eye irritation/damage potential of Centella asiatica dry ext.

Centella asiatica dry ext. resulted irritating to the eyes, but not irritating to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

july 2016

Deviations:

no

Principles of method if other than guideline:

Experimental Procedure
Upon receipt of the EpiDerm TM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37  1°C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.
60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37  1°C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37  1°C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 h at 37  1°C, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.

After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

GLP compliance:

yes

Test system:

human skin model

Source species:

other: stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm, a reconstituted three-dimensional human epidermis model

Vehicle:

other: water and isopropanol (1:1 v/v)

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

25mg in 25 µL of water

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Irritation / corrosion parameter:

other: NSC living (%)

Run / experiment:

3 min

Value:

ca. 0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: NSC living (%)

Run / experiment:

60 min

Value:

ca. 0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.995	2.067	0.344	0.357	1.862	1.870
	1.891	2.057	0.361	0.340	1.852	1.887
	2.008	2.080	0.341	0.359	1.839	1.897
Mean Absolute OD570	2.016****		0.350		1.868
OD570- Blank Corrected	1.951	2.023	0.300	0.314	1.818	1.827
	1.847	2.013	0.317	0.297	1.809	1.844
	1.965	2.037	0.297	0.316	1.795	1.854
Mean OD570of 3 Aliquots (Blank Corrected)	1.921	2.024	0.305	0.309	1.807	1.841
SD OD570 of 3 Aliquots	0.064	0.012	0.011	0.010	0.011	0.014
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.973*		0.307		1.824
SD OD570 of 2 Replicate Tissues	0.073		0.003		0.024
Mean Relative Tissue Viability [%]	100.0		15.6		92.5
Coefficient Of Variation [%]^***	3.7		0.9		1.3

^*              corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^***            coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is£ 30%.

^****          The mean absolute OD₅₇₀of the negative control is ≥ 0.8 and ≤ 2.8

Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.871	1.889	0.155	0.162	1.502	1.423
	1.904	1.888	0.158	0.154	1.497	1.423
	1.845	1.900	0.155	0.163	1.490	1.443
Mean Absolute OD570	1.883****		0.158		1.463
OD570- Blank Corrected	1.827	1.846	0.112	0.118	1.459	1.379
	1.860	1.844	0.115	0.110	1.454	1.380
	1.802	1.857	0.111	0.120	1.446	1.400
Mean OD570of 3 Aliquots (Blank Corrected)	1.830	1.849	0.113	0.116	1.453	1.386
SD OD570 of 3 Aliquots	0.029	0.007	0.002	0.005	0.006	0.012
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.839*		0.114		1.420
SD OD570 of 2 Replicate Tissues	0.014		0.002		0.047
Mean Relative Tissue Viability [%]	100.0		6.2**		77.2
Coefficient Of Variation [%]^***	0.7		2.1		3.3

^*              corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^**             mean relative tissue viability of the 60 min positive control < 15%,

^***            coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is£ 30%.

^****          The mean absolute OD₅₇₀of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no corrosive effects.
The mean relative tissue viability (% negative control) was <= 50% (92.5%) after 3 min treatment and >=15% (77.2%) after 60 min treatment.
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was >= 0.8 and ≤ 2.8 for each exposure period (2.016, 1.883). The mean relative tissue viability (% negative control) of the positive control was < 15% (6.2%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was <= 30% (0.7% – 3.7%).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The test was carried out with the reconsttued three-dimensional human modle EPISKIN-DM TM (SkinEthic). This skin model consists of normal (non-cancerous), adult huma-derived epidermal keratinocytes (NHEK) which have been cultured to form a moltilayered, highly differentiated model of the huma epidermidis. The NHEK are cultured on chemically modified, collagen-coated cellculture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative control: 10 uL PBS
Positive control: 10 uL 5% SDS solution
Test item: 10 mg + 5 uL aqua dest.

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

negative control / mean

Value:

100

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control / mean

Value:

7

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item / mean

Value:

94

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 minutes of exposure and 42 hours post-incubation was > 50%. The test item i therefore classified as "non-irritant" in accordance with UN GHS and EU CLP "No Category".

Executive summary:

In the present study the skin irritant potential of Centella asiatica purified dry extract was analysed. The EPISKIN-Standard Model TM (EPISKIN-SM TM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS and EU CLP "Category 2" skin irritating test substances and not categorized test substances ("No Category") which may be considered as non-irritant. hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after 15 min exposure and 42 h post incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 minutes of exposure and 42 hours post-incubation was > 50%. The test item i therefore classified as "non-irritant" in accordance with UN GHS and EU CLP "No Category".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Sept 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

27.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

18.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

52.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.523

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1.173

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

The eye irritancy potential of Centella asiatica purified dry extract, was investigated in the bovine corneal opacity and permeability assay. The test item was diluited with physiological saline 0.9% NaCl to gain a 20% concentration. The mean in vitro irritation score was calculated: 53,23.
Therefore the test item was classified as moderate irritant.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

not specified

GLP compliance:

yes

Irritation parameter:

morphological effects

Run / experiment:

mean

Value:

0.851

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

The study has been conducted in order to asses in vitro, on a Human Corneal Epitelium model (HCE), the eye irritation potential of Centella asiatica purified drye extract. According to the adpted model based on MTT results the chemicals have been clessified as reported in the Results: Irritant for the eye

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the in vitro study results, Centella asiatica dry ext. is classified as Eye Irrit. 2, H319.