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Administrative data

Description of key information

skin irritation (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:
29 Sep - 05 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
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:
TEST SKIN MODEL EpiDerm™ (Epi-200-SIT Kit)
- Source: MatTek Corporation, Bratislava, Slovakia
- Lot No.: 21697

TEST METHOD
The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
0.9 mL assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further 22 - 23 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95 ± 5

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: After the end of the treatment interval, tissues were gently rinsed with DPBS at least 15 times. After rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
- Post-treatment incubation period: 42 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 37 ± 1 °C and 5 ± 0.5% CO2. After aspiration of the MTT solution, wells were rinsed three times with DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. The formazan salt was extracted for nearly 70 h without shaking in the refrigerator. The optical density (OD) was measured at 570 ± 1 nm wave length in a microplate reader (Versamax Molecular Devices).

EVALUATION OF RESULTS
The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(OD-test item/positive control) / (OD-negative control)] x 100

ACCEPTABILITY OF THE ASSAY
1. OD 570 nm of the negative control ≥ 0.8 and ≤ 2.8
2. Mean relative tissue viability of the positive control ≤ 20%
3. Rel. standard deviation of 3 identical replicates < 18%
OD values should not be below historically established boundaries.
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% in deionised water
Duration of treatment / exposure:
60 min (35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH; thereafter, plates were removed from the incubator and placed in a sterile bench at room temperature until the end of treatment)
Duration of post-treatment incubation (if applicable):
about 42 h
Number of replicates:
The test was performed in triplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 min exposure
Value:
60.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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.
- 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 lead to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.607, 1.622 and 1.669) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment.
- 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 5.9 % thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance, the positive and negative control, were < 18% (2.0, 9.2 and 3.6%), thus ensuring the validity of the study.

Table 2. Results after 60 min incubation time.

Test group

Absorbance at 570 nm

Mean absorbance

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

Rel. SD (%)

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

Tissue 1*

Tissue 2*

Tissue 3*

Negative control

1.607

1.622

1.669

1.633

98.4
99.3
102.2

2.0

100.0

Positive control

0.107

0.092

0.091

0.097

6.5
5.7
5.6

9.2

5.9

Test substance

0.994

1.022

0.952

0.989

60.9
62.6
58.3

3.6

60.6

* 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 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). Each three human skin tissues (EpiDermTM) with a tissue size of 0.6 cm² were treated with 30 µL of the test substance, the negative control (DPBS buffer) or the positive control (5% sodium lauryl sulphate) for 60 minutes. After a 42-hours post-incubation period, the cytotoxic (irritancy) effect was assessed. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 60 minutes treatment with the test substance compared to the negative control tissues was 60.6%. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 60 min treatment the test substance was considered to be non-irritant to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, 5% sodium lauryl sulphate, revealed a mean cell viability of 5.9% (required ≤ 20%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was not irritating to the skin under the conditions of the test.

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:
30 Aug - 03 Sep 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Feb. 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Jan. 1997
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.3 - 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

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
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 (corneal opacity)
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: 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: not applicable
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of 4 animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal: #2 and #4
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Irritant / corrosive response data:
One hour after application of the test substance two animals showed some conjunctival vessels definitely injected. Some conjunctival vessels definitely injected and some swelling above normal were seen in one animal. Some conjunctival vessels definitely injected, some swelling above normal and amount of discharge different from normal were observed in one animal. After 24 hours some conjunctival vessels definitely injected were observed in three animals. One animal was free of any signs of eye irritation. After 48 hours some conjunctival vessels definitely injected were observed in one animal. Two animals were free of any signs of eye irritation. After 72 hours all animals 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

1

0

0

0

24

1

0

0

0

48

1

0

0

0

72

0

0

0

0

Average 24+48+72

0.7

0

0

0

2

1

1

0

0

0

24

1

0

0

0

48

0

0

0

0

72

0

0

0

0

Average 24+48+72

0.3

0

0

0

3

1

1

1

0

0

24

0

0

0

0

48

0

0

0

0

72

0

0

0

0

Average 24+48+72

0

0

0

0

4

1

1

1

0

0

24

1

0

0

0

48

0

0

0

0

72

0

0

0

0

Average 24+48+72

0.3

0

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.
Executive summary:

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 material 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 hours after application. Conjunctival redness (score 1) was observed in all animals 1 hour after treatment. Additionally, chemosis (score 1) was observed in two animals and discharge (score 1) was noted in one animal 1 hour after treatment. Conjunctival redness (score 1) was noted in three animals at 24 hours and in one animal at 48 hours after application. After 72 hours all animals were free of any signs of eye irritation. The overall mean score after 24, 48 and 72 hours out of four rabbits for conjunctival redness was 0.3. The overall mean irritation score over 24, 48 and 72 hours for chemosis, iris and corneal effects was 0. Based on the results of this study, the test substance was not irritating to the eyes under the conditions of the test.

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 using a human skin model according to OECD Guideline 439 and in compliance with GLP (2016). Each three human skin tissues (EpiDermTM) with a tissue size of 0.6 cm² were treated with 30 µL of the test substance, the negative control (DPBS buffer) or the positive control (5% sodium lauryl sulphate) for 60 minutes. After a 42-hours post-incubation period, the cytotoxic (irritancy) effect was assessed. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 60 minutes treatment with the test substance compared to the negative control tissues was 60.6%. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 60 min treatment the test substance was considered to be non-irritant to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, 5% sodium lauryl sulphate, revealed a mean cell viability of 5.9% (required ≤ 20%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was not irritating to the skin under the conditions of the test.

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 material 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 hours after application. Conjunctival redness (score 1) was observed in all animals 1 hour after treatment. Additionally, chemosis (score 1) was observed in two animals and discharge (score 1) was noted in one animal 1 hour after treatment. Conjunctival redness (score 1) was noted in three animals at 24 hours and in one animal at 48 hours after application. After 72 hours all animals were free of any signs of eye irritation. The overall mean score after 24, 48 and 72 hours out of four rabbits for conjunctival redness was 0.3. The overall mean irritation score over 24, 48 and 72 hours for chemosis, iris and corneal effects was 0. 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 of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.