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Description of key information

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:
yes
Remarks:
The Negative Control narrowly failed the required acceptance criterion in the first run of the assay. Therefore the test was repeated with a fresh batch of EPISKINTM tissues
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not applicable
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other: Human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Cell source:
other: A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum from an adult human.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface
Duration of treatment / exposure:
Tissues were treated with the test item for an exposure period of 15 minutes
Duration of post-treatment incubation (if applicable):
42-Hour post-exposure incubation period
Number of replicates:
Triplicate tissues were treated with the test item.
Details on study design:
Pre-Test Procedure
To identify this possible interference, the test item is checked for the ability to directly reduce MTT.

Pre-incubation (Day 0: Tissue Arrival)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main test

Application of Test Item and Rinsing (Day 1)

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Irritation / corrosion parameter:
% tissue viability
Value:
98.6
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: The test item was classified as non-irritant
Conclusions:
The test item was classified as non-irritant.EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

The skin irritation potential of the 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate was evaluated using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the post-exposure incubation period tissues were taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made for extraction of formazan crystals. At the end of the formazan extraction period the optical density was measured at 570 nm. The percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) was calculated. The relative mean viability of the test item treated tissues was 98.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The relative mean viability of negative control was 100% and for positive control was 12.8%.

The test item was classified as non-irritant.The test item is not requiring classification for skin irritancy under either UN GHS or EU CLP.

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 Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch: AH03201
Purity: 100%
Physical state/Appearance: clear colorless liquid
Expiry Date: 01 February 2019
Storage Conditions: approximately 4o C in the dark
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.
Duration of treatment / exposure:
Treatment of Corneas

The EMEM (Eagle’s Minimum Essential Medium) was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.
Number of animals or in vitro replicates:
Three
Details on study design:
Preparation of Corneas

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM (Eagle’s Minimum Essential Medium) was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology

Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Irritation parameter:
in vitro irritation score
Value:
0.7
Negative controls validity:
valid
Remarks:
Gave Irritancy Score of 0.4
Positive controls validity:
valid
Remarks:
Gave Irritancy Score of 42.1
Interpretation of results:
other: No category. Not requiring classification to UN GHS or EU CLP.
Remarks:
No category. Not requiring classification to UN GHS or EU CLP.
Conclusions:
No category. Not requiring classification to UN GHS or EU CLP.
Executive summary:

An in vitro BCOP test was conducted to identify test item that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The In Vitro irritancy scores reported as 0.7 for 2-ethylhexyl 7-oxabicyclo [4.1.0] heptane-3-carboxylate and 0.4 and 42.1 for negative and positive control. Based on irritancy score the test item is not classified as eye irritant. The test item is not requiring classification to UN GHS or EU CLP.

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

Respiratory irritation

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

Additional information

Eye irritation

Anin vitro eye irritation study was conducted according to EU Method B.47 and OECD Guideline No. 437. This study was selected as a key study because it is the most modern validated study available, which was performed under GLP conditions. The undiluted 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate was applied to corneas for 10 minutes to followed by an incubation period of 120 minutes.The corneas treated with the test item were clear post treatment and post incubation. Based on In Vitro irritancy score the test item is not classified as eye irritant. The substance does not require classification for UN GHS or EU CLP.

Justification for classification or non-classification

For eye and skin irritation, the in vitro testing resulted in no potential irriation, and as such, no classification.