Savory, Satureja hortensis, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Justification for type of information:

An in vitro skin corrosion study of Summer Savory oil: Reconstructed human epidermis (RhE) test method has been performed. According to the test results the substance Summer Savory oil fulfils the criteria for classification as Skin Corrosion Sub Category 1B/1C.

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:

26 April 2019 – 23 June 2019

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:

OECD Guideline 431 Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

EssentÍal oil of Summer Savory/batch No. SSVI.ES.sample
Date of manufacture: 08.01.2019
Expration date of the batch: 08.01.2021
UVCB substance;
Purity test date: 18.01.2019

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Three-dimensional RhE models are obtained commercially (epiCS® supplied by CellSystems®). Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

Source strain:

other: human

Justification for test system used:

According to Annex VII of REACH Regulation, if new test data is required those must be derived from in vitro methods only.
Human skin models (epiCS®, SkinEthic™) (OECD 431) are accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

other:  Four killed control tissue models (two by exposure time) were added to the study, which underwent the entire testing procedure to generate a non-specific MTT reduction control.

Amount/concentration applied:

Preliminary test
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). The preparation turned into two phases, one being yellow and one being purple, after 1 hour of incubation between 37.1°C and 37.4°C, 5% CO2.
As two phases appeared with a coloured part, direct interaction with MTT could be masked.
That is why four killed control tissue models (two by exposition time) were added to the study, which underwent the entire testing procedure to generate a non-specific MTT reduction control.

Main test
50 µL of the test item as supplied on 0.6 cm² human skin model
50 µL for positive control 8N KOH– Fisher Scientific
50 µL for negative control of distilled water– ADL Prochilab.

Duration of treatment / exposure:

3 minutes and 1 hour for test material
3 minutes and 1 hour for negative and positive control

Duration of post-treatment incubation (if applicable):

During 3 minutes at room temperature
During 1 hour at 370 C ± 10 C, 5%CO2

Number of replicates:

Two tissue replicates.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes and 1 hour after the test item application

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosion

Other effects / acceptance of results:

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 431 for in vitro skin corrosion: Reconstructed human epidermis (RhE) test method.

Acceptability criteria:
The difference of viability between the 2 tissues treated with the negative control during 3 minutes was higher than 30% (40.9%).
Considering the results obtained (both epidermises have a viability much higher than 50% and are clearly classified as “Non-corrosive”), this deviation is considered as without impact on the conclusion of the study.

ASSESSMENT OF THE SKIN CORROSION

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

TEST ITEM: Summer Savory oil

 

Application: 50 μL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

 

Table 1

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability, %	Mean Viability, %	Viability difference between replicates, %
Negative control	SPL 1       SPL 2	0.716 0.704 0.719   0.456 0.461 0.494	0.713       0.471	0.592	120.44       79.56	100.00	40.9
Positive control	SPL 3       SPL 4	0.100 0.116 0.131   0.106 0.110 0.115	0.116       0.111	0.114	19.59       18.75	19.17	0.8
Test item PH-19/0208	SPL7       SPL8	0.240 0.241 0.245   0.190 0.218 0.220	0.242       0.210	0.0226	40.88       35.47	38.18	5.4
Test item PH-19/0208 NSMTT	SPL7       SPL8	0.084 0.089 0.094   0.088 0.091 0.087	0.089       0.089	0.089	15.03       15.03	15.03	0.0
Test item PH-19/0208 corrected						23.14

Note

#: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

 

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model.

- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

 

ASSESSMENT OF THE SKIN CORROSION

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

TEST ITEM: Summer Savory oil

 

Application: 50 µL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

Table 2

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability, %	Mean Viability, %	Viability difference between replicates, %
Negative control	SPL 11       SPL 12	0.667 0.832 0.807   0.653 0.664 0.730	0.769       0.683	0.726	105.92       94.08	100.00	11.8
Positive control	SPL 13       SPL 14	0.002 0.000 0.001   0.005 0.005 0.005	0.001       0.005	0.003	0.14       0.69	0.41	0.6
Test item PH-19/0208	SPL 17       SPL 18	0.194 0.204 0.207   0.187 0.191 0.195	0.202       0.191	0.197	27.82       26.31	27.07	1.5
Test item PH-189/0208 NSMTT	SPL 19       SPL 20	0.063 0.059 0.062   0.061 0.056 0.062	0.062       0.060	0.061	8.54       8.26	8.40	0.3
Test item PH-19/0208 corrected						18.66

Note

#: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model.

- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

ASSESSMENT OF THE SKIN CORROSION

CONCLUSION AFTER 3 MINUTES AND AFTER 1 HOUR EXPOSURE

 

TEST ITEM: Summer Savory oil

 

Application: 50 μL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

 

Table 3

	Mean viability (%)
	3 min exposure	1 hour exposure	Conclusion
Positive control	19.17	0.41	Corrosive sub-category 1B/1C
Test item PH-19/0208	23.14	18.66	Corrosive sub-category 1B/1C

Note

 

The item is to be classified as non-corrosive:

-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 %;

 

The item is to be classified as corrosive:

- If the viability after 3 minutes’ exposure is strictly 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 strictly less than 15 %

 

Summary of Negative Control data after a treatment of 3 minutes

Date	Negative control item	Replicate num1ber	OD	Mean OD	Viability (%)	Viability difference between replicates (%)
10/01/2018	Distilled water	1   2	0.578   0.562	0.570	101.4   98.6	2.8
24/01/2018	Distilled water	1   2	0.710   0.750	0.730	97.26   102.74	5.5
21/02/2018	Distilled water	1   2	0.686   0.695	0.691	99.35   100.65	1.3
21/03/2018	Distilled water	1   2	0.667   0.805	0.736	90.63   109.38	18.8
05/04/2018	Distilled water	1   2	0.655   0.637	0.646	101.39   98.61	2.8
11/04/2018	Distilled water	1   2	0.787   0.643	0.715	110.07   89.93	20.1
23/05/2018	Distilled water	1   2	0.821   0.669	0.745	110.2   89.80	20.4
04/07/2018	Distilled water	1   2	0.649   0.641	0.645	100.62   99.38	1.2
08/08/2018	Distilled water	1   2	0.476   0.464	0.470	101.28   98.72	2.6
05/09/2018	Distilled water	1   2	0.570   0.727	0.649	87.90   112.10	24.2
10/10/2018	Distilled water	1   2	0.629   0.616	0.623	101.04   98.96	2.1
17/10/2018	Distilled water	1   2	0.612   0.635	0.624	98.16   101.84	3.70
05/12/2018	Distilled water	1   2	0.722   0.636	0.679	106.33   93.67	12.70

OD: optical density

Negative control item = Dulbecco's phosphate-buffered saline (DPBS) or distilled water.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Negative Control data after a treatment of 1 hour

Date	Negative control item	Replicate number	OD	Mean OD	Viability (%)	Viability difference between replicates (%)
10/01/2018	Distilled water	1   2	0.772   0.604	0.688	112.21   87.79	24.4
24/01/2018	Distilled water	1   2	0.793   0.652	0.723	109.76   90.24	19.5
05/12/2018	Distilled water	7   8	0.655   0.668	0.662	99.02   100.98	2.0
21/03/2018	Distilled water	7   8	0.583   0.563	0.573	101.75   98.25	3.5
05/04/2018	Distilled water	9   10	0.640   0.656	0.648	98.77   101.23	2.5
11/04/2018	Distilled water	9   10	0.662   0.765	0.714	92.78   107.22	14.4
23/05/2018	Distilled water	9   10	0.713   0.830	0.772	92.42   107.58	15.2
04/07/2018	Distilled water	11   12	0.679   0.682	0.681	99.78   100.22	0.4
08/08/2018	Distilled water	15   16	0.455   0.457	0.456	99.78   100.22	0.4
05/09/2018	Distilled water	13   14	0.694   0.676	0.685	101.31   98.69	2.6
10/10/2018	Distilled water	7   8	0.683   0.634	0.659	103.72   96.28	7.4
17/10/2018	Distilled water	9   10	0.719   0.764	0.742	96.97   103.03	6.1

OD: optical density

Negative control item = Dulbecco's phosphate-buffered saline (DPBS) or distilled water.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Positive Control data after a treatment of 3 minutes

Date	Positive control item	Replicate number	OD	Mean OD	Viability (%)	Viability difference between replicates (%)
10/01/2018	KOH 8N	3   4	0.046   0.185	0.116	8.07   32.46	24.4
24/01/2018	KOH 8N	3   4	0.562   0.420	0.491	76.99   57.53	19.5
21/02/2018	KOH 8N	3   4	0.025   0.059	0.042	3.62   8.54	4.9
21/03/2018	KOH 8N	3   4	0.048   0.033	0.041	6.52   4.48	2.0
05/04/2018	KOH 8N	3   4	0.030   0.036	0.033	4.64   5.57	0.9
11/04/2018	KOH 8N	3   4	0.034   0.041	0.038	4.76   5.73	1.0
23/05/2018	KOH 8N	3   4	0.144   0.244	0.194	19.33   32.75	13.4
04/07/2018	KOH 8N	3   4	0.044   0.041	0.043	6.82   6.36	0.5
08/08/2018	KOH 8N	3   4	0.035   0.042	0.039	7.45   8.94	1.5
05/09/2018	KOH 8N	3   4	0.001   0.003	0.002	0.15   0.46	0.3
10/10/2018	KOH 8N	3   4	0.260   0.154	0.207	41.77   24.74	17.0
17/10/2018	KOH 8N	3   4	0.055   0.054	0.055	8.82   8.66	0.2
05/12/2018	KOH 8N	3   4	0.050   0.226	0.138	7.36   33.28	25.9

