Registration Dossier

Administrative data

Description of key information

Application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation. The test substance was classified as non-irritant to rabbit skin. Additionnaly, two in vitro test were performed (irritation and corrosion tests according to OECD TG 431 and 439), none of these test showed irritant properties of the test substance. Hence, according to CLP criteria, the test substance was not classified for skin irritation.

N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate was found to be irritating when instilled undiluted (without eye rinse) to rabbit’s eye. Indeed, according to the results of the three key studies, the test item was classififed as Category 2A for Eye irritation according to CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 20, 1979 to Feb. 23, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, comparable to scientific standards/principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was performed to determine the skin irritation potential of test substance on the rabbit skin. The aqueous slurry of test substance was applied to the shaved skin and the irritation potential was determined.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
EXPERIMENT INITIATION DATE: Feb. 20, 1979

EXPERIMENT COMPLETION DATE: Feb 23, 1979
No details on test animals and environmental conditions were provided in the study report.
Type of coverage:
not specified
Preparation of test site:
other: shaved intact and abraded skin
Vehicle:
other: aqueous slurry of test substance
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount applied : 500 mg (aqueous slurry)
- Concentration: Undiluted
Duration of treatment / exposure:
24 hours
Observation period:
24 and 72 h after treatment
Number of animals:
Shaved intact skin: 6 animals (4 female and 2 male)
Shaved abraded skin: 6 animals (4 female and 2 male)
Details on study design:
TEST SITE: The test site was shaved. No other information on test site was provided in the study report.

REMOVAL OF TEST SUBSTANCE: No details on the removal of test substance are provided in the study report.

SCORING SYSTEM: The irritation index used to classify the test substance was as follows:
0-2………………………………Mild irritant
2.1-4.9………………………..Moderate irritant
5-8……………………………… Severe (primary skin irritation) irritant
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24 and 72 h
Score:
0
Reversibility:
other: No erythema observed
Remarks on result:
other: On the both shaved intact and abraded skin
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24 and 72 h
Score:
0
Reversibility:
other: No edema observed
Remarks on result:
other: On the both shaved intact and abraded skin
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritant / corrosive response data:
- No signs of irritation were observed in any of treated animals (shaved intact and abraded skin) during the study period.
Interpretation of results:
not irritating
Remarks:
expert judgment
Conclusions:
Application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation. The test substance was classified as non-irritant to rabbit skin.
Executive summary:

The purpose of this in-vivo skin irritation test was to evaluate skin irritation potential of N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate when applied to rabbit’s shaved intact and abraded skin.

6 rabbits (4 female and 2 male) were used in each treatment group (shaved intact and abraded skin). 500 mg of test substance was applied to the test site of each rabbit.

The treated skin site was observed for edema and erythema for 24 h and 72 h after treatment.

No signs of irritation were observed in any of the treated animals (shaved intact and abraded skin) during the study period.

Based on above, application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation. The test substance was classified as a non-irritant to rabbit skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 February 2017 to
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:
The test item was found to have the potential to cause color interference with the MTT endpoint, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.
This deviation was considered to have not affected the integrity or validity of the study.
Deviations:
yes
Remarks:
See "Version / remarks" section above
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch No . 7215609434
- Expiration date of the lot/batch: 01 February 2018
- Purity test date: 15 December 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable, the test item was used as supplied
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) The test item was used as supplied
Test system:
human skin model
Source species:
human
Cell type:
other: three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes (highly differentiated and stratified epidermis model )
Cell source:
other: not applicable
Source strain:
other: not applicable
Justification for test system used:
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 17-EKIN-021
- Production date: not detailed
- Shipping date: not detailed
- Delivery date: 23 May 2017
- Date of initiation of testing: 22 May 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No modification of the SOP

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: the Labtech LT 4500 microplate reader
- Wavelength: 570nm
- Filter: no filter was used
- Filter bandwidth: no filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Historical data was not mentionned in the report. However, the test system was considered conform by the supplier

