Registration Dossier

Administrative data

Description of key information

The substance is not irritating to the skin in animals and humans. It is slightly irritating to the eye in animals, without classification being required. 
For skin and eye irritation the conclusion is “Not classified - based on specific, valid data on the substance”.
Considering these results in high-sensitivity assays, there is no reason to suspect any relevant respiratory irritation, especially as the granulometry of the substance (as the substance is in the form of pearls) leads to negligible exposure of terminal airways.
For this route, the conclusion is “Not classified - based on weight-of-evidence analysis”.
Value used for CSA:
Skin irritation/corrosion: not irritating
Eye irritation: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23/11/2010 until 25/01/2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The protocol used in this study is based on the OECD guideline for the testing of chemicals, draft proposal for a new guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method” (11th of December 2009, version 4). the study is aloso perfomed following GLP.
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstituted human epidermis
Type of coverage:
other: reconstituted epidermis incubated
Preparation of test site:
other: reconstituted epidermis incubated
Vehicle:
water
Controls:
not required
Amount / concentration applied:
10μL of H2O + 16 mg of test item (LCE10041) are dispensed on the surface of three tissue replicates
Duration of treatment / exposure:
The treated tissues are then incubated for 42 minutes (± 1 minute) at room temperature. At the end
of the incubation period, the treated tissues are rinsed with 25 ml of PBS buffer, dried with cotton
swabs, transferred in a 6-well plate pre-filled with 2 ml SkinEthic growth culture medium and
incubated for 42h at 37°C, 5% CO2 for recovering. Negative (PBS buffer) and positive (SDS 5% aq.)
controls are applied in parallel using the same procedure.
Observation period:
42h
Number of animals:
not applicable.
For the application of the test item, the SkinEthic RHE reconstituted epidermis (3 replicates by test
item and test references) are transferred in a 24-well plate containing 300 μl of pre-warmed
SkinEthic maintenance culture medium. A single 24-well plate is used per test item to avoid crosscontamination
between tissues.
Details on study design:
In order to assess if the test item interferes with MTT, a preliminary contact experiment is performed
using the SkinEthic Irritation Test-42bis conditions. A 24-well plate is filled with 300 μl of MTT (1
mg/ml). An aliquot (16 mg +/- 0.5 mg) of the test item (LCE10041) is then added, mixed with MTT
and incubated at 37°C, 5% CO2 for 3 hours (± 5 minutes).
If the MTT solution colour becomes blue or purple after this 3h incubation period, it means that the
product interacts with MTT. Consequently, the part of O.D. due to the non-specific reduction of MTT
needs to be evaluated using SkinEthic killed epidermis.


At the end of the 42h post-incubation period, the excess medium on the unit bottom of the tissues is
removed with absorbent paper and the treated tissues are transferred in 24-well plates containing
300 μl of MTT solution at 1 mg/ml and incubated for 3h (± 5 minutes) at 37 °C, 5% CO2.
After the 3h MTT incubation period, the excess MTT on the unit bottom of the tissues is removed
with absorbent paper and the treated tissues are transferred in new 24-well plates containing 800 μl
of isopropanol. 700 μl of isopropanol are then added on the top of each tissue to completely
immerse the tissues in isopropanol. Afterwards, the treated tissues are incubated at room
temperature for 2h (± 5 minutes) with gentle agitation.
Irritation parameter:
other: change of colour
Basis:
other: O.D parameter
Remarks on result:
other: no induce any change
Irritant / corrosive response data:
The test item (LCE10041) does not induce a change in colour of the MTT solution to blue or purple
appearance. As a consequence, it does not interfere with MTT.--> no irritating
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
According to the irritation prediction model, the test item must be considered as non-irritant.
Executive summary:

Negative control (NC) acceptance: VALID. The mean OD value of the 3 tissues exposed to NC is ≥ 1.2 at 570 nm and the standard deviation value is ≤18%.

Positive control (PC) acceptance: VALID.The mean viability, expressed as % of the NC, is < 40 % and the standard deviation value is ≤ 18%.

