Registration Dossier

Administrative data

Description of key information

RHE test in vitro, OECD 439: no skin irritation (reference 7.3.1 -1)

BCOP assay in vitro, OECD 437: no conclusion can be made (reference 7.3.2.-1)

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:
2014-10-02 to 2015-01-29
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)
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)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended system by the guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis (RHE Kit)
- Tissue batch number: 14-RHE-024

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing with minimum of 25 mL
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours +/- 5 min

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: well
- Barrier function: checked
- Morphology: well
- Contamination: none
- Reproducibility: well

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 42 minutes exposure is less or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 42 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 µL

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
- Concentration: 5 % aqueous solution
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
63.77
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: not applicable
- Colour interference with MTT: not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1 Results obtained after in RHE assay

Dose
group

Treatment
Interval

Optical
density
Tissue 1

Optical
density
Tissue 2

Optical
density
Tissue 3

Mean

optical
density

Mean relative
viability in %

Standard

deviation (SD)

in %

Negative
Control

42 min

2.445

2.588

2.510

2.515

100.00

2.85

Positive
Control

42 min

0.022

0.021

0.021

0.021

0.83

2.77

Test item

42 min

1.571

1.634

1.606

1.604

63.77

1.97

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the test item is considered to be not irritating to human skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test according to OECD TG 438. The test consisted of a topical administration of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer), positive control (5% aqueous solution of sodium dodecyl sulfate) or test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.515. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 0.83%. Therefore, the study fulfilled the validity criteria.

The tissue viability after treatment with the test item was 63.77 % and, thus, higher than 50 %, i.e. according to UN GHS classification the test item is considered as non-irritant to skin.

In conclusion, under the conditions of this study, the test item is not considered to possess an irritant potential to 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:
2014-10-09 to 2015-01-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals:
age: 18-30 months
corneal diameter: 24-26 mm
- Storage, temperature and transport conditions of ocular tissue:
Freshly isolated bovine eyes of donor cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice.
Transport medium: HBSS (Hanks' Balanced Salt Solution)

- Time interval prior to initiating testing:
The corneas were prepared immediately after delivery of the eyes to the laboratory.

- 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 or scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 mL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were prepared immediately after delivery of the eyes to the laboratory. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (prewarmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, 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 form.

QUALITY CHECK OF THE ISOLATED CORNEAS :
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES : 3


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 washing steps

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Corneal permeability: passage of sodium fluorescein dye

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
12.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1 Summary of results of BCOP assay

 

 

 

 

IVIS

 

 

Opacity

Permeability

per cornea

per group
(mean value)

Standard
deviation

Negative
control

 

 

0.9% sodium
chloride solution

3.702

-0.002

3.667

28

18

 

4.050

-0.002

4.020

 

0.629

0.002

0.659

Positive
control

 

N,N-

Dimethylformamide

86.937

0.821

99.254

1050

5.8

 

84.609

1.339

104.696

 

84.439

1.766

110.925

Test Item

 

12.089

-0.001

12.037

12.2

2.8

9.391

0.008

9.508

15.008

0.007

15.119

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of this study alone no conclusion can be made in regards to eye irritation/corrosion properties of the test item.
Executive summary:

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay). Therefore, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used. Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 2.8. Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 105.0. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 12.2 and, thus, according to OECD 437 no prediction can be made regarding the eye hazard potential.

In conclusion, under the conditions of the present study, no prediction can be made for the test item with regard to the eye hazard potential.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation in vitro, reference 7.3.1-1

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test according to OECD TG 439. The test consisted of a topical administration of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer), positive control (5% aqueous solution of sodium dodecyl sulfate) or test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.515. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 0.83%. Therefore, the study fulfilled the validity criteria. The tissue viability after treatment with the test item was 63.77 % and, thus, higher than 50 %, i.e. according to UN GHS classification the test item is considered as non-irritant to skin.

In conclusion, under the conditions of this study, the test item is not considered to possess an irritant potential to skin.

Eye irritation in vitro, reference 7.3.2 -1

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay), according to OECD 437. Therefore, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used. Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 2.8. Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 105.0. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 12.2 and, thus, according to OECD 437 no prediction can be made regarding the eye hazard potential.

In conclusion, under the conditions of the present study, no prediction can be made for the test item with regard to the eye hazard potential.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item does not require classification as skin irritanting according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.

Regarding eye irritation, no final conclusion can be made based on the available data.