Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: weight of evidence approach out of two studies (OECD 404 and OECD 439): not irritating

Eye irritation (OECD 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 Jun - 18 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation 440/2008, 1st ATP 2009)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23345
- Delivery date: 12 July 2016
- Date of initiation of testing: 12 July 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.783 ± 0.027 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.16 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solultion
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approximately 43 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 minutes exposure
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.892, 1.821 and 1.717) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the 3 identical replicates of the test substance, positive and negative control in the main test were below 8% (threshold of OECD 439: <18%), thus ensuring the validity of the study.

Table 2. Results after treatment with the test substance and controls

Test group

Mean absorbance at 570 nm*

Rel. absorbance (%)**

SD (%)

Rel. absorbance (% of negative control)***

Tissue 1

Tissue 2

Tissue 3

Tissue 1

Tissue 2

Tissue 3

Negative control

1.892

1.821

1.717

104.5

100.6

94.8

4.9

100.0

Positive control

0.051

0.053

0.046

2.8

2.9

2.5

7.6

2.8

Test substance

1.838

1.821

1.936

101.6

100.6

107.0

3.3

103.0

* Mean of three replicate wells after blank correction (blank = 0.041)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

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

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the reconstructed human epidermis test the test substance does not possess any skin irritating potential.
Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with 30 µL of the test substance the mean relative absorbance value was 103.0% compared to the negative control (threshold for classification <50%).

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 Oct - 7 Nov 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted Jan 1997
Deviations:
yes
Remarks:
5 test substance concenrations were tested in parallel; 4 rabbits instead of 3 rabbits were used
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministry of Agriculture, Environmental Protection and Regional Planning, Potsdam, Germany
Species:
rabbit
Strain:
other: Chbb:HM(SPF) - Littlerussian
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.4 - 2.9 kg
- Housing: individually in PPO cages (floor area: 2576 cm²) with perforated floor
- Diet: Altromin 2123 (Altromin, Lage, Germany), ad libitum
- Water: domestic quality drinking water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: at least one week

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: ethanol/diethylphthalat 1:1
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration: 1, 10, 25, 50 and 100%
Duration of treatment / exposure:
4 h
Observation period:
21 days
Reading time points: 1, 24, 48 and 72 h
Number of animals:
4
Details on study design:
TEST SITE
- Area of exposure: on the back (10 x 10 cm)
- Type of wrap: The test substance was applied to each of two 16-layer gauze patches (2.5 x 2.5 cm) and the patches were hold in place with adhesive Gothaplast tape (2.5 cm wide) and fixed with Gothaplast tape 85 cm width) loosely wound round the trunk.

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

OBSERVATION TIME POINTS
1, 24, 48 and 72 h and 7, 14 and 21 days

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Remarks:
at 100%
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 21 days
Remarks:
a slight redness and a slight formation of scales was observed in animal #1 after 21 days
Irritation parameter:
erythema score
Remarks:
at 100%
Basis:
animal #4
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
edema score
Remarks:
at 100%
Basis:
animal: #1 and #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Remarks:
at 100%
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Remarks:
at 100%
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
7 days after termination of exposure animal #1 showed thick, white scales distributed all over the anterior left (100%) and middle left (50%) test field. In animal #2, white scales distributed all over the anterior left test field (100%) as well as isolated, little white scales on the middle left test field were observed. Animal #3 showed thick, white scales distributed all over the anterior left test field (100%) as well as little, white scales on the middle left test field (50%). The posterior left (25%) and the right test fields (0, 1 and 10%) of animals #1, #2 and #3 were free of skin reactions. In animal #4, thick, white scales on the whole anterior left test field (100%), little white scales on the whole middle left test field (50%) as well as teeny-weeny scales on approximately 30% of the posterior left test area (25%) were observed. The right test fields (0, 1 and 10%) of this animal showed no skin reactions.

14 days after termination of exposure in animals #1 and #2 the scales were falled down on approximately 40% of the anterior left test area (100%). On the middle left test field (50%) intact skin was observed under the falled down scales crust. The posterior left (25%) and the right test fields (0, 1 an d10%) were free of any skin reactions. In animal #3, scales on approximately 30% of the anterior left test area (100%) were observed. The scale were falled down on the middle left test field (50%). Animal #4 showed white scales on approximately 50% of the anterior left test area (100%) as well as little, white scales on approximately 40% of the middle left test area (50%). The posterior left (25%) and the right test fields (0, 1 and 10%) of animals #3 and #4 were free of skin reactions.

21 days after termination of exposure animal #1 showed a slight redness and a slight formation of scales on the anterior left test field (100%). On the middle left test field (50%) few scales were observed. The posterior left (25%) and the right test fields (0, 1 and 10%) were free of skin reactions in this animal. Animals #2, #3 and #4 showed no signs of a skin reaction.

Table 1. Results of the skin irritation study.

Rabbit no.

