Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: 9-Octadecenoic acid (9Z)-, propyl ester
Product Description: Propyl oleate
CAS No.: 111-59-1
Physical state: colourless to yellow liquid at 20 °C
Batch No.: 37584
Re-certification date of batch: 08 March 2018
Purity: > 95 % (mono constituent substance, water content 0.33 % (w/w))
Free Fatty Acid, % Oleic: 0,1
Color, Gardner: 3
Iodine Value, cg I2/g 75
Cloud Point, degrees C 0
Saponification Number, mg KOH/g 177
Moisture, % 0,07
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Specific details on test material used for the study:
Name: 9-Octadecenoic acid (9Z)-, propyl ester
Product Description: Propyl oleate
CAS No.: 111-59-1
Physical state: pale yellow to yellow liquid at 20 °C
Batch No.: 37584
Re-certification date of batch: 08 March 2018
Purity: > 99 % (fatty acid propyl esters, max water content 0.3 % (w/w))
Free Fatty Acid, % Oleic: 0.1
Color, Gardner: 3
Iodine Value, cg I2/g 75
Cloud Point, degrees C 0
Saponification Number, mg KOH/g 177
Moisture, % 0,07
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Amount/concentration applied:
The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item was dispensed directly atop the EpiDerm tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette.
Duration of treatment / exposure:
In the present study the test item was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Duration of post-treatment incubation (if applicable):
In the present study the test item was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Number of replicates:
3 replicate tissues per group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean relative tissue viability [%]
Value:
113.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
Dulbecco’s phosphate buffered saline (DPBS)
Positive controls valid:
yes
Remarks:
5% sodium dodecyl sulfate in H2O
Remarks on result:
no indication of irritation

Any other information on results incl. tables

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 equalled 0%. The mixture of 30 µL of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

Results

Table 1: Result of the Test Item

 Name  NC        PC        Test item      
 Tissue  1  2  3  1  2  3  1  2  3
 absolute OD570

1.923

1.901

 2.198

2.146

 2.195

2.236

 0.100

0.099

 0.125

0.128

 0.132

0.131

 2.391

2.323

 2.466

2.424

 2.343

2.293

 OD570 (blank-corrected)

 1.882

1.860

 2.156

2.195

 2.154

2.195

 0.059

0.058

 0.084

0.087

 0.091

0.090

 2.350

2.281

 2.425

2.383

 2.302

2.252

mean OD570 of the duplicates (blank-corrected)

 1.871  2.131  2.174  0.059  0.085  0.090  2.316  2.404  2.277

total mean OD570 of 3 replicate tissues (blank-corrected)

 2.059* 0.078 2.332
relative tissue viability [%]  90.9  103.5  105.6  2.8  4.1  4.4  112.5  116.8  110.6
SD OD570 0.164 0.017 0.065
 mean relative tissue viability [%] 100.0 3.8**  113.3

SD tissue viability [%]***

8.0  0.8  3.2
CV [% viabilities]  8.0  21.8    2.8

* Blank-corrected mean OD570 nm of 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%

Discussion

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm comprising a reconstructed epidermis with a functional stratum corneum. In the present study the test item was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. 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 test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (113.3%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was >= 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was <= 20% (3.8%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.8% - 8.0%).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the OECD 439 study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

In the in vitro skin irritation study (OECD 439) under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.