NUMBER OF REPLICATE TISSUES: triplicates were used per condition

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified in the study, no water killed tissue was used.
- N. of replicates : triplicates
- Method of calculation used: Relative mean viability (%)= [(MeanOD570 test item) / (MeanOD570 control)] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 independant test sequences were performed : pre test (assessment for direct MTT interference and color interference) and main test (with test substance, including MTT assay)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post-exposure incubation is equal or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post-exposure incubation is greater than 50% .
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: no different cut off point
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10mg (26.3 mg/cm2)
- Concentration (if solution): not applicable

VEHICLE
No vehicle was sued, however,5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates were used per condition
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - negative control
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - positive control
Value:
4.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - test substance
Value:
72.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: no
- Colour interference with MTT: The solution containing the test item was a pale pink color therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Positive control condition showed proficiency for test system to measure decrease of cellular viability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.802 and the standard deviation value of the viability was 10.2%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 4.6%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: not specified

Table 1: Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD570of tissues

Mean OD570 of triplicate tissues

± SD of OD570

Relative  individual tissue viability(%)

Relative mean viability(%)

± SD of Relative mean viability (%)

Negative Control Item

0.721

0.802

0.082

89.9

100*

10.2

0.800

99.8

0.885

110.3

Positive Control Item

0.033

0.039

0.007

4.1

4.9

0.8

0.039

4.9

0.046

5.7

Test Item

0.623

0.581

0.037

77.7

72.4

4.6

0.555

69.2

0.566

70.6

 


OD= Optical Density

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of thestudy, the test substance N,N-Bis(2-Hydroxyethyl)-P-Phenylenediamine Sulfate (A050) did not induced irritant effect on the EpiSkinTM model. Hence, according to CLP criteria, the test substance was not classified as skin irritant.
Executive summary:

The purpose of this GLP-compliant in vitro test was to evaluate the skin irritation potential of the test item N,N-Bis(2-Hydroxyethyl)-P-Phenylenediamine Sulfate (A050) using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours according to OECD TG 439 method.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.  The test item was found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours.  At the end of the post exposure incubation period each tissue was taken for MTT assay in order to determine the cellular viability.  

The relative mean viability of the test item treated tissues was 72.4% after the 15 Minute exposure period and 42 Hours post exposure incubation period.

Under the experimental conditions of thestudy, the test substance N,N-Bis(2-Hydroxyethyl)-P-Phenylenediamine Sulfate (A050) did not induced irritant effect on the EpiSkinTM model. Hence, according to CLP criteria, the test substance was not classified as skin irritant.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 February 2017 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, batch No . 7215609434
- Expiration date of the lot/batch: 01 February 2018
- Purity test date: 15 December 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature in the dark
- Stability under test conditions:
not applicable
- Solubility and stability of the test substance in the solvent/vehicle:
not applicable, no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
not applicable, the test item was used as supplied
- Preliminary purification step (if any):
not applicable
- Final dilution of a dissolved solid, stock liquid or gel:
not applicable
- Final preparation of a solid:
not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was used as supplied
Test system:
human skin model
Source species:
human
Cell type:
other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium
Cell source:
other: not applicable
Source strain:
other: not applicable
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Tissue batch number(s): 25806
- Production date: not detailed
- Shipping date: not detailed
- Delivery date: 19 April 2017
- Date of initiation of testing: 20 April 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No modification

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570nm
- Filter: not filter was used
- Filter bandwidth: no filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No historical data was mentionned in the report

NUMBER OF REPLICATE TISSUES: duplicates were used per condition

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : not specified
- Procedure used to prepare the killed tissues (if applicable): not specified
- N. of replicates : duplcates
duplicates
- Method of calculation used: % Relative mean viability : [(Mean OD570 of test item) / (Mean OD570 of negative conrol)] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
2 independant tests : Assessment of direct test item reduction of MTT and Color Interference ; Main test including MTT assessment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: no different cut-off points
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): used as supplied

VEHICLE
No vehicle was used but 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution):pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution):8.0N
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
No post treatement incubation was performed but a pre incubation of 1 hour was performed.
Number of replicates:
duplicates were used
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - negative control - 3 and 60 minutes
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - positive control - 3 minutes
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - positive control - 60 minutes
Value:
4.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - test item - 3 minutes
Value:
99.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - test item - 60 minutes
Value:
77.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction:The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT
- Colour interference with MTT: The solution containing the test item was a light pink color, therefore additional color correction tissues were incorporated into the testing procedure. The results of these tissues were used to correct the mean OD570 values of the test item treated tissues

