Matricaria recutita, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August to 27 November, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September 2017 to 05 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 431 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 30-31 January 2017 / signed on 27 April 2017)

Specific details on test material used for the study:

Date received; 25 July 2017
Test item was identified under the following code number: PH-17/0469

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not precised

Source strain:

other: Not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Following the REACH bottom-up strategy, as the result of the OECD test guideline No. 439 was positive, the epiCS® Reconstructed Human Epidermis Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The test item was applied, as supplied, at the dose of 50 µL to 2 living and 4 killed Human skin model surfaces during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.
- Living human skin model used: 0.6 cm² reconstituted epidermis epiCS® (CellSystems®). Product number: CS-1001
- Living tissue batch number(s): 100-AG1830-1
- Killed tissues: killed Human skin model epiCS® CellSystems® - batch No. 100-AF2056
- Production date (living tissues): 09.10.2017
- Shipping date (living tissues): Not reported
- Delivery date (living tissues): 11 October 2017
- Date of initiation of testing: 11 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2
- Temperature of post-treatment incubation: not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 x 1 mL of DPBS (Dutscher - Batch No. 1310817)
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and presented blue residue of test item.
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm
- Filter: Not applicable
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function/viability (MTT, 2h Triton X-100): 58.64%
- Morphology: 3-D, fully differentiated human epidermis equivalent
- Contamination: absence of virus, bacteria, yeast and mycoplasma
- Reproducibility: Not reported

NUMBER OF REPLICATE TISSUES: 3

ADDITIONAL CONTROL TISSUES
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were defrozen the day of treatment.
- 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) were treated in the same manner as during both 3 minutes and 1 hour, but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of test item
- Concentration (if solution): Not applicable: undiluted

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of distilled water
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of potassium hydroxide 8N (8N KOH)
- Concentration (if solution): Not applicable

Duration of treatment / exposure:

3 min at room temperature or 1 hour at 37°C ± 1°C, 5% ± 1% CO2

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2 tissues per condition
Triplicate measurement of OD570 for each tissue.

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected

Run / experiment:

3 min

Value:

80.19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean OD of negative control tissues after 3 minutes exposure was 1.186 instead of ≤ 0.9.

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected

Run / experiment:

1 hour

Value:

84.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean OD of negative control tissues after 3 minutes exposure was 1.112 instead of ≤ 0.9.

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
The mean OD of negative control tissues after 3 minutes exposure and after 1 hour exposure were1.186 and 1.112 respectively instead of ≤ 0.9 as initially scheduled. Considering the fact that these values are included in the historical range for negative controls performed at Phycher and that the results obtained with the positive control are conformed to what was expected (mean viability <20%), this deviation is considered as without impact on the conclusion of this study.
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Yes

Table 7.3.1/1: Individual and average values after 3 minutes exposure

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD
Negative control	1	1.003 1.195 1.062	1.087	1.186	91.65	100.0	16.7
	2	1.408 1.300 1.148	1.285		108.35
Positive control	3	0.515 0.425 0.459	0.466	0.461	39.29	38.87	0.8
	4	0.451 0.460 0.457	0.456		38.45
Test item	13	0.995 1.073 1.061	1.043	1.040	87.94	87.69	0.5
	14	0.956 1.052 1.104	1.037		87.44
Test item NSMTT	15	0.082 0.080 0.081	0.081	0.078	6.83	6.58	0.5
	16	0.075 0.076 0.075	0.075		6.32
Test item NSC living control	17	0.014 0.016 0.017	0.016	0.022	1.35	1.85	1.0
	18	0.028 0.029 0.028	0.028		2.36
Test item NSC killed control	19	0.017 0.015 0.017	0.016	0.011	1.35	0.93	0.8
	20	0.005 0.006 0.006	0.006		0.51
Test item corrected						80.19

#: mean of 3 values

OD: optical density

Table 7.3.1/2: Individual and average values after 1 hour exposure

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD
Negative control	1	1.077 1.107 1.093	1.092	1.112	98.20	100.0	3.6
	2	1.303 1.102 0.992	1.132		101.80
Positive control	3	0.002 0.002 0.013	0.006	0.005	0.54	0.4	0.3
	4	0.003 0.003 0.003	0.003		0.27
Test item	5	1.054 1.114 1.093	1.087	0.979	97.75	87.99	19.5
	6	0.905 0.869 0.835	0.870		78.24
Test item NSMTT	7	0.025 0.027 0.025	0.026	0.033	2.34	2.92	1.2
	8	0.037 0.038 0.041	0.039		3.51
Test item NSC living control	9	0.006 0.006 0.005	0.006	0.008	0.54	0.67	0.3
	10	0.010 0.009 0.009	0.009		0.81
Test item NSC killed control	11	0.009 0.005 0.006	0.007	0.007	0.63	0.58	0.1
	12	0.005 0.007 0.007	0.006		0.54
Test item corrected						84.98

#: mean of 3 values

OD: optical density

Table 7.3.1/3: Conclusion

	Mean viability (%)
	3 min exposure	1 hour exposure	Conclusion
Positive control	38.87	0.40	Corrosive sub-category 1B/1C
Test item	80.19	84.98	Non Corrosive

Interpretation of results:

other: non-corrosive

Conclusions:

Under the experimental conditions of this study, the test item cannot be classified in Cateogry 1 "Corrosive to skin" according to the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431, the EU Method B.40 and in compliance with GLP, using the epiCS® (CellSystems^®) Reconstructed Human Epidermis Model.

