Registration Dossier

Administrative data

Description of key information

Skin irritation (in vitro, EPISKIN): irritant

Eye irritation (in vitro, BCOP): non-corrosive/non-severe eye irritant

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:
03 April 2013 to 09 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and in compiance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Remarks:
Certificate of Compliance from UK GLP Monitoring Authority included in report.
Test system:
human skin model
Remarks:
EPISKIN TM human epidermis skin constructs.
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
On receipt, the EPISKINTM kit contents were checked and the inserts with tissues on agar were stored at room temperature until use. Procedures requiring viable tissues, dosing,
42 hour incubation and incubation with MTT were conducted within the expiry date indicated by the supplier (expiry date: 08 April 2013). The maintenance medium was pre-warmed to
37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for
a minimum of 24 hours at 37° +/- 2°C in a humidified atmosphere of 5% CO2 in air.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37° +/- 2°C


After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for minutes with the test substance, negative or positive control at room temperature.
A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. The test substance, MTF, was spread over the
surface of the tissue using a curved spatula. On application of 10 microL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat
spatula after 7 minutes application time.
After 15 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Tissues treated with test
substance, MTF, were gently swabbed to remove remaining test material. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a
well containing 2 mL maintenance medium and incubated for 42 hour at 37°C in a humidified atmosphere of 5% CO2 in air.

After 42 hours , each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours at 37°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours, all the inserts were blotted on absorbent paper. The tissue was removed from the insert using a biopsy punch and the epidermis was separated from the
collagen matrix using forceps. The epidermis and the collagen matrix were placed together in a micro tube.
When all the tissues had been punched, the tissues (epidermis and collagen matrix) were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration) to solubilize and
extract the formazan product. The formazan product was extracted from the tissues by storing at 2-8 ºC, protected from light, for 48 - 70 hours.
After formazan extraction, duplicate 200 microlitre aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by
vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Duration of treatment / exposure:
Triplicate tissues were dosed for 15 +/- 0.5 minutes with the test substance, negative or positive control at room temperature.
Number of replicates:
3 replicates.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of three tissues
Value:
27.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Negative control:
The mean absorbance of the triplicate negative control values was 0.778 which was between the minimum and maximum values of 0.6 and 1.5. The standard
deviation (SD) of the % viability was 5.2 which was below the maximum value of 18.

Positive control:
The percentage mean viability of the positive control was 22.8 ± 1.4 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

EPISKIN Results:
?Sample Tissue viability as percentage of mean OD negative control
Replicate Tissues Mean ± SD Prediction MTT endpoint
a b c
Negative Control 94.0 102.8 103.1 100.0 ± 5.2 Not applicable
Positive Control 21.3 23.0 24.2 22.8 ± 1.4 Irritant
MTF 25.1 24.8 33.7 27.9 ± 5.1 Irritant
Interpretation of results:
Category 2 (irritant)
Conclusions:
It was concluded that the test substance, MTF, with a mean tissue viability of 27.9 ± 5.1%, was predicted as irritant to the skin.
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:
21 March 2013 - 19 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to the OECD test Guideline and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eyes
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
10 mins (+/- 30 seconds) at 32°C 1°C
Observation period (in vivo):
two hour (+/- 10 minutes) incubation period
Number of animals or in vitro replicates:
n/a
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
ca. -1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
ca. -1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
other: non-corrosive/non-severe eye irritant
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test substance, MTF, elicited an In Vitro Irritancy Score of -1.0 ± 0.1 and was predicted to be a non-corrosive/non-severe eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

An in vitro skin irritation study has been performed (Huntingdon Life Sciences, 2013, VHJ0080) to assess the skin irritation potential, of the test substance, MTF.The test was conducted in accordance with the Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009), supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the OECD 439 Guideline For The Testing of Chemicals: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (OECD 439) and in compliance with GLP.

The test substance, MTF, elicited a mean tissue viability of 27.9 ± 5.1% and was predicted as irritant to the skin.

An in vitro eye irritation study has been performed (Huntingdon Life Sciences, 2014, VHJ0079) to assess the ocular irritancy potential of the test substance, MTF. The test was conducted in accordance with the following testing guideline:

OECD 437. OECD Guideline for the Testing of Chemicals: Bovine Corneal Opacity and Permeability (BCOP) Test method for Identifying Corrosives and Severe Irritants, 7 September 2009 and in compliance with GLP.

The test substance, MTF, elicited an In Vitro Irritancy Score of -1.0 ± 0.1 and was predicted to be a non-corrosive/non-severe eye irritant.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

On the basis of an in-vitro skin irritation test, MTF was predicted to be a skin irritant (Skin Irritation Category 2 according to the CLP Regulation). The resulting Hazard Phrase "H315: Causes skin irritation" will therefore be applied. The corresponding Risk phrase under the Dangerous Substances Directive is R38.

On the basis of an in-vitro eye irritation test, MTF was concluded not to be irritant to the eyes; it is therefore concluded that no classification is required.