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Administrative data

Description of key information

Based on test results listed above, substance is non-irritating to skin and eyes.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP, OECD TG 439 and other similar study guidelines without deviations.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Principles of method if other than guideline:
Protocol deviations: The 12 wells plates with tissues were preincubated for 2 hours instead of 24 hours.
Evaluation: Shipment of the tissue kit took longer than expected. After consultation with the
supplier the advice was to transfer the Episkin tissues to maintenance medium for at least
2 hours instead of 24 hours. Since all acceptability criteria were met this has no adverse effect
on the study integrity.
GLP compliance:
yes
Species:
other: in vitro: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009,
Details on test animals and environmental conditions:
Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009, See APPENDIX 4).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Source
SkinEthic Laboratories, Lyon, France.
Type of coverage:
open
Preparation of test site:
other: in vitro
Controls:
yes
Amount / concentration applied:
The solid test substance (12.7 to 14.2 mg) was applied directly on top of the skin tissue. TRANTOS was spread to match the size of the tissue.
Duration of treatment / exposure:
After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C
Observation period:
not applicable
Number of animals:
3 tissues per test substance together with negative and positive controls
Details on study design:
Tissues
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.


MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Irritation / corrosion parameter:
other:
Value:
103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Remarks: The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% TRANTOS is considered to be non-irritant.. (migrated information)
Irritant / corrosive response data:
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% TRANTOS is considered to be non-irritant.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
TRANTOS is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with TRANTOS using a human skin model. This report describes the ability of TRANTOS to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of TRANTOS was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch CQE299K1DL2 of TRANTOS was a white solid with a purity of 85-95%. Skin tissue was moistened with 10 μl of Milli-Q water and 12.7 to 14.2 mg of TRANTOS was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with TRANTOS compared to the negative control tissues was 103%. Since the mean relative tissue viability for TRANTOS was above 50% after 15 minutes treatment TRANTOS is considered to be non-irritant. The positive control had a mean cell viability of 19% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 13%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that TRANTOS is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:
2013-02-20 to 2013-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP, OECD 437, EU Method B. 47 and other similar study guidelines without deviations.
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
Qualifier:
equivalent or similar to
Guideline:
other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the NICEATM, The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
Deviations:
no
GLP compliance:
yes
Species:
other: in vitro
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. TRANTOS was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (314 to 374 mg). Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
9 eyes total were used for the study; 3 negative control tests, 3 positive control tests, and 3 tested with Trantos.
Details on study design:
SCORING SYSTEM: In vitro irritancy score:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints. A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

TOOL USED TO ASSESS SCORE: fluorescein
Irritant / corrosive response data:
The individual in vitro irritancy scores for the negative controls ranged from -0.2 to 0.1.

The individual positive control in vitro irritancy scores ranged from 100 to 132. All three corneas treated with the positive control were turbid after the 240 minutes of treatment and on one cornea a spot was observed.

The corneas treated with TRANTOS showed opacity values ranging from -2 to 0 and permeability values ranging from 0.021 to 0.034. The corneas were clear after the 240 minutes of treatment with TRANTOS. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.7 to 0.3 after 240 minutes of treatment with TRANTOS.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 115 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
TRANTOS did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.6 after 240 minutes of treatment.
Finally, it is concluded that this test is valid and that TRANTOS is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to GLP, OECD TG 439 and other similar study guidelines without deviations.

Justification for selection of eye irritation endpoint:
The study was conducted according to GLP, OECD 437, EU Method B. 47 and other similar study guidelines without deviations.

Justification for classification or non-classification

Substance is non-irritating to skin and eyes. Based on these results: - according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, TRANTOS does not have to be classified and has no obligatory labelling requirement for irritation or corrosion.