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control showed proficiency for the test system to determine cellular viability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.580 for the 3 Minute exposure period and 1.404 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: not specified

Table1 :Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

Mean OD570of individual tissues (tvt)

Corrected OD570(tvt- (cct–ccu))

Mean OD570of duplicate tissues

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.469

 

1.580

0.156

9.9

100*

1.690

 

60 Minutes

1.480

 

1.404

0.107

7.7

1.328

 

Positive Control

3 Minutes

0.055

 

0.063

0.011

N/A

 

4.0

0.071

 

60 Minutes

0.065

 

0.069

0.006

N/A

4.9

0.073

 

Test Item

3 Minutes

1.721

1.688

1.569

0.168

10.7

99.3

1.483

1.450

60 Minutes

1.160

1.127

1.088

0.056

5.1

77.5

1.081

1.048

 

3 minute exposure corrected mean OD570= 0.040  (Cct) – 0.007 (Ccu) = 0.033

60 minute exposure corrected mean OD570=           0.044  (Cct) – 0.011 (Ccu) = 0.033

Tvt = Treated viable tissue

Cct = Treatedcolourcorrection tissue

Ccu = Untreatedcolourcorrection tissue

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of thestudy, the test substance N,N-Bis(2-Hydroxyethyl)-P-Phenylenediamine Sulfate (A050) did not induced corrosive effect on the EpiDermTM model. Hence, according to CLP criteria, the test substance was not considered as skin corrosive substance.
Executive summary:

The purpose of this GLP compliant in vitro test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model according to OECD TG 431 method.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.  The test item was found to have the potential to cause color interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT assay to determine potential skin corrosive properties.

The test item when applied on EpiDermTM skin model induced relative cellular viability values at 99.3% and 77.5% respectively for 3 and 60 minutes.

Under the experimental conditions of thestudy, the test substance N,N-Bis(2-Hydroxyethyl)-P-Phenylenediamine Sulfate (A050) did not induced corrosive effect on the EpiDermTM model. Hence, according to CLP criteria, the test substance was not considered as skin corrosive substance.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 13, 1979 to Feb. 20, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, comparable to scientific standards/principles
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was performed to determine the eye irritation potential of test substance in the rabbit eye. The test substance was applied undiluted to one eye. The eyes were evaluated for signs of toxicity/irritation.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
EXPERIMENT INITIATION DATE: Feb. 13, 1979
EXPERIMENT COMPLETION DATE: Feb. 20, 1979
No details on test animals and environmental conditions were provided in the study report.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied : 100 mg
Duration of treatment / exposure:
Once (unwashed)
Observation period (in vivo):
Day 1, 2, 3 and 4 following treatment
Number of animals or in vitro replicates:
6 animals (2 males and 4 females)
Details on study design:
REMOVAL OF TEST SUBSTANCE: Eyes were not washed after treatment.

SCORING SYSTEM: The parameters observed for scoring were density, area of corneal opacity, iris irritation, conjuctivae redness, lid swelling and discharge of treated eye. The details on the scoring system were not provided in the study report.

CLASSIFICATION OF TEST SUBSTANCE: The criteria used for classification of test substance was as follows:
Non-irritant: 0 or 1 rabbit with positive score during study
Irritant: 4 or more rabbits with positive score
Questionable: 2 or 3 rabbits with positive score. Repeat test indicated.

TOOL USED TO ASSESS SCORE: Not reported
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: Day 4-1
Score:
0.16 - 1.34
Reversibility:
not fully reversible within: 4 d
Remarks on result:
other: 100 mg of test substance, Unwashed
Irritation parameter:
iris score
Basis:
mean
Time point:
other: Day 4-1
Score:
0.34 - 1.167
Reversibility:
not fully reversible within: 4 d
Remarks on result:
other: 100 mg of test substance, Unwashed
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: Day 4-1
Score:
1 - 2
Reversibility:
not fully reversible within: 4 d
Remarks on result:
other: 100 mg of test substance, Unwashed
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.6
Max. score:
4
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3.6
Max. score:
4
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
4
Max. score:
4
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal #5
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal #5
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #5
Time point:
24/48/72 h
Score:
3.6
Max. score:
4
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
animal #6
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #6
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal #6
Time point:
24/48/72 h
Score:
3.3
Max. score:
4
Reversibility:
not specified
Irritant / corrosive response data:
- Treatment related symptoms of corneal opacity, iris irritation, conjuctivae redness, lid swelling and discharge were observed in all animals during the study.
The details on result are provided in the ‘Table 1’ in the section of ‘Any other information on results incl. tables’.

