Registration Dossier

Administrative data

Description of key information

skin irritation (OECD 404): irritating
eye irritation (OECD 492): not irritating; (OECD 437): non-corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Sep - 14 Oct 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study (reversibility of the effects was observed only up to 7 days after removal of the patches)
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
National Board of Health, BronshØj, Denmark
Species:
rabbit
Strain:
other: Mol:Russian
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: MØllegaard Breeding and Research Centre A/S, Lille Skensved, Denmark
- Weight at study initiation: 2.3-2.5 kg
- Housing: individually in PPO/HIPS cages (2576 cm²) with perforated floor
- Diet: Altromin 2123, pelleted complete rabbit diet (Chr. Petersen, Ringsted, Denmark), ad libitum
- Water: domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%): 55±15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: 1:1 (w/w) ethanol/diethylphthalate
Controls:
other: adjacent areas of skin treated with vehicle served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
- Concentration: 5, 10, 25, 50 and 100%
Duration of treatment / exposure:
4 h
Observation period:
7 days
Reading time points: 1, 24, 48 and 72 h
Number of animals:
4 females
Details on study design:
TEST SITE
- Area of exposure: Appropriate test site on the back (10 cm x 10 cm) divided into two anterior, two centrally and two posterior located test sites. The different test concentrations were applied to the different skin areas simultaneously.
- Type of wrap: The test substance of one of the test concentrations or the vehicle was applied to the skin, using gauze patches of 2.5 cm x 2.5 cm. The patches were secured with 2.5 cm wide adhesive tape and fixed with Scanpor tape (5 cm width). Thereafter Nobifix (6 cm width) was loosely wound round the trunk.

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance using lukewarm water and mild soap.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
animal: #1, #2 and #4
Time point:
other: mean after 24/48/72 h
Score:
2
Max. score:
4
Reversibility:
no data
Remarks on result:
other: test concentration: 100%
Irritation parameter:
erythema score
Basis:
animal: #3
Time point:
other: mean after 24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
no data
Remarks on result:
other: test concentration: 100%
Irritation parameter:
edema score
Basis:
animal: #1, #2 and #4
Time point:
other: mean after 24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
no data
Remarks on result:
other: test concentration: 100%
Irritation parameter:
edema score
Basis:
animal: #3
Time point:
other: mean after 24/48/72 h
Score:
2
Max. score:
4
Reversibility:
no data
Remarks on result:
other: test concentration: 100%
Irritant / corrosive response data:
None to well-defined skin reactions were seen at the test sites with 25, 50 and 100% test substance.
None to slight skin reactions were seen at the test sites with vehicle and with 10% test substance. No skin reactions were seen at the test sites with 5% test substance.
On Day 8 scabs were observed in animal No. 1 at the test sites with 25% and 50% test substance, and in animal No. 2 at the test site with 25% test substance.
On Day 8 few scattered scabs were observed in animal No. 1 at the test site with 10% test substance.
On Day 8 crust was observed in animal No. 1 at the test site with 100% test substance and in animal No. 2, 3 and 4 at the test sites with 50% and 100% test substance.
The skin of the other test fields was intact on Day 8.

Table 1. Results of the skin irritation study.

Rabbit no.

Test concentration

Erythema

Individual mean values (mean after 24/48/72 h)

Edema

Individual mean values (mean after 24/48/72 h)