Test concentration

Erythema

Individual mean values 24/48/72 h

Edema

Individual mean values

24/48/72 h

1 h

24 h

48 h

72 h

1 h

24 h

48 h

72 h

1

AL

100%

2

2

2

2*

2.0

1

1

1

0

0.7

AR

10%

0

0

0

0

0

0

0

0

0

0

ML

50%

1

2

2

1*

1.7

0

1

1

0

0.7

MR

1%

0

0

0

0

0

0

0

0

0

0

PL

25%

0

1

1

0

0.7

0

0

0

0

0

PR

V%

0

0

0

0

0

0

0

0

0

0

2

AL

100%

2

2

2

2*

2.0

2

2

2

1

1.7

AR

10%

0

0

0

0

0

0

0

0

0

0

ML

50%

1

2

2

2*

2.0

2

2

2

1

1.7

MR

1%

0

0

0

0

0

0

0

0

0

0

PL

25%

0

1

1

1

1.0

0

1

0

0

0.3

PR

V%

0

0

0

0

0

0

0

0

0

0

3

AL

100%

2

2

2

2*

2.0

2

1

1

1*

1.0

AR

10%

0

0

0

0

0

0

0

0

0

0

ML

50%

1

1

1

1*

1.0

1

0

0

0

0

MR

1%

0

0

0

0

0

0

0

0

0

0

PL

25%

0

1

0

0

0.3

0

0

0

0

0

PR

V%

0

0

0

0

0

0

0

0

0

0

4

AL

100%

1

1

2

2*

1.7

1

1

0

1*

0.7

AR

10%

0

0

0

0

0

0

0

0

0

0

ML

50%

0

1

2

2*

1.7

0

1

0

0

0.3

MR

1%

0

0

0

0

0

0

0

0

0

0

PL

25%

0

0

0

0

0

0

0

0

0

0

PR

V%

0

0

0

0

0

0

0

0

0

0

AL: anterior left treatment site

AR: anterior right treatment site

ML: middle left treatment site

MR: middle right treatment site

PL: posterior left treatment site

PR: posterior right treatment site

V: Vehicle (1:1 ethanol/diethylphthalate)

*: patch area sharp demarcated

Interpretation of results:
other: borderline result between no classification and Skin Cat. 2 (H315) according to Regulation (EC) No 1272/2008 for 100% test substance concentration.
Conclusions:
Under the conditions of this skin irritation study in rabbits scales were observed in all 4 test fields treated with 100% after 14 days. Slight scale formation was persistent in 1 animal (100%) until 21 days. Test substance concentrations of 1, 10, 25 and 50% were considered not irritating to the skin according to CLP criteria.
Executive summary:

The skin irritation potential of the test substance was determined by an in vivo skin irritation test according to OECD Guideline 404 (adopted in 1992) and in compliance with GLP (2000). Test substance concentrations of 100, 50, 25, 10, 1 and 0% (vehicle only) were applied to the skin of 4 rabbits under semi-occlusive conditions for 4 hours. Scales were observed in all 4 test fields treated with 100% after 14 days. Slight scale formation persisted in 1 animal on the test field treated with 100% until 21 days. Since scales were still evident at the end of the observation period for the 100% test subtsance concentration, this result is considered borderline between no classification and skin irritation. Test substance concentrations of 1, 10, 25 and 50% were not irritating to the skin.

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:
17 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL

NEGATIVE CONTROL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
in triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. After equilibration for about 1 hour, the anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

NUMBER OF REPLICATES: 3 corneae per test group

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.
- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = opacity value + (15x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
0.29
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 83.03) corresponding to a classification as serious eye damaging.
Relative to the negative control, the test substance did neither cause an increase of the corneal opacity nor permeability (mean IVIS = 0.29).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for postive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 10 min incubation time.

Test group

Opacity value =
Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

Mean

 

Mean

 

 

Negative

control

0

0

0.062

0.066

0.93

0.99

0

0.058

0.87

0

0.078

1.17

Positive

control

51*

1.268*

70.02

83.03

57*

1.821*

84.32

72*

1.517*

94.76

Test substance

1*

-0.006*

0.91

0.29

1*

-0.006*

0.91

-1*

0.003*

-0.96

*: corrected values

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the BCOP assay the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.29.
Executive summary:

The eye irritation/corrosion potential of the test substance was determined by a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2016). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.29 and thus the test substance was not considered as irritating to the eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vivo skin irritation test according to OECD Guideline 404 (adopted in 1992) and in compliance with GLP (2000). Test substance concentrations of 100, 50, 25, 10, 1 and 0% (vehicle only) were applied to the skin of 4 rabbits under semi-occlusive conditions for 4 hours. Scales were observed in all 4 test fields treated with 100% after 14 days. Slight scale formation persisted in 1 animal on the test field treated with 100% until 21 days. Since scales were still evident at the end of the observation period for the 100% test subtsance concentration, this result is considered borderline between no classification and skin irritation. Test substance concentrations of 1, 10, 25 and 50% were not irritating to the skin.

One further study was considered for the evaluation of the endpoint skin irritation/corrosion.

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with 30 µL of the test substance the mean relative absorbance value was 103.0% compared to the negative control (threshold for classification <50%).

Based on a weight of evidence taking into account the available data described above, the test substance is not considered as skin irritant and thus not classified for skin irritation/corrosion according to Regulation (EC) No 1272/2008.

Eye

The eye irritation/corrosion potential of the test substance was determined by a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2016). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.29 and thus the test substance was not considered as irritating to the eye.

Justification for classification or non-classification

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