The test item was applied as supplied, at the dose of 50µL, to 2 living and 4 killed Human skin model surface during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

Additionally, 2 living and 4 killed Human skin model surfaces were treated in the same manner during both 3 minutes and 1 hour, but they were incubated in MTT assay medium instead of MTT solution in order to generate non-specific living and killed colour controls. 

 

3 minutes and 1 hour after the test item application, the mean corrected percent viability of the epidermis skins treated with test item were 80.19 and 84.98%, versus 38.87% and 0.40%, respectively, with the positive control item (potassium hydroxide 8N).

Under the experimental conditions of this study, the test item cannot be classified in Cateogry 1 "Corrosive to skin" according to the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 30-31 January 2017 / signed on 27 April 2017)

Test material

Test material information

Constituent 1

Reference substance name:

Matricaria recutita, ext.

EC Number:

282-006-5

EC Name:

Matricaria recutita, ext.

Cas Number:

84082-60-0

Molecular formula:

Not relevant, UVCB substance

IUPAC Name:

Essential oil of Matricaria recutita (Asteraceae) obtained from flower tops by steam distillation of Egypt origin

Test material form:

liquid: viscous

Details on test material:

Common name: Chamomile Blue essential oil
Botanical name: Chamomilla recutita (L.) Rauschert
Family: Asteraceae
No CAS TSCA: 8002-66-2
No CAS EINECS: 84082-60-0
Part of the plant distilled: flowering tops
Manufacture date: March 2017
Best use before: March 2020
Color: Inky blue to black
Odor: Intense sweet, herbaceous aroma
Miscibility: Miscible in alcohol and other oils
Conditions of storage: In the refrigerator (at +2 - +8° C), under nitrogen

Specific details on test material used for the study:

Date received; 25 July 2017
Test item was identified under the following code number: PH-17/0469

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: Not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model (Episkin)
- Tissue batch number(s): 17-RHE-088
- Production date: Not reported
- Shipping date: Not specified
- Delivery date: 29 August 2017
- Expiry date: 04 September 2017
- Date of initiation of testing: 23 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617)
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek
- Wavelength: 570 nm
- Filter: Not applicable
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: No data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570= 1.4, CV= 4.3% (specification: OD > 0.7)
- Barrier function: ET50 (Exposure time inducing 50% viability using Triton X-100 1%)= 4.3h (specification: 4.0h =< ET50 =< 10.0h)
- Morphology: well differenciated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE / COLOUR INTERFERENCE
MTT direct interference
- Killed tissues: SkinEthic RHE® model RHE/S/17
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were defrozen the day of treatment. Then, they were treated in the manner as the living RHE in order to generate non-specific MTT reduction.
- N. of replicates : two tissues
Colour interference
- 2 living and 2 killed Human skin model surfaces were treated in the same manner in order to generate non-specific living and killed colours controls.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes of exposure and 42 hours of post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL of the test item was then applied to the epidermal surface.
- Concentration (if solution): Not appicable: undiluted

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of DPBS
- Concentration (if solution): Not applicable: undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of SDS
- Concentration (if solution): 5 % w/v aqueous solution

Duration of treatment / exposure:

Living and killed tissues were treated with the test item for an exposure period of 42 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 41 hours in fresh medium.

Number of replicates:

- 3 living tissues for test item, negative and positive controls
- 2 killed tissues for MTT direct interference controls
- 2 killed and 2 living tissues for colour interference controls
Triplicate measurement of OD570 for each tissue.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

corrected 'True viability'

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

1.2%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Provided in the study report

Any other information on results incl. tables

Table 7.3.1/1: Individual and average values of OD₅₇₀ after 42 minutes exposure

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	0.750 0.851 0.872	0.824	0.787	104.7	100.0	5.2
	2	0.770 0.795 0.813	0.793		100.8
	3	0.759 0.745 0.726	0.743		94.4
Positive control	4	0.008 0.009 0.010	0.009	0.009	1.1	1.2	0.2	Irritant
	5	0.009 0.009 0.008	0.008		1.0
	6	0.010 0.010 0.013	0.011		1.4
Test item	13	0.033 0.037 0.037	0.036	0.038	4.6	4.9	1.6
	14	0.028 0.026 0.026	0.027		3.4
	15	0.053 0.051 0.052	0.052		6.6
Test item NSC Control living tissues	27	0.003 0.003 0.002	0.003	0.002	0.4	0.3	0.2
	28	0.001 0.000 0.002	0.001		0.1
Test item NSC Control killed tissues	1 (plate 2)	0.002 0.001 0.001	0.001	0.002	0.1	0.2	0.1
	2 (plate 2)	0.001 0.002 0.002	0.002		0.3
Test item killed tissues MTT control	3 (plate 2)	0.021 0.025 0.022	0.023	0.018	2.9	2.3	0.9
	4 (plate 2)	0.014 0.013 0.013	0.013		1.7
Test item corrected						2.5		Irritant

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item has to be classified in Category 2 "Irritating to skin" according to the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439, the EU Method B.46 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

The test item was applied, as supplied, at the dose of 16 μL to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Two additional killed control tissues were used for MTT direct interference and two living and two killed tissues for colour interference.

 

The mean corrected percent viability of the treated tissues was 2.5%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).

The mean OD negative control obtained after a 1:2 dilution of the formazan extracts in isopropanol is include in the acceptability criteria range which is ≥ 0.4 ≤ 1.5.

The mean percent tissue viabilites obtained with the negative control and positive controls are within the range of historical data.

Under the experimental conditions of this study, and with the classification non-corrosive obtained with the in vitro skin corrosion test (HSMC-PH-17/0469), the test item has to be classified in Category 2 "Irritating to skin" according to the Regulation (EC) No.1272/2008 (CLP) and to the GHS.