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Description of key information

Skin Irritation

Under the conditions of the study, the relative mean viability of tissues treated with the test material was 30.0%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as skin irritant. 

However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Eye Irritation

Under the conditions of the study, the relative mean tissue viability of tissues treated with the test material was 30.0%, less than 50%.

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:
07 February 2018 to ****
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
GLP compliance:
yes
Specific details on test material used for the study:
Purity: >94% (nominal); This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB).
Sponsor Description: Brown paste to very viscous liquid
Envigo Description: Amber colored viscous liquid
Storage Conditions: Room temperature (15-25°C) in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
The EPISKINTM Kit consisted of one EPISKIN™ plate containing 12 reconstructed human epidermis tissues (0.38 cm2), maintenance medium (used for tissue pre-conditioning and testing purposes) and assay medium (used for MTT assay).

The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint therefore, as a precaution, additional tissues were incorporated into the testing to correct for this.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (26.3 µL/cm2)
Duration of treatment / exposure:
Triplicate 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:
Three.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 hours post exposure
Value:
ca. 30
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the study, the relative mean tissue viability of tissues treated with the test material was 30.0%, less than 50%.
Executive summary:

Under the conditions of the study, the relative mean viability of tissues treated with the test material was 30.0%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as skin irritant. 

However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
22 February 2018 to 19 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
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:
no
GLP compliance:
yes
Specific details on test material used for the study:
- CAS RN 1412893-77-6
- Purity: >94% (UVCB)
- Description: Brown paste to very viscous liquid
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 prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test material or control items were applied to the appropriate corneas
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
Preparation of Corneas
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.

Treatment of Corneas
The EMEM 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 3 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.

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.
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control (Neat Ethanol) In Vitro Irritancy Score was within the historical range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied.

The negative control (0.9% (w/v) Sodium Chloride) gave opacity of ≤2.3 and permeability ≤0.041. The negative control acceptance criteria were therefore satisfied.

Test Item
The in vitro irritancy score of the test material was 31, based on this result, no prediction of eye irritation can be made for the test material.

The In Vitro irritancy scores are summarized as follows:

Treatment

In VitroIrritancy Score

Test Item*

31.0

0.9% (w/v) Sodium Chloride (Negative Control)

0.1

Neat Ethanol (Positive Control)

61.4

Note: * Phosphoric acid, methyl ester, compds. with branched dodecylbenzenamine (CAS RN 1412893-77-6)

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item according to UN GHS or EU CLP.
Executive summary:

In a study performed to the standardized guidelines OECD 437 and EU Test Method B.47., under GLP conditions, the undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) 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) of 31. Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item according to UN GHS or EU CLP.

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