Table 1: Eye irritation observations (Study # 6A050)

Animal

Parameters observed

Days after instillation

1

2

3

4

1

Density

2

2

2

1

Area of corneal opacity

3

2

2

1

Iris irritation

2

2

2

1

Conjuctivae redness

2

2

2

1

Lid swelling

4

4

3

2

Discharge

3

3

3

2

2

Density

2

1

1

0

Area of corneal opacity

1

1

1

0

Iris irritation

1

1

1

1

Conjuctivae redness

2

2

2

1

Lid swelling

4

4

3

2

Discharge

3

3

3

2

3

Density

1

1

1

0

Area of corneal opacity

1

1

1

0

Iris irritation

1

1

1

0

Conjuctivae redness

2

2

2

1

Lid swelling

4

4

4

2

Discharge

3

3

2

1

4

Density

1

1

1

0

Area of corneal opacity

1

1

1

0

Iris irritation

1

1

1

0

Conjuctivae redness

2

2

2

1

Lid swelling

3

3

3

2

Discharge

2

2

2

1

5

Density

1

1

1

0

Area of corneal opacity

1

1

1

0

Iris irritation

1

1

1

0

Conjuctivae redness

2

2

2

1

Lid swelling

4

4

3

2

Discharge

2

2

2

1

6

Density

1

0

0

0

Area of corneal opacity

1

0

0

0

Iris irritation

1

1

1

0

Conjuctivae redness

2

2

2

1

Lid swelling

4

3

3

1

Discharge

2

2

2

1

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Remarks:
expert judgment
Conclusions:
N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate was found to be irritating when instilled undiluted (without eye rinse) to rabbit’s eye. According to CLP criteria, the registered substance was classified as Category 1 for eye irritation.
Executive summary:

The purpose of this in-vivo eye irritation test was to evaluate eye irritation potential of N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate when applied once to rabbit eye.

Six rabbits (2 male and 4 female) were used in the study. The 100 mg test substance was applied to the left eye of each rabbit. The test substance was not removed after treatment.

The animals were observed for signs of corneal opacity, iris irritation, conjuctivae redness, lid swelling and discharge on Day 1-4 of study.

The signs of irritation were observed on Day 1-4 after treatment.

Based on above, N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate was found to be irritating when instilled undiluted (without eye rinse) to rabbit’s eye. According to CLP criteria, the registered substance was classified as Category 1 for eye irritation.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 November 2016 to 17 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by the sponsor, Batch No. 7213133638 (DRDU2011S103)
- Expiration date of the lot/batch: 16 September 2018
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No treatment, Approximately 50 mg of the test item (83.3 mg/cm² according to guideline) were tested topically on duplicate EpiOcular tissues.
Species:
human
Strain:
other: Reconstructed human Cornea-like Epithelium (RhCE)
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Reconstructed ocular tissue model containing normal human keratinocytes. All used cells were screened for potential contaminant. The samples were free of HIV-1, Hepatitis B and C virus, bacteria, yeast and other fungi.
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
Approximately 50 mg of the test item (83.3 mg/cm² according to guideline) were tested topically on duplicate EpiOcular™ tissues

VEHICLE
No vehicle was used
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
duplicates were used
Details on study design:
- Details of the test procedure used:
The test item proved to be an MTT reducer in the MTT pre-test, and it changed colour (reddish) in the presence of water in the colour interference pre-test. Therefore, additional tests with freeze-killed and with viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment.
Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.
After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

- RhCE tissue construct used, including batch number
EpiOcularTM (MatTek Corporation), Batch No. 23764