Batch acceptance: VALID. Both negative and positive controls data fulfil the NC and PC acceptance criteria requirements.

Test substance data acceptance: VALID. The standard deviation of the three tissues treated with the test item (LCE10041) is ≤ 18%.

The test item (LCE10041) does not induce a change in colour of the MTT solution to blue or purple appearance. As a consequence, it does not interfere with MTT.

MTT VIABILITY TESTING

A MTT assay was performed to measure the viability of the reconstituted epidermis after a 42 min exposure to the test item (LCE10041) and a 42h recovery period.

The cell viability is expressed as the percentage of the mean viability of tissues exposed to the negative control (PBS buffer), which is arbitrarily set to 100 %. As the mean viability of the tissues treated with the test item (LCE10041) is above 50%, the test item is qualified as non-irritant.

IL-1a QUANTIFICATION

After application of the test item and references and 42 hours recovery, culture supernatants were collected and the IL-1α release was measured by ELISA.

As expected, the negative reference of the test (PBS buffer) doesn’t induce any IL-1a release, while the positive reference (SDS 5%) induces a high release in the tissue culture medium. The mean release by tissues treated with the test item (LCE10041) is 10pg/ml (6.7 pg/ml).

According to the irritation prediction model, the test item must be considered as non-irritant.

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:
from 2010-06-08 to 2010-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been conduced according to an accepted OECD guideline and in compliance with the GLP.
Qualifier:
according to
Guideline:
other: OECD protocol n°437
GLP compliance:
yes (incl. certificate)
Remarks:
2009-12-09
Vehicle:
physiological saline
Remarks:
NaCl to 0.9%
Amount / concentration applied:
Preparation: test item diluted to 20% (W/W) in NaCl to 0.9%
Quantity applied : 750 uL per cornea
Duration of treatment / exposure:
Contact timepoint : 4 hours ± 10 minutes
Number of animals or in vitro replicates:
not applicable
Irritation parameter:
other: OPT2 - OPT0
Basis:
mean
Time point:
other: 4 hours
Score:
ca. 2.7
Max. score:
2.7
Irritation parameter:
other: OD
Basis:
mean
Time point:
other: 4 hours
Score:
ca. 0.14
Max. score:
0.161
Irritant / corrosive response data:
Score (4 hours): 4.8 ± 0.4
Classification: Test item not classified corrosive or severe irritant
Interpretation of results:
other: not corrosive or severe irritant
Remarks:
Criteria used for interpretation of results: other: OECD guideline 437
Conclusions:
From the results obtained under the experimental conditions adopted, the test item designated as "LCE10042 (batch n° T92235 of 27/05/2009)", applied diluted to 20 % (W/W) in NaCl to 0.9%, is "not classified corrosive or severely irritant" for the isolated bovine cornea, after 4 hours of contact.
Executive summary:

The aim of this study was to assess the corrosive or severe irritant potential of LCE10042 using the ocular primary irritation BCOP.

This study has been conduced according to an accepted OECD guideline (n°437) and in compliance with the GLP.

The bovine corneal opacity and permeability method (BCOP) is based on the Muir method (1984, 1985). This technique is adapted from the one described by Gautheron and coll., 1992 to determine the ocular irritant potential of diverse substances.

Thanks to this method, two parameters such as corneal opacity and permeability can be measured.

The test item was diluted to 20% (W/W) in NaCl to 0.9%.

Results:

IVIS = (OPT2-OPT0) + (15 x O.D.)

Classification

IVIS ≥ 55.1àcorrosive or severe irritant test item 

IVIS < 55.1àtest item not classified corrosive or severe irritant

score= 4.8 ± 0.4

Conclusion

From the results obtained under the experimental conditions adopted, the test item designated as "LCE10042(batch n° T92235 of 27/05/2009)", applied diluted to 20 % (W/W) in NaCl to 0.9%, is "not classifiedcorrosive or severely irritant" for the isolated bovine cornea, after 4 hours of contact.

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