Registration Dossier

Administrative data

Description of key information

The test item is a Cat. 2 skin irritant, but is not irritant to the eyes.

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:
31.08.2016 - 09.09.2016
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. However, because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SITKit
- Tissue batch number(s): 23353
- Delivery date: 06.09.2016
- Date of initiation of testing: 06.09.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The substance is considered skin irritant category 2 according to UN GHS (published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
- Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: The rel. SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system / test kit.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 tissue runs
Value:
6.4
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The acceptance criteria were met.

Results

 

Dose Group

Tissue No.

Absorbance 570 nm Well 1

Absorbance 570 nm Well 2

Absorbance 570 nm Well 3

Mean Absorbance of 3 Wells

Mean Absorbance
 of 3 wells blank corrected

Mean Absorbance
of 3 tissues after blank correction *

Rel. Absorbance [%] Tissue 1, 2 + 3**

Relative Standard Deviation [%]

Mean Rel. Absorbance [% of Negative Control]***

Blank

 

0.038

0.038

0.037

0.038

0.000

 

 

 

 

Negative Control

1

2.064

2.078

2.207

2.116

2.079

1.927

107.9

8.2

100.0

2

1.955

2.003

1.978

1.979

1.941

100.7

3

1.768

1.784

1.849

1.800

1.762

91.4

Positive Control

1

0.119

0.122

0.112

0.117

0.080

0.084

4.1

5.2

4.4

2

0.126

0.125

0.128

0.126

0.088

4.6

3

0.129

0.118

0.120

0.122

0.084

4.4

Test Item

1

0.159

0.161

0.164

0.161

0.123

0.124

6.4

1.2

6.4

2

0.165

0.168

0.158

0.164

0.126

6.5

3

0.160

0.160

0.163

0.161

0.123

6.4

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue [rounded values]: 100 × (absorbancetissue) / (mean absorbancenegative control)

*** Relative absorbance per treatment group [rounded values]: 100×(mean absorbancetest item / positive control) / (mean absorbancenegative control)

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is to be considered as irritant to skin.
Executive summary:

In this in vitro study the irritation potential of the test item was assessed by means of the Human Skin Model Test. The study was according to OECD 439 and GLP.

The test item did not reduce MTT (test for direct MTT reduction), and did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive (colourless to yellow). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The main study consists of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured (as optical density (OD)) after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and used for the purpose of classification as irritating or non-irritating. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.

Three tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes.

Hereafter the skin tissues are washed and further incubated for about 42 hours, whereafter the tissues were treated with MTT for 3 hours following about 2.7 hours 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 within the required 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 sufficient decrease to 4.4% in the relative absorbance as compared to the negative control for the 60 minutes treatement interval, thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 9% (threshold of the OECD TG: < 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 6.4% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is to be considered as irritant to 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:
02.09.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: bovine
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 month old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): in HBSS at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were 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 (vascularization, pigmentation, opacity and scratches) were discarded. 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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Each isolated cornea was mounted in a cornea holder which 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 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. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

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

NUMBER OF REPLICATES: 3

POST-INCUBATION PERIOD: 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) - the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The test is accepted if:
- The positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- The negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
0.31
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The acceptance criteria were met.
Positive control: IVIS = 113.06
Negative control: IVIS = 0.82

Results after 10 Minutes Incubation Time

Test Group

Opacity value = Difference (t130 - t0) of Opacity

Permeability at 490 nm (OD 490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

(IVIS)

Negative Control

0

0.00

0.053

0.054

0.80

0.82

Not categorized

0

0.055

0.83

0

0.055

0.83

Positive Control

112.00*

1.060*

127.90

113.06

Category 1

88.00*

0.987*

102.80

90.00*

1.233*

108.49

Test item

0.00*

0.024*

0.36

0.31

Not categorized

0.00*

0.014*

0.21

0.00*

0.024*

0.36

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
According to the current study and under the experimental conditions reported, the test item is not eye irritant.
Executive summary:

In the current study the corneal damage potential of the test item was assessed by means of the BCOP assay using fresh bovine corneae. The study was according to OECD 437 and GLP.

This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein and the opacity before and after the exposure to the test item is compared to determine the damaging effect of the test item. For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item is measured and the results of both criteria were combined. The resulting in vitro irritation factor is used for classification as “no category (GHS)”, “no prediction can be made”, and "serious eye damaging" (CLP/EPA/GHS (Cat 1)).

