Matricaria recutita, ext.

Eye irritation

Administrative data

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.

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 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)

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

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.