- Doses of test chemical and control substances used
Deionised water was used as negative control. 50 µL were applied on duplicate tissues (negative control).
Methyl acetate was used as positive control. 50 µL were applied to duplicate tissues (positive control)
Approximately 50 mg of the test item (83.3 mg/cm² according to guideline) were tested topically on duplicate (test item)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 37°C

- Description of any modifications to the test procedure: Not specified

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): 50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : duplicates were used

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- Description of the method used to quantify MTT formazan :
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions. Inserts were removed from the 24-well plate after 180 minutes; then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were and were extracted overnight at 2-8 °C in the dark and shaken for 2 hours at room temperature (main experiment) afterwards or immediately extracted (shaken for 2 hours at room temperature; additional viable and freeze-killed tissues), respectivelyThe extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s). The absorbance at 570 nm (OD570) of each well was measured with a plate reader

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria Yes, See attached table 1

- Complete supporting information for the specific RhCE tissue construct used : Yes
- Reference to historical data of the RhCE tissue construct : Yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not specified
- Positive and negative control means and acceptance ranges based on historical data: yes
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes
Irritation parameter:
other: Viability (Percent)
Run / experiment:
Negative Control
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (Percent)
Run / experiment:
Positive Control
Value:
17.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (Percent)
Run / experiment:
Test Item
Value:
6.3
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed blue colour. Therefore, an additional test with freeze-killed tissues was necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 6.3% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye. Since the test item proved to be clearly eye irritant, correction of the viability using the determined correction factors derived from the additional tests was not necessary.

Results after treatment for 6 hours withN,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050)and the controls

Dose Group

Ab-sorbance
Well 1
(Tissue 1/2)

Ab-sorbance
Well 2 (Tissue 1/2)

MeanAbsor-bance(Tissue 1/2)

Mean Absorbance Tissue 1 and 2 minus Mean Blank

Mean Absorbance of
2 Wells

Rel. Absorbance [%]
Tissue 1 and 2*

Absolute Value of the Difference of the Rel.Absorbances[%]
Tissue 1 and 2

Mean Rel. Absorbance

[% of Negative Control]*

 

0.038

0.038

0.038

 

 

Negative Control

1.742

1.751

1.747

1.709

1.703

100.3

0.7

100.0

1.767

1.703

1.735

1.697

99.7

Positive Control

0.340

0.363

0.351

0.313

0.303

18.4

1.2

17.8

0.332

0.331

0.332

0.293

17.2

Test Item

0.152

0.151

0.152

0.113

0.108

6.7

0.6

6.3

0.141

0.141

0.141

0.102

6.0

Blank

0.038

0.038

0.038

0.000

 

Negative Control
Add. Viable Tissue

0.041

0.040

0.040

0.003

0.003

0.1

0.0

0.2

0.041

0.041

0.041

0.003

0.2

Test Item
Add. Viable Tissue

0.042

0.043

0.042

0.005

0.006

0.3

0.2

0.4

0.045

0.046

0.046

0.008

0.5

Negative Control
Add. Freeze-Killed Tissue

0.093

0.092

0.093

0.055

0.056

3.2

0.2

3.3

0.095

0.096

0.096

0.058

3.4

Test Item
Add. Freeze-Killed Tissue

0.122

0.132

0.127

0.089

0.097

5.2

0.9

5.7

0.142

0.142

0.142

0.104

6.1

*         Relative absorbance [rounded values]: (100x(Absorbance test item/positive control))/Abosrbancenegative control

 

Interpretation of results:
other: eye irritating potential
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050) possesses an eye irritating potential.
Executive summary:

This in vitro GLP-compliant study was performed to assess the eye irritation potential of N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050) by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test, and it changed colour (reddish) in the presence of water in the colour interference pre-test. Therefore, additional tests with freeze-killed and with viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment. Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

Irritating effects were observed following incubation with N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased to 6.3% (threshold for irritancy: < 60%). Since the test item was classified as irritant in the main experiment due to a viability being lower than the cut-off of 60%, it was not necessary to correct the viability value in the main experiment with the determined correction factor.

