Matricaria recutita, ext.

Endpoint summary

Description of key information

- Skin irritant Category 2 (OECD 439, K, Rel.1: positive ; OECD 431, K, Rel.1: negative)

- Eye irritant Category 2 (OECD 492, GLP, K, Rel.1: positive ; HET-CAM, K, Rel.2: negative)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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:

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.

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

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

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.

Reference 2

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

26 September 2017 - 05 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 492 without any deviation.

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)

Version / remarks:

Adopted 28 July 2015

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)

Species:

other: Reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to liquids and semi-solids, so is considered to be applicable to the test item.

CELL SYSTEM:
- EpiOcular™ OCL-212-ver2.0, supplied by MatTek Corporation (Bratislava, Slovakia).
- Lot No.: 27008
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.208, within the acceptance criteria (1.1-3.0)
- Barrier function: 12.84 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile
- Transport: Not specified
- Storage: On day of receipt of the EpiOcular™ tissues in their well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 100917MGKA) and incubated during 20 hours at standard culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other:

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): not applicable

VEHICLE: Not applicable

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

- 12-minute immersion period at room temperature
- 2-hour incubation at 37°C, 5% CO2

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used: procedure for liquids
* Pre-treatment: after an overnight incubation, tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS. Then tissues were incubated for 30 minutes at standard culture conditions.
* Treatment: 50 µL of test item, positive or negative control was applied to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. Additionally, 2 killed RhCE (EpiOcularTM tissue model) were treated in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed RhCE tissue replicates were treated in the same manner in order to generate non-specific living and killed colour controls.
* Post-exposure incubation period: after treatment, tissues were carefully washed by extensive rinsing with Ca2+Mg2+Free-DPBS. Rinsed tissues were checked for any coloration: residual test item with blue coloration was noted on the epidermis. The rinsing step was followed by a 12-minute immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. Then the RhCE constructs were incubated for 2 hours at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of pre-treatment (30 minutes), exposure (30 minutes), post-exposure immersion (2 hours)

- Description of any modifications to the test procedure: None

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Since the test item was identified as a direct MTT reducer, two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non­specific MTT reduction control. The test item was identified as causing colour interference with the viability assay, thus two viable control tissues were added to the study.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled): 2

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The OD values obtained with the replicate tissue extracts for each test item were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results are interpreted as follows:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified potentially requiring classification and labelling according to UN GHS (Category 2 of Category 1).
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes, attached to the study report.
* Historical negative control ranges 83.17 - 116.83%
* Historical positive control ranges 6.50 - 60.03%

- Complete supporting information for the specific RhCE tissue construct used: Yes, attached to the study report.

- Reference to historical data of the RhCE tissue construct: No

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes, attached to the study report.

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: % Tissue viability

Remarks:

mean corrected percent

Run / experiment:

1

Value:

44.39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.00%

Positive controls validity:

valid

Remarks:

32.69%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Yes

Table 7.3.2/1:Mean OD₅₇₀Values and Percentage Viabilities for the Negative Control Item, Positive Control Item, Control Tissues and Test Item

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Difference of viability	Conclusion
Negative control	1	1.036 1.012 1.022	1.023	1.034	98.94	100.00	2.1
	2	1.063 1.036 1.037	1.045		101.06
Positive control	3	0.357 0.360 0.353	0.357	0.338	34.53	32.69	3.7	UN GHS Category 2 or 1
	4	0.394 0.352 0.210	0.319		30.85
Test item	7	0.571 0.581 0.581	0.578	0.495	55.90	47.87	16.1	UN GHS Category 2 or 1
	8	0.415 0.412 0.409	0.412		39.85
Test item - NSMTT	9	0.005 0.004 0.004	0.004	0.029	0.39	2.80	4.8
	10	0.054 0.055 0.054	0.054		5.22
Test item - NSC living control	11	0.014 0.012 0.014	0.013	0.021	1.26	2.03	1.5
	12	0.033 0.027 0.028	0.029		2.80
Test item NSC killed control	13	0.015 0.015 0.016	0.015	0.014	1.45	1.35	0.2
	14	0.013 0.013 0.013	0.013		1.26
Test item corrected						44.39

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

OD: optical density

Note: As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.25 for the negative control.

Interpretation of results:

other: UN GHS Category 2 or Category 1

Conclusions:

Under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item Camomille HE Egypte BLH (Essential Oil of Blue Chamomilla) has to be identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 492 and in compliance with GLP, using the in vitro reconstructed human cornea-like epithelium tissues (EpiOcular^TMtissue model).

Test item Camomille HE Egypte BLH (Essential Oil of Blue Chamomilla) was applied as applied at the dose of 50 µL to 2 living DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcular^TM tissue model) were treated in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed RhCE (EpiOcular^TM tissue model) were treated in the same manner but they were incubated in assay medium instead of MTT solution in order to generate non-specific living and killed colour controls.

The mean corrected percent tissue viability of the RhCE replicates treated with test item Camomille HE Egypte BLH (Essential Oil of Blue Chamomilla) was 44.39%, versus 32.69% in the positive control (Methyl acetate).