After a first opacity measurement of the fresh bovine corneae, the neat test item, the positive, and the negative controls are applied to the corneae for 10 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls are washed off and the corneae are further incubated for 120 minutes at 32 ± 1 °C. Afterwards, the opacity is measured a second time.

The opacity measurements are used to determine the permeability of the corneae by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 minutes at 32 ± 1 °C.

The negative control (0.9% (w/v) NaCl solution in deionised water) did not increase the opacity or alter the permeability of the corneae ( mean in vitro irritancy score (IVIS): 0.82) and the positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS: 113.06) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The controls indicated the validity of the test method.

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability and had a calculated in vitro irritancey score (mean IVIS) of 0.31. According to the GHS criteria the test item is not eye irritant as the threshold for serious eye damage is IVIS ≥ 55.

In conclusion, according to the current study and under the experimental conditions reported, the test item is not eye irritant.

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

Additional information

Skin Irritation / Corrosion

For this endpoint there are 2 studies available, a skin irritation test according to OECD 439 and a skin corrosion test according to OECD 431. Both were GLP.

In the first in vitro study the irritation potential of the test item was assessed by means of the Human Skin Model Test. The test item did not reduce MTT and did not change colour when mixed with deionised water and its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

In the main study the test item is topically applied to the human reconstructed epidermis model and the cell viability is measured by dehydrogenase conversion of MTT into a blue formazan salt that is quantitatively measured. The percentage of reduced cell viability is used to predict the skin irritation potential and used for classification. The test chemical is considered to be irritant to the skin (Category 2) if the tissue viability is ≤ 50%.

Three tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes and afterwards treated with MTT for 3 hours.

The negative control absorbance values were well within the required acceptability criterion showing the quality of the tissues and the positive control induced a sufficient decrease in the relative absorbance ensuring the validity of the test system. The relative standard deviations were below 9% ensuring the validity of the study.

The test item had a mean relative absorbance value decreased to 6.4% compared to the negative control. This value is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to possess an irritant potential.

In the second study the corrosive potential of the test item was assessed by means of the Human Skin Model Test using the EpiDermTM tissue model. In this study the tissues are exposed to the test item for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect measured by formazan production from MTT. The threshold for corrosivity is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. The negative control was deionised water and the positive control was 8.0 N KOH.

The negative control absorbance values met the required acceptability criterion confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance confirming the validity of the test system and the specific batch of tissue models. The coefficient of variation (CV) in the range 20 – 100% viability between the tissue replicates is <= 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

The test item did not show a decrease in absorbance value after 3 minutes exposure (105.9%) nor after 1 hour exposure (136.2%). Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that the test item is irritant but not corrosive to the skin.

Eye Irritation / Corrosion

For this endpoint there is 1 study available in which the corneal damage potential of the test item was assessed in a BCOP assay using fresh bovine corneae according to OECD 437 and GLP. In this test the opacity and permeability of the cornea are measured before and after exposure to the test item and the irritation potential of the test item is expressed as mean in vitro irritancy score (IVIS) and used for classification purposes.

Exposure of the cornea to the test item, the positive or the negative controls occurs for 10 minutes, whereafter the test item, the positive or negative controls are washed off and the corneae are further incubated for 120 minutes. The opacity is measured before and after exposure.

The permeability of the corneae is measured spectrophotometrically using sodium fluorescein.

The negative control (0.9% (w/v) NaCl solution in deionised water) did not increase the opacity or alter the permeability of the corneae (mean IVIS: 0.82), while the positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS: 113.06) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The controls indicated the validity of the test method.

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability and had a mean IVIS of 0.31. According to the GHS criteria the test item is not eye irritant as the threshold for serious eye damage is IVIS ≥ 55.

Justification for classification or non-classification

Skin Irritation / Corrosion

In a skin irritation test according to OECD 439 the test item had a mean relative absorbance value of 6.4% which is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to possess an irritant potential. In a skin corrosion test according to OECD 431 the test item did not show a decrease in absorbance after 3 minutes nor 1 hour of exposure. Therefore, the substance should be classified as Category 2 Skin Irritant.

Eye Irritation / Corrosion

In a BCOP assay using fresh bovine corneae according to OECD 437 and GLP, the mean IVIS of the test item was 0.31. The test item did not cause an increase of the corneal opacity or permeability and according to the GHS criteria the test item is not eye irritant as the threshold for serious eye damage is IVIS ≥ 55.