In conclusion, it can be stated that in this study and under the experimental conditions reported, N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050) possesses an eye irritating potential.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 August 2016 to 29 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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 : 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by HFC Prestige Service, Batch No. DRDU20110103
- Expiration date of the lot/batch: 16 September 2018
- Purity test date: 2 May 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at ambient temperature (15-25 deg celsius)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: the test item was used pure
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used neat
- Final dilution of a dissolved solid, stock liquid or gel: no dilution was used
- Final preparation of a solid:not specified
Species:
chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bor poultry slaughterhouse (Netherlands)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 1.5 - 2.5 kg

ENVIRONMENTAL CONDITIONS
The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30mg of test item ; 30 µL of positive control
- Concentration (if solution): the positive control was used neat (ground)

VEHICLE
The test item was used pure

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit):30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): pure
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
0, 30, 75, 120, 240 minutes
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
Number of animals or in vitro replicates
1 for Negative Control
3 for Positive Control
3 for Test group
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using t
he following procedure: First the eye lids were carefully removed without damaging the cornea and a
small drop of Fluorescein sodium 2.0% w/v (Minims, England) was applied to the corneal surface for
a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the h
ead with the fluorescein treated cornea was examined with a slit lamp microscope to ensure that the
cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤
0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was take
n to remove the eye ball from the orbit without cutting off the optical nerve too short. The enucleated
eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a
chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that
the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate
of 0.10 0.15 mL/min (peristaltic pump set at speed 5.00). The chambers of the superfusion apparat
us as well as the saline were temperature controlled at approximately 32 deg Celsius (water pump se
t at 36.4 deg Celsius). After placing in the superfusion apparatus, the eyes were examined again wi
th the slit lamp microscope to ensure that they were not damaged. Corneal thickness was measured
using the Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope, set at 0.095
mm. Corneal thickness was expressed in instrument units. An accurate measurement was taken at
the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average
corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably
stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that
showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein re
tention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the
eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
1 for Negative Control ; 3 for Positive Control ; 3 for Test group

NEGATIVE CONTROL USED
Physiological Saline NaCl

POSITIVE CONTROL USED
Benzalkonium Chloride (BAC)

APPLICATION DOSE AND EXPOSURE TIME
30 μL of negative control or 30 mg of postive control and test item were applied for 10 seconds

OBSERVATION PERIOD
0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eye was rinsed 10 seconds after treatment with test item or control. They were rinsed with 20 mL saline solution.
- Indicate any deviation from test procedure in the Guideline : no deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope
- Damage to epithelium based on fluorescein retention: Slit lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: No
- Others (e.g, histopathology): Histopathological examination

SCORING SYSTEM:
Corneal swelling, expressed as a percentage, is calculated according to the following formula:
“Corneal thickness at time t minus corneal thickness at time t = 0, divided by corneal thickness at time
t = 0 and multiplied by 100”.
The mean percentage of swelling for the three test eyes will be calcula¬ted for each of the observa
tion time points of 30, 75, 120, 180, and 240 minutes. The maximum mean percentage (can be at any
of the time points) will be used for classification into one of four categories

- Mean maximum opacity score
Opacity degree of density (area most dense taken for scoring)
0 = no opacity
0.5 = very faint opacity (= very slight)
1 = scattered or diffuse areas, details of iris clearly visible (= slight)
2 = easily discernible translucent area, details of iris slightly obscured (= moderate)
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely discernible (= severe)
4 = complete corneal opacity, iris invisible (= very severe)
The mean corneal opacity value for all test eyes is calculated for the observation time points of 30, 75
, 120, 180, and 240 minutes. The maximum mean opacity score (can be at any of the time points) will
be used for classification into one of four categories

- Mean fluorescein retention score at 30 minutes post-treatment
0 = no fluorescein retention
0.5 = very minor single cell staining (= very slight)
1 = single cell staining scattered throughout the treated area of the cornea (= slight)
2 = focal or confluent dense single cell staining (= moderate)
3 = confluent large areas of the cornea retaining fluorescein (= severe)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