 

In conclusion, under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item Camomille HE Egypte BLH (Essential Oil of Blue Chamomilla) has to be identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

HET-CAM assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Basic data given, but considered sufficiently reliable for the purpose of hazard assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: HET-CAM assay

Principles of method if other than guideline:

- Principle of test: HET-CAM assay (hen’s egg test – chorioallantoic membrane) was used to determine the irritation potential of test item.
- Short description of test conditions: Test item was applied to the chorioallantoic membrane of fertile eggs at incubation day 9.
- Parameters analysed / observed: vascular lysis, hemorrhage, coagulation

GLP compliance:

not specified

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Test system: Fresh, fertile eggs
- 1st incubation: 37.8°C and 70% humidity for 72 h
- 2nd incubation: 96-h

Vehicle:

other: olive oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Concentrations ranging from 10% to 100% (undiluted)

Duration of treatment / exposure:

Not reported

Duration of post- treatment incubation (in vitro):

Not reported

Number of animals or in vitro replicates:

six replicates, repeated three times

Details on study design:

The HET-CAM assay (hen’s egg test – chorioallantoic membrane) was used to determine the irritation potential of chamomile oil (Möller et al 2006). Fresh, fertile eggs were
incubated at 37.8°C and 70% humidity for 72 h. Five millilitres of albumin were removed from the eggs with a syringe followed by another 96-h period of incubation.
For the HET-CAM assay the eggs were used on day 9 of incubation. A 50% mixture of cream and water was injected under the CAM to increase visibility of the CAM’s vessel system. Essential oils were applied to the CAM at different concentrations until the irritation threshold of the individual oils were detected. For documentation, the eggs were placed under a stereo microscope, equiped with a digital camera. Olive oil, the non-irritating solvent for essential oils, served as negative control. HET-CAM assays were performed with six replicates and repeated three times.
Test item was applied to the chorioallantoic membrane of embryonated eggs at concentrations ranging from 10% to 100%.

Irritation parameter:

other: observation of the chorioallantoic membrane

Run / experiment:

up to 100%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no indication of irritation

Other effects / acceptance of results:

Scores are not reported.
- Test item did not reveal any irritating behaviour, when applied either diluted or undiluted to the highly sensitive chorioallantoic membrane.
- Olive oil, the solvent for essential oils, served as negative control and caused no irritation of the chorioallantoic membrane.
- 0.5% SDS-solution represents a positive control and induces visible haemorrhage.

See attached 'Illustration'.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test item is considered to be non-irritant to the eyes according to the Regulation (EC) N°1272/2008 and to the GHS.

Executive summary:

In a in vitro study performed following the Chorioallantoic Membrane (HET-CAM) Test Method, test item was applied at concentrations from 10 to 100% to the chorioallantoic membrane of fertile eggs at incubation day 9.

Test item did not cause any irritation even when applied undiluted to the highly sensitive chorioallantoic membrane.

Negative control (olive oil) caused no irritation of the chorioallantoic membrane whereas positive control (0.5% SDS-solution) induces visible haemorrhage.

Under the test conditions, the test item did not induce irritation and is therefore not classified according to the Regulation (EC) N°1272/2008 and to the GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

Two key studies were identified for the skin irritation/corrosion.

Study N°1 :

In an in vitro skin irritation study performed according to the OECD Guideline 439, the EU Method B.46 and in compliance with GLP (Phycher, 2017), 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.

 

Study N°2 :

In an in vitro skin corrosion study performed according to the OECD Guideline 431, the EU Method B.40 and in compliance with GLP (Phycher, 2018), the test item was applied as supplied, at the dose of 50 µL, 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).

 

CONCLUSION : According to the available data for skin irritation, the substance is considered to be a skin irritant.

 

 

Eye irritation/corrosion

Two key studies were identified for the eye irritation/corrosion.

Study N°1

In an in vitro skin irritation study performed according to the OECD Guideline 492 and in compliance with GLP (Phycher, 2018), test item was applied as applied at the dose of 50 µL to 2 living DPBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcular^TMtissue model) were treated in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed RhCE (EpiOcular^TMtissue model) were treated in the same manner but they were incubated in assay medium instead of MTT solution in order to generate non-specific living and killed colour controls.

 

The mean corrected percent tissue viability of the RhCE replicates treated with test item was 44.39%, versus 32.69% in the positive control (Methyl acetate).

 

Study N°2

In an in vitro study performed following the Chorioallantoic Membrane (HET-CAM) Test Method (Koch et al. 2008), the test item was applied at concentrations from 10 to 100% to the chorioallantoic membrane of fertile eggs at incubation day 9.

Test item did not cause any irritation even when applied undiluted to the highly sensitive chorioallantoic membrane.

Negative control (olive oil) caused no irritation of the chorioallantoic membrane whereas positive control (0.5% SDS-solution) induces visible haemorrhage.

 

CONCLUSION : According to the available data for eye irritation, the substance is considered to be an eye irritant.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on the available data, the substance is classified as Skin irr. Category 2 (H315: Causes skin irritation) and Eye irrit. Category 2 (H319 : Causes serious eye irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available regarding respiratory irritation.