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Diss Factsheets

Administrative data

Description of key information

Skin irritation (OECD 439): not irritating

Eye irritation / Serious eye damage (OECD 437): 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:
11 Apr - 5 May 2017
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 (EC) No 440/2008 of 30 May 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™ (EPI-200-SIT)
Source strain:
other: 00267
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number(s): 25810
- Shipping date: 3 May 2017
- Delivery date: 3 May 2017
- Date of initiation of testing: 3 May 2017

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
- Temperature of post-treatment incubation: Tissues were incubated for 24.7 hours at 37 ± 1.5 °C. After change of medium, tissues were again incubated for another 17.5 hours at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Inserts were removed from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. 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 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 1.556 ± 0.088 (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 5.23 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.
- Reproducibility: Absorbance of positive and negative control were within the range of historical controls.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT in a pre-test, 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 ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg ± 2 mg (~ 39 mg/cm2) of the test item, wetted with 25 μL DPBS

NEGATIVE CONTROL
- Amount applied: 30 μL

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5%
Duration of treatment / exposure:
35 min at 37 ± 1.5 °C and 25 min at RT
Duration of post-treatment incubation (if applicable):
42.2 h
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
102.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values in the main test were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: ≤18%), thus ensuring the validity of the study.
- Range of historical values if different from the ones specified in the test guideline: The relative standard deviations of historical positive controls were with 20.12% higher than standard deviations ≤ 18% in the test guideline. In the study, the relative standard deviations of the positive control were 5.1% and therefore ≤ 18%.

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

1.504

1.372

1.266

108.9

99.4

91.7

8.6

100.0

Positive control

0.062

0.059

0.065

4.5

4.3

4.7

5.1

4.5

Test substance

1.281

1.401

1.578

92.8

101.5

114.3

10.8

102.9

* Mean of three replicate wells after blank correction (blank = 0.037)

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

*** Relative absorbance per treatment group (rounded values): 100 × (mean 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 reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with the test substance for 60 min the tissue viability was 102.9% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is not considered to be 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:
18 April 2017
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:
adopted July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No 1152/2010
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, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The freshly isolated eyes were stored in Hank's Buffered Salt Solution (HBSS) containing 1% (v/v) Penicillin/Streptomycin in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- 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:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in saline

VEHICLE / NEGATIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 0.9% NaCl (w/v) in deionised water (saline)

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 10 % (w/v) in 0.9% NaCl in deionised water

Duration of treatment / exposure:
240 min
Number of animals or in vitro replicates:
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 240 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.

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: After incubation with fluorescein dye at 32 ± 1 °C in the water-bath for 90 min in a horizontal position, passage of sodium fluorescein dye was measured using 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
Remarks:
mean value of 3 corneas
Run / experiment:
240 min
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.01). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =102.27) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

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 positive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 240 min incubation time.

Test group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

 

Mean

 

Mean

Vehicle Control

0

0.00

0.061

0.067

0.92

1.01

0

0.080

1.20

0

0.061

0.92

Positive Control

115.0*

0.014*

115.21

102.27

82.00*

0.040*

82.60

109.00*

0.001*

109.01

Test substance

-1.00*

0.029*

0.00**

0.00

0.00*

0.010*

0.15*

0.00*

0.002*

0.03*

*corrected values

**negative values are set to zero

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 Bovine Corneal Opacity and Permeability Test (BCOP) the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.00.
Executive summary:

The eye irritation 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 (2017). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.00. Thus, the test substance is not considered to be 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 using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with the test substance for 60 min the tissue viability was 102.9% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is not considered to be irritating to the skin.

Eye

The eye irritation 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 (2017). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.00. Thus, the test substance is not considered to be irritating to the eye.

Justification for classification or non-classification

The available data on skin and eye irritation are conclusive but not sufficient for classification.

The data do not meet the criteria for classification according to Regulation (EC) 1272/2008.