1 h

24 h

48 h

72 h

1 h

24 h

48 h

72 h

1

LA

100%

1

2

2

2

2.0

1

1

2

2

1.7

RA

50%

1

2

2

2

2.0

1

1

2

2

1.7

LM

25%

1

2

2

2

2.0

1

1

2

2

1.7

RM

10%

1

1

1

1

1.0

1

1

1

1

1.0

LP

5%

0

0

0

0

0.0

0

0

0

0

0.0

RP

V%

0

0

0

0

0.0

0

0

0

0

0.0

2

LA

50%

1

2

2

2

2.0

1

1

2

2

1.7

RA

25%

1

2

2

2

2.0

1

1

2

2

1.7

LM

10%

1

1

1

1

1.0

1

1

1

1

1.0

RM

5%

0

0

0

0

0.0

0

0

0

0

0.0

LP

V

0

0

0

0

0.0

0

0

0

0

0.0

RP

100%

1

2

2

2

2.0

1

1

2

2

1.7

3

LA

25%

1

1

2

2

1.7

1

1

1

1

1.0

RA

10%

1

1

0

0

0.3

1

1

0

0

0.3

LM

5%

0

0

0

0

0.0

0

0

0

0

0.0

RM

V

0

0

0

0

0.0

0

0

0

0

0.0

LP

100%

2

2

2

3

2.3

2

2

2

2

2.0

RP

50%

1

2

2

3

2.3

1

1

2

2

1.7

4

LA

10%

1

1

0

0

0.3

1

1

0

0

0.3

RA

5%

0

0

0

0

0.0

0

0

0

0

0.0

LM

V

0

1

1

1

1.0

0

0

0

0

0.0

RM

100%

1

2

2

2

2.0

1

1

2

2

1.7

LP

50%

1

2

2

2

2.0

1

1

2

2

1.7

RP

25%

1

2

2

2

2.0

1

1

1

1

1.0

LA: Left anterior treatment site

RA: Right anterior treatment site

LM: Left middle treatment site

RM: Right middle treatment site

LP: Left posterior treatment site

RP: Right posterior treatment site

V: Vehicle (1:1 (w/w) ethanol/diethylphthalate)

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this skin irritation study the test substance was irritating 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:
23- 25 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 492 ( Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage) (2015)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
TEST MODEL (EpiOcular™ Kit)
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 21572

TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. Irritant materials are identified by their ability to damage the underlying cell layers which is determined through a decrease in cell viability as determined by MTT reduction.

ADAPTATION TO CELL CULTURE CONDITIONS
1.0 mL assay medium (37 °C) was aliquoted into 6-well plates. The inserts with EpiOcular™ tissues were transferred aseptically into the plates and pre-incubated at standard culture conditions for 1 h. Afterwards, the medium was replaced by 1 mL fresh assay medium and the EpiOcular™ tissues were incubated at standard culture conditions overnight (18 h). After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca²+ Mg²+ free DPBS. The tissues were incubated at standard culture conditions for 30 min.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95
Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control was deionised water and methyl acetate was used as positive control.
Amount / concentration applied:
TEST MATERIAL
- Applied volume: 50 µL

POSITIVE SUBSTANCE
- Substance: methyl acetate
- Applied volume: 50 µL

NEGATIVE CONTROL
- Substance: deionised water
- Applied volume: 50 µL
Duration of treatment / exposure:
30 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
The test was performed in duplicate for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment time, the test substance was removed by extensively rinsing the tissues with Ca²+Mg²+ free DPBS in clean beakers.
- Post-treatment incubation period: 2 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, a volume of 300 µL MTT solution was added to each well for 3 h at standard culture conditions. After removal of the MTT solution, wells were rinsed three times with Ca²+Mg²+ free DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. At the end of the extraction period the optical density (OD) was measured.
Irritation parameter:
other: cell viability (%)
Basis:
other: mean values of 2 tissues
Time point:
other: 30 min
Score:
95.1
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: cell viability (%)
Basis:
other: mean values of 2 tissues
Time point:
other: 30 min
Score:
6.9
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: cell viability (%)
Basis:
other: mean value of 2 tissues
Time point:
other: 30 min
Score:
100
Reversibility:
other: not applicable
Remarks on result:
other: Negative control

Table 1. Results after 30 min incubation time

Test group

Absorbance*

Mean absorbance of 2 tissues*

Rel. absorbance (%)**

Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2

Rel. absorbance (% of negative control)**

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative control

1.568

1.653

1.611

97.4

102.6

5.3

100.0

Positive control

0.107

0.114

0.110

6.6

7.1

0.5

6.9

Test substance

1.459

1.606

1.533

90.6

99.7

9.1

95.1

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour. Therefore, additional tests with viable or freeze-killed tissues were not performed.

All acceptance criteria were met.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the conducted test, the test substance did not exhibit irritating properties towards human-derived epidermal keratinocytes in the EpiOcular TM model.
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:
There is only one study available.

Justification for selection of eye irritation endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

The available data on skin irritation meet the criteria for classification as Skin Irrit. Cat. 2 (H315) according to Regulation (EC) 1272/2008.

The available data on eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.