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Description of key information

in vitro EPISKIN(TM) 4h Skin Corrosion Test (OECD431):
Conclusion: WS400104 is not corrosive to the skin.
in vitro EPISKIN(TM) 15 min Skin Irritation Test (OECD439):
Conclusion: WS400104 is irritating to the skin.
in vitro Eye Irritation, Isolated Chicken Eyes (OECD 438):
Conclusion: WS400104 is not an ocular corrosive or severe eye irritant.
in vivo Eye Irritation, Rabbit with Neat WS400104 (OECD 405):
Conclusion: No requirement of classification regarding eye irritation.

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: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Reconstructed Human Epidermis Test Method of 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test 15min - 42 hours, Standard Operating Procedure: February 2009 Version 1.8.
Deviations:
yes
Remarks:
Dose volume per tissue sample was at least 50 µL (132 µL/cm2) instead of 10 µL to adequately cover the entire epidermis surface
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
of 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
Episkin
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
Test System
EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes and forming a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum (matrix: collagen type 1 coated with type IV collagen).

Principle of the Test – Main Test
Irritant substances are sufficiently cytotoxic to cause cell deaths in the cell layers. Therefore, cell viability of the multilayers was determined by measurement of mitochondrial dehydrogenase activity assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, blue coloured, formazan salt. The degree of formazan salt formation (positively correlated with the degree of cell viability) was measured photometrically (i.e. determination of the optical density of formazan extracts from tissue at 540 nm).

Depending on the percentage of tissue viability attained (compared to negative control viability) a test substance is classified as skin irritating or not skin irritating.

Pre-Tests – Checking for Interference of the Test Substance with the Assay
It was demonstrated, that the test material WS400104 itself did not interact with MTT, i.e. possible false estimation of tissue viability caused by direct interaction of WS400104 with MTT could be ruled out. However, intrinsic colour of WS400104 was evident. Therefore, non specific colour % (NSC%) was determined using one additional yyyyyliveyyyyy control tissue (treated and processed as test material treated tissue but incubated with fresh assay medium instead of MTT) and accounted for when calculating the tissue viability of the test item treated tissue of the main test.

Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 live (viable) tissue samples placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. In addition the above oneyyyyyliveyyyyy control tissue was included in the main test for determination of non specific colour % (NSC%) induced by the test material.
Incubation of these tissues before treatment in maintenance medium: ≥ 24 h at 37°C, 5% CO2 in air, in humidified atmosphere (>95% r.h.), in wells
each containing 2 mL fresh pre-warmed maintenance medium.
Test material administration: Spreading of thin even layer over the epidermal surface.
Termination of 15 ± 0.5 minute exposure period: Removal of residual test material or positive control substance by thorough
rinsing of each epidermis unit with phosphate buffered saline 1x solution (0.9%)
Removal of remaining PBS by use of a Pasteur pipette linked to a vacuum.
Posttreatment incubation (all tissue samples) : 42 ± 1 h at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL fresh pre-warmed maintenance medium
Then MTT incubation (all except NSC sample): 3 hours (± 5 minutes) at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL of 0.3 mg/mL MTT.
NSC sample: 3 hours (± 5 minutes) at 37°C , 5% CO2 & >95% r.h., in wells each containing
2 mL fresh pre-warmed maintenance medium.
Formazan extraction (all tissue samples) : Further processing of tissue samples & formazan extraction by vortexing in
acidic isopropanol, 500 µL/sample and then storing the vortexed samples with
gentle agitation at room temperature in the dark for ca. 3 hours.
Qantitative determination of optical density: At 540 nm with acidified isopropanol (0.04 N HCl final concentration,
6 x 200 µL) as blanks.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
14
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
0.2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid

  

Tabelle 1:  Results of in vitro EpiSkin (TM) Skin Irritation Test

Exposure Period: 15 ± 0.5 minutes

 

OD 540*
Tissue 1

OD 540*
Tissue 2

OD 540*
Tissue 3

OD 540
Mean of Tissue 1, 2 + 3

Tissue Viability
[% of Negative Control
± s.d.]

Negative Control

1.048

0.902

1.014

0.988

 100 ± 7.94

WS400104

0.331

0.191

0.212

0.056**

    5.5 ± 7.44**

Positive Control

0.226

0.171

0.130

0.176

  18 ± 5.03

 

*  OD 540 of individual tissues = Mean Optical Density [wavelength 540 nm] of 2 measurements minus Mean OD of six blanks

   Mean OD of six blanks ± standard deviation (s.d.) = yyyy ± yyyyy

 

** Derived from “true” OD 540 values, the direct colouring potential of WS400104 has already been accounted for.

  OD of Non Specific Colour (NSC) tissue sample = 0.189, NSC% = 19%

Hence assay validity was confirmed both, for the negative and the positive controls, and the test material, WS400104, was irritating.

