Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-06 to 2018-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Absolute of Jasmine (Jasminum grandiflorum) obtained from Jasmine concrete by ethanol extraction
EC Number:
948-021-2
Molecular formula:
n.a.
IUPAC Name:
Absolute of Jasmine (Jasminum grandiflorum) obtained from Jasmine concrete by ethanol extraction
Test material form:
liquid
Details on test material:
- Batch: AH17.001
- Purity: 100%
- Physical state: liquid
- Colour: pale yellow-orange to orange reddish-brown
- Storage Conditions: ≤ 15 °C
- Expiry date: 30 June 2019
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 30 µL of the test item were dispensed directly atop the EpiDerm tissue

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model (MatTek)
- Tissue batch number(s): 28623

EpiDerm Kit:
The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 28623)
- 2x 24-well plates
- 8x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 060718MSB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 051518ISA)
- 1x 1 vial 5% SDS Solution (TC-SDS-5%)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h +/- 5 min
- Spectrophotometer: Yes
- Wavelength: 570 nm
- Filter bandwidth: +/- 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (Gibco, Cat. No.14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% sodium dodecyl sulfate (MatTek, CAS No.: 151-21-3, Lot No.: 040418SVA)
Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 h post-incubation
Number of replicates:
3 tissues per dose group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
40.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD570 nm ≥ 0.8 and ≤ 2.8 (1.806)
- Acceptance criteria met for positive control: Yes, mean relative tissue viability of the three positive control tissues was ≤ 20% (3.7%)
- Acceptance criteria met for variability between replicate measurements: Yes, standard deviation (SD) of viability of replicate tissues of all dose groups was ≤ 18%. (0.2-6.0%).

For detailed results see Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Results of the Pre-Experiments:

The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT was determined to be 0%.

The mixture of 30 µL of the test item per 300 μL isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm.

Results of the main experiment:

Table 1: Result of the Test Item

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.830

1.783

1.804

0.103

0.103

0.108

0.678

0.879

0.703

1.827

1.772

1.822

0.113

0.110

0.119

0.687

0.877

0.717

OD570(Blank Corrected)

1.786

1.739

1.760

0.059

0.059

0.064

0.634

0.835

0.659

1.783

1.728

1.778

0.067

0.066

0.075

0.643

0.834

0.666

Mean OD570of the Duplicates (Blank Corrected)

1.785

1.734

1.769

0.063

0.062

0.070

0.638

0.834

0.66

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.762*

0.065

0.713

SD OD570

0.026

0.004

0.106

Relative Tissue Viability [%]

101.3

98.4

100.4

3.6

3.5

4.0

36.2

47.3

37.8

Mean Relative Tissue Viability [%]

100.0

3.7**

40.4

SD Tissue Viability [%]***

1.5

0.2

6.0

CV [% Viabilities]

1.5

6.4

14.9

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:
other: The test item is identified as requiring classification and labelling according to UN GHS (“Category 2” or “Category 1”).
Conclusions:
In conclusion, in this in vitro skin irritation study (OECD 439), Jasmine absolute is considered to be irritant to the skin (UN GHS category 1 or 2).
Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to Jasmine absolute (100% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (40.4%) after 60 min treatment and 42 h post-incubation. Based on this result, Jasmine absolute is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" .