Registration Dossier

Administrative data

Description of key information

Skin irritation: 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”.

Eye irritation: The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated to 0.93. Therefore the test item was classified into UN GHS No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

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

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%).

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”.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye 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
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
yes
Remarks:
SD of positive control slightly increased in comparison to historical mean.
Qualifier:
equivalent or similar to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
SD of positive control slightly increased in comparison to historical mean.
GLP compliance:
yes (incl. certificate)
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
Details on test animals or tissues and environmental conditions:
Preparation of the Corneas

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +- 1 °C.

Test Groups:

- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive controls treated with ethanol 100%
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL of the test substance or the control substance was introduced into the anterior chamber. As the viscosity of the test item was relatively high, it was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment. After 10 minutes incubation at 32 +- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
Duration of treatment / exposure:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test substance or the control substance was introduced into the anterior chamber. As the viscosity of the test item was relatively high, it was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment. After 10 minutes incubation at 32 +- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber expect of cornea no. 8 was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 +- 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
Duration of post- treatment incubation (in vitro):
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.
Number of animals or in vitro replicates:
Test Groups:

- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive controls treated with ethanol 100%
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
physiological saline 0.9% NaCl; IVIS: 1.18
Positive controls validity:
valid
Remarks:
ethanol 100%; IVIS: 74.52
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Validity

The in vitro irritation score obtained with the positive control is slightly increased (deviation of 5.47 SD) in comparison to the two standard deviations of the current historical mean. The deviation of the positive control did not influence the quality or integrity of the present study and therefore the assay is considerd to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Interpretation of results:
GHS criteria not met
Conclusions:
The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated to 0.93. Therefore the test item was classified into UN GHS No Category.
Executive summary:

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated to 0.93. Therefore the test item was classified into UN GHS No Category.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification