Tagetes minuta, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 09 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

222-081-3

EC Name:

(Z)-3,7-dimethylocta-1,3,6,-triene

Cas Number:

3338-55-4

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Constituent 2

Reference substance name:

(5Z)-2,6-dimethylocta-2,5,7-trien-4-one (5E)-2,6-Dimethyl-2,5,7-octatrien-4-oneChl

Cas Number:

502-33-0

Molecular formula:

C10H14O

IUPAC Name:

(5Z)-2,6-dimethylocta-2,5,7-trien-4-one (5E)-2,6-Dimethyl-2,5,7-octatrien-4-oneChl

Constituent 3

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-1-methyl-4-(prop-1-en-2-yl)cyclohexene

Constituent 4

Reference substance name:

2,6-dimethyloct-7-en-4-one

EC Number:

217-532-6

EC Name:

2,6-dimethyloct-7-en-4-one

Cas Number:

1879-00-1

Molecular formula:

C10H18O

IUPAC Name:

2,6-dimethyloct-7-en-4-one

Constituent 5

Reference substance name:

2,6-dimethylocta-5,7-dien-4-one

EC Number:

245-971-3

EC Name:

2,6-dimethylocta-5,7-dien-4-one

Cas Number:

23985-25-3

Molecular formula:

C10H16O

IUPAC Name:

2,6-dimethylocta-5,7-dien-4-one

Test material form:

liquid

Details on test material:

-Name: Tagete Essential Oil
-Other identifiers: Tagete Minuta Essential Oil
-Batch no.: 2563305
-Appearance: mobile liquid with characteristic odour
-CAS No.: 91770-75-1 / 8016-84-0
-EINECS-No.: 294-862-7
-Purity: 100% wt (UVCB substance)
-Homogeneity: 100%
-Production date: 07 Sep. 2016
-Expiry date: 07. Mar. 2018
-Storage Room Temprature (20 ± 5°C); Keep away from light/humidity and under inert gas

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 2563305
- Appearance: Yellow to orange liquid
- Manufacturing date: 07 September 2016
- Date received: 27 January 2017
- Expiration date of the lot/batch: 07 March 2018
- Purity test date: 21 December 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, darkness

Test animals / tissue source

Species:

other: Reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs: The 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23770) were received on 08 March 2017.
The killed Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23706) were defrost on 03 March 2017, re-freeze on 03 March 2017 and defrost to be used on 08 March 2017.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions).

Duration of post- treatment incubation (in vitro):

After 30 minutes of exposure, extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 02 minutes post-exposure incubation at standard culture conditions.

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues (2 living RhCE tissue replicates and 2 killed DPBS pre-treated Reconstructed human Cornea-like Epithelia tissue replicates).

Details on study design:

- RhCE tissue construct used, including batch number: Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs: The 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23770) were received on 08 March 2017. The killed Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23706) were defrost on 03 March 2017, re-freeze on 03 March 2017 and defrost to be used on 08 March 2017.
The same day, the 12 tissues in their 24-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. 030617MGKA) and incubated during 19 hours and 55 minutes at standard culture conditions.

- Evaluation of direct interaction with MTT: The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL. A purple solution was observed after 3 hours of incubation between 36.4 °C and 37.8 °C, 5% CO2. Therefore, the test item was identified as a direct MTT reducer and two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

- Doses of test chemical and control substances used: 50 µL of test substance, negative or positive controls used.

- Pre-treatment: After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.

Treatment of tissues
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
- The test item was applied, as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates and 2 killed DPBS pre-treated Reconstructed human Cornea-like Epithelia tissue replicates during 30 minutes at standard culture conditions. In the same experimental conditions, the positive and negative controls were carried out.
- After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish). This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2 hours and 02 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Spectral analysis of the test item in isopropanol: The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol (same conditions as in the main test). A yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.024 which is less than 0.08 (value corresponding to approximately twice the OD (Optical Density) of the extracting solvent). Therefore the test item was considered not to interfere with the MTT assay and there was no need to add non-specific coloration controls to the study.

- Cell viability measurements: Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Optical Density measurements: The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours and 05 minutes at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 28 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD at 570 nm was measured in triplicate samples of formazan extracts. The measured OD are proportional to the number of living cells. The measurement of OD was performed using the 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.

- 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:
The test item is identified as not requiring classification and labelling according to UN GHS No Category: if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. In this case, no further testing in other test methods is required.
The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%.
When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test - 30 minutes

Value:

69.55

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MEAN PERCENT TISSUE VIABILITIES
- The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 69.55 %, versus 39.16% in the positive control (Methyl acetate).

ACCEPTANCE OF RESULTS:
- The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

Any other information on results incl. tables

Table 7.3.2/1: Assessment of the eye irritation potential individual and average values of OD after 30 minutes exposure

 

Items	Tissues	OD	Mean OD/disc#	Mean OD / product	Viability%	Mean viability%	Difference of viability%	Conclusion
Negative control	1	0.947	0.912	0.959	95.10	100.00	9.80	-
		0.902
		0.887
	2	1.162	1.006		104.90
		0.966
		0.891
Positive control	3	0.318	0.366	0.376	38.16	39.16	1.98	UN GHS category 2 or 1
		0.372
		0.408
	4	0.382	0.385		40.15
		0.394
		0.379
Test item	7	0.749	0.733	0.726	76.434	75.70	1.46	-
		0.728
		0.722
	8	0.710	0.719		74.974
		0.720
		0.727
Test item killed control	11	0.060	0.059	0.059	6.152	6.15	0.00
		0.059
		0.057
	12	0.060	0.059		6.152
		0.059
		0.058
Test item	-					69.55	-	No category

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

OD: optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

Executive summary:

An in vitro eye irritation test, reconstructed human cornea-like epithelium tissues (EpiOcular^TM tissue model) was performed according to the OECD Guideline 492 and in compliance with GLP to evaluate the eye hazard potential of the test item Tagete Essential Oil.

 

The test item was applied, as supplied, at the dose of 50 µL, to 2 living and 2 killed 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 and 02 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 69.55 %, versus 39.16% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

 

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.