OD: optical density

Positive control item = 8N potassium hydroxide.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Positive Control data after a treatment of 1 hour

Date	Positive control item	Replicate number	OD	Mean OD	Viability (%)	Viability difference between replicates (%)
10/01/2018	KOH 8N	3   4	0.001   -0.001	0.0001	0.15   -0.15	0.3
24/01/2018	KOH 8N	3   4	0.009   0.008	0.009	1.25   1.11	0.1
21/02/2018	KOH 8N	17   18	0.004   0.002	0.003	0.52   0.26	0.3
21/03/2018	KOH 8N	9   10	0.001   0.002	0.002	0.17   0.35	0.2
05/04/2018	KOH 8N	11   12	0.003   0.004	0.004	0.46   0.62	0.2
11/04/2018	KOH 8N	11   12	0.002   0.004	0.003	0.28   0.56	0.3
23/05/2018	KOH 8N	11   12	0.003   0.003	0.003	0.39   0.39	0.0
04/07/2018	KOH 8N	13   14	0.009   0.015	0.012	1.32   2.20	0.9
08/08/2018	KOH 8N	17   18	0.003   0.003	0.003	0.66   0.66	0.0
05/09/2018	KOH 8N	15   16	0.007   0.005	0.006	1.02   0.73	0.3
10/10/2018	KOH 8N	9   10	0.000   0.000	0.000	0.00   0.00	0.0
17/10/2018	KOH 8N	11   12	0.002   0.001	0.002	0.27   0.13	0.10
05/12/2018	KOH 8N	9   10	0.001   0.002	0.002	0.15   0.3	0.20

OD: optical density

Positive control item = 8N potassium hydroxide.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

 

Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

 

The aim of the study was to evaluate the possible corrosive effects of the test item Summer Savory oil after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Summer Savory oil was applied as supplied, at the dose of 50 µL, to two tissue Human skin model surfaces (epiCS®, CellSystems®) 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 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

The experimental protocol was established in accordance with the OECD. Test Guideline No. 431 dated 29 July 2016 and the EU method Method B.40.bis. In vitro skin corrosion: Human skin model test of the Council Regulation No. 440/2008 dated 30 May 2008.

Three minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 23.14% and 18.66%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.

The corresponding hazard statement H314: Causes severe skin burns and eye damage” has to be assigned.

Interpretation of results:

other: Corrosive sub-category 1B/1C

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.
The corresponding hazard statement is “H314: Causes severe skin burns and eye damage”.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item Summer Savory oil after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Summer Savory oil was applied as supplied, at the dose of 50 µL, to two tissue Human skin model surfaces (epiCS®, CellSystems®) 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 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

 

The experimental protocol was established in accordance with the OECD. Test Guideline No. 431 dated 29 July 2016 and the EU method Method B.40.bis. In vitro skin corrosion: Human skin model test of the Council Regulation No. 440/2008 dated 30 May 2008.

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 23.14% and 18.66%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.

 

The corresponding hazard statement H314: Causes severe skin burns and eye damage” has to be assigned.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Savory, Satureja hortensis, ext.

EC Number:

283-922-8

EC Name:

Savory, Satureja hortensis, ext.

Cas Number:

84775-98-4

IUPAC Name:

Essential oil of Savory, Satureja hortensis, obtained from whole, dried herb by steam distillation

Test material form:

liquid

Specific details on test material used for the study:

EssentÍal oil of Summer Savory/batch No. SSVI.ES.sample
Date of manufacture: 08.01.2019
Expration date of the batch: 08.01.2021
UVCB substance;
Purity test date: 18.01.2019

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The Summer savory oil is classified as Serious eye damage, Category 1.

Executive summary:

On the basis of the results of an in vitro test for skin corrosion: Reconstructed human epidermis(RhE)) test method (OECD guideline 431) and according to the classification criteria of Regulation (EC) 1272/2008, the Summer Savory oil is classified as Skin corrosion, sub-category 1B/1C.The study for Serious eye damage/eye irritation does not need to be conducted since the substance is classified as skin corrosion, which leads to classification as serious eye damage (Category 1).