Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion (OECD 439): not irritating

Eye irritation (OECD 405): not irritating

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:
23 Feb - 21 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(Commission Regulation EC (No) 440/2008 of 30 May 2008, 1st ATP 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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™ (Epi-200)
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 lot number: 23324
- Delivery date: 15 March 2016
- Date of initiation of testing: 15 March 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 hood
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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 was prepared on the day of testing using MTT-100 Assay Kit Components.
- MTT concentration:1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 ± 1 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 1.862 ± 0.078 (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 5.6 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 solution
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approximately 42 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 tissues
Run / experiment:
60 min exposure
Value:
71.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour. Thus, the test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test substance's colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.635, 1.976 and 1.715) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment, thus showing the quality of 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 6.2% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variabilities of the test substance, the positive and negative controls were <14%, thus ensuring the validity of the study.

Table 2. Results after 60 min incubation time.

Test group

Mean absorbance at 570 nm

Mean absorbance of three tissues

Rel. absorbance (%) Tissue 1, 2 and 3**

Rel. standard deviation (%)

Mean rel. absorbance (% of negative control)***

Tissue 1*

Tissue 2*

Tissue 3*

Negative control

1.635

1.976

1.715

1.775

92.1
111.3
96.6

10.0

100.0

Positive control

0.124

0.095

0.111

0.110

7.0
5.3
6.3

13.2

6.2

Test substance

1.369

1.170

1.285

1.275

77.1
65.9
72.4

7.8

71.8

* Mean of 3 replicate wells after blank correction

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

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 - 26 June 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 1.6 - 2.0 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

Vehicle:
unchanged (no vehicle)
Controls:
other: The right eye remained untreated and served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
72 hours
Reading time points: 1, 24, 48 and 72 hours after treatment
Number of animals or in vitro replicates:
4 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: 20 mL 0.9% sodium chloride solution was used
- Time after start of exposure: 24 hours

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility: not applicable
Irritation parameter:
iris score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal: #1 and #4
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal: #2 and #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
One hour after application of the test material a diffuse, crimson red conjunctiva with individual vessels not easily discernible, obvious swelling with partial eversion of lids and some amount of discharge different from normal were seen in animal No. 1. The remaining three animals showed some conjunctival vessels definitely injected, obvious swelling with partial eversion of lids and some amount of discharge different from normal. After 24 h all animals showed some conjunctival vessels definitely injected and some swelling of the conjunctiva above normal. After 48 h animal No. 1 and 4 showed some conjunctival vessels definitely injected. Animal No. 2 and 3 were free of any signs. After 72 h animal No. 1 and 4 were free of any signs of eye irritation.

Table 1. Results of the eye irritation study

Animal

Time (h)

Conjunctivae

Cornea

Iris

Redness

Chemosis

1

1

2

2

0

0

24

1

1

0

0

48

1

0

0

0

72

0

0

0

0

Average 24+48+72

0.67

0.33

0

0

2

1

1

2

0

0

24

1

1

0

0

48

0

0

0

0

72

0

0

0

0

Average 24+48+72

0.33

0.33

0

0

3

1

1

2

0

0

24

1

1

0

0

48

0

0

0

0

72

0

0

0

0

Average 24+48+72

0.33

0.33

0

0

4

1

1

2

0

0

24

1

1

0

0

48

1

0

0

0

72

0

0

0

0

Average 24+48+72

0.67

0.33

0

0

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 this eye irritation study in rabbits the test substance was not 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 vitro skin irritation test in human keratinocytes according to OECD Guideline 439 and in compliance with GLP (2016). The test substance did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). 30 μL of the test substance, 30 µL of the negative control (DPBS) or 30 µL of the positive control (5% aqueous solution of sodium lauryl sulphate) were applied to each tissue and spread to match the surface of triplicate tissue each. The test substance and the positive and negative controls were washed off the skin tissues after 60 min treatment. After further incubation for about 42 h the tissues were treated with the MTT solution for 3 h following about 67.25 h extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.2%, and thus assuring the validity of the test system.

After treatment with the test substance the mean relative absorbance value decreased to 71.8% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test substance is not considered to possess an irritant potential.

Eye

The eye irritation potential of the test substance was investigated in four albino rabbits according to OECD Guideline 405 and in compliance with GLP (1999). 0.1 mL of the test substance was placed in the left eye of each animal. The right eye remained untreated and served as control. The eyes were examined and scored according to Draize 1, 24, 48 and 72 h after application. One hour after application of the test substance a diffuse, crimson red conjunctiva (score 2) with individual vessels not easily discernible, obvious swelling (score 2) with partial eversion of lids and some amount of discharge different from normal (score 1) were seen in animal No. 1. The remaining three animals showed some conjunctival vessels definitely injected (score 1), obvious swelling with partial eversion of lids (score 1) and some amount of discharge different from normal (score 1). After 24 h all animals showed some conjunctival vessels definitely injected (score 1) and some swelling of the conjunctiva above normal (score 1). After 48 h some conjunctival vessels definitely injected (score 1) was noted in animal No. 1 and 4. Animal No. 2 and 3 were free of any signs of eye irritation after 48 h. After 72 h animal No. 1 and 4 were also free of any signs of eye irritation. The overall mean score out of four rabbits after 24/48/72 h for conjunctival redness, chemosis, iris lesion and corneal opactiy was 0.5, 0.33, 0 and 0, respectively. Based on the results of this study, the test substance was not irritating to the eyes under the conditions of the test.


Justification for classification or non-classification

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