 

Interpretation of results:
Category 2 (irritant)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Remarks:
in vivo study performed prior to change in information requirements
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
of 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
of 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
of 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS

- Number and Sex: 3 males
- Animal supplier: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
- Age on day of dosing (Day 0): Ca. 11 weeks old (young adult)
- Weight prior to dosing (Day 0): Minimum 2744 g, maximum 2931 g
- Housing: Individual housing in AAALAC approved metal wire rabbit cages allowing for some
social interaction with rabbit(s) in adjacent cages
- Diet (ad libitum): Commercially available rabbit diet, AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Hungary
- Water (ad libitum): Tap water from municipal supply
- Acclimation period: 7 days prior to study start under laboratory conditions.

Water was regularly analysed for contaminants, detailed information on the diet was provided by the supplier. Water and diet used in the present study were not considered to adversely affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS

Controlled environment, environmental conditions were:
- Ventilation, air changes per hour: 15-20
- Temperature (°C): 19.7 to 27.1°C
- Relative Humidity (%): 32 to 83%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
Relative humidity and temperature at times slightly exceeding the upper target limits of 70% and 23°C, respectively, were not considered to have compromised the integrity or validity of the study.

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1 mL of the undiluted wax-like test material was administered into the conjunctival sac of one eye per rabbit (left eye). The contralateral eye (right eye) remained untreated to serve as a control.
Duration of treatment / exposure:
Residual test material was seen in the treated eyes. Therefore, the eyes were rinsed with physiological saline at 1 hour after test material instillation.
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3 adult male rabbits
Details on study design:
EYE EVALUATION:

Within 2.5 hours before treatment start both eyes of each animal were investigated to ensure that there were no pre-existing corneal damage, eye irritation or ocular defects.

One animal was initially treated and, in the absence of a severe effect at 1 hour post instillation, the remaining two animals were committed to the study.

Eyes were evaluated in all animals at approximately 1, 24, 48 & 72 hours and 7, 14 & 21 days after test material instillation adopting the numerical scoring system listed in Table 1 in the field below. Grades attained at 24, 48 and 72 hours after instillation were included in the mean gradings of ocular lesions and used for evaluation of the necessity of eye irritation/corrosion classification.

????Equipment used for eye evaluation: Ophthalmoscope or pencil beam torch
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
over 3 animals and 3 time points
Time point:
other: 24, 48, 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Corneal findings were confined to 1 animal
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: This was the only animal with corneal opacity findings
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
other: Corneal ulceration or opacity were not evident
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
other: Corneal ulceration or opacity were not evident
Irritation parameter:
other: corneal area affected
Basis:
animal #1
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
3.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: This was the only animal with corneal opacity findings
Irritation parameter:
iris score
Basis:
mean
Remarks:
over 3 animals and 3 time points
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
2
Reversibility:
other: Iridic changes were not evident
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Remarks:
over 3 animals and 3 time points
Time point:
other: 24, 48, 72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
over 3 animals and 3 time points
Time point:
other: 24, 48, 72 h
Score:
0.8
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 h
Irritation parameter:
other: Discharge
Basis:
mean
Remarks:
over 3 animals and 3 time points
Time point:
other: 24, 48, 72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: Discharge
Basis:
animal #1
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
2.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: Discharge
Basis:
animal #2
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
other: Discharge
Basis:
animal #3
Remarks:
mean over 3 time points
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
3
Reversibility:
fully reversible within: 24 h
Irritant / corrosive response data:
Iridic lesions were not evident throughout the study, and corneal lesions (opacity grade 1) were confined to one animal (#1) from 1 to 72 hours after test material instillation entirely disappearing thereafter. The area of cornea involved was scored grade 4 to 0. Conjunctival redness grade 2, chemosis grade 2 or 3 and discharge, grade 2 or 3, were seen in all animals at 1 h after instillation, therafter gradually decreasing in severity and incidence and having fully disappeared in all animals by three weeks after instillation. Control eyes were without ocular findings throughout the 21 day observation period.
Other effects:
Observation of the animals for defined behavioural criteria led to the conclusion that instillation of the test material induced slight initial pain (grade 2). Any other signs of systemic toxicity or ill health were not evident and bodyweight was unaffected by treatment with the test material.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The outcome of the present in vivo study does not necessitate any labelling regarding eye irritation according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008]. The findings noted were fully reversible and their incidence is within the category “not irritating to eyes”.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Based on the results of the in vitro and in vivo irritation tests WS400104 is classified as follows:

“Category 2 (skin irritant)" [REGULATION (EC) 1272/2008]. WS400104 is not corrosive.

The incidence and severity of in vivo eye irritation (OECD 405) do not necessitate any classification and labelling of WS400104 regarding eye irritation or corrosion according to EU classification rules [REGULATION (EC) 1272/2008].