On the basis of the severity of the observed findings for corneal swelling, corneal opacity and fluoresc
ein retention, the effects are divided into four categories, viz. I = no effect; II = slight effect; III = m
oderate effect; IV = severe effect.
Interpretation of corneal swelling, corneal opacity, and fluorescein retention and categorisation into
the four categories is done according the following methodology:
Corneal swelling:
Mean corneal swelling (%) Category
0 5 I
>5 12 II
>12 - 18 (>75 min. after treatment) II
(≤75 min. after treatment) III
>18 26 III
>26 - 32 (>75 min. after treatment) III
(≤75 min. after treatment) IV
>32 IV
Corneal opacity:
Max. mean opacity score Category
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 4.0 IV
Fluorescein retention:
mean fluorescein retention score Category
at 30 min after treatment:
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 3.0 IV
Irritation parameter:
percent corneal swelling
Run / experiment:
Test item
Value:
10
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Test item
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Test item
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
90
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: NaOH was used as positive control and provided informations about suitability of the test system

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050; GTS119107) caused slight corneal swelling (mean swelling 10%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0).
Microscopic examination of the corneas treated with N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050; GTS119107) revealed very slight (3/3 corneas) erosion and very slight (2/3 corneas; top region) vacuolation of the epithelium.

Table 1 - Summary results of the slit-lamp examination

Test material

 

Maximum mean score for:

 

 

Irritation

catego­ries1

Irritation

Index2

Classifi­ca­tions

(EU-CLP3/UN-GHS4)

 

Swelling %

Opaci­ty

Fluores­cein

retention

N,N-Bis(2-Hydroxyethyl)-p-PhenylenediamineSulfate (A050; GTS119107)

10

2.0

2.0

II;III;III

90

2/2A

NaOH(positive control)

45

4.05

3.0

IV;IV;IV

185

1/1

1 I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2 Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonized System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5 Immediate iris constriction and severe loosening of epithelium

 

Table 2 - Individual histopathological findings

 

 

Test Material

 

Eye no.

 

Epithelium

Notes

Stroma

Endothelium

Erosion

Necrosis

Vacuolation

 

 

Disorder of fibers

 

 

Pyknoticnuclei

Necrosis

top

mid

low

outer region (adjacent to epithelium)

inner region (adjacent to endothelium)

N,N-Bis(2-Hydroxyethyl)-p-PhenylenediamineSulfate (A050; GTS119107)

4

½

-

-

-

-

 

-

-

-

-

5

½

-

½

-

-

 

-

-

-

-

6

½

-

½

-

-

 

-

-

-

-

NaOH

(positive control)

13

3

3

-

-

-

-

-

-

P

14

3

3

-

-

-

-

-

-

P

15

3

3

-

-

-

-

-

-

P

Saline

(negative control)

16

-

-

-

-

-

 

-

-

-

-

 

- = not observed; P = present; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe;

= scored in the top/mid/low section of the epithelium; ‡ = stromal necrosis



 

Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Conclusions:
Under experimental conditions of the study, the registered substance N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate caused slight corneal swelling (mean swelling 10%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas revealed very slight (3/3 corneas) erosion and very slight (2/3 corneas; top region) vacuolation of the epithelium. According to the CLP regulation and OECD, the ICE test cannot be used for classify Eye irritation Category 2 due to false negative rate of the test (including alcohol).
However, microscopic observation was performed on histopathologic slide of the chicken eye. Hence, by caution, the substance was considered as irritant for the eyes due to erosion, vacuolation of the epithelium in this test, and classified as Category 2.
Executive summary:

This GLP-compliant study was performed to assess the potential irritation/corrosion property of the registered substance N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate in a Isolated Chicken Eye test (ICE test) according to OECD guideline 438 method.

N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050; GTS119107) was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

Under experimental conditions of the study, the registered substance N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate caused slight corneal swelling (mean swelling 10%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas revealed very slight (3/3 corneas) erosion and very slight (2/3 corneas; top region) vacuolation of the epithelium. According to the CLP regulation and OECD, the ICE test cannot be used for classify Eye irritation Category 2 due to false negative rate of the test (including alcohol). However, microscopic observation was performed on histopathologic slide of the chicken eye. Hence, by caution, the substance was considered as irritant for thee eyes due to erosion, vacuolation of the epithelium in this test, and classified as Category 2.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

According to the results of the availables key studies, the test item, when applied in vivo in rabbit showed irritant property. When tested in in vitro assays (ICE assay : Prinsen, 2005, Klimisch 1, GLP, OECD 439) (HCM Test : Duschl, 2007, GLP, Klimisch 1, OECD 492), the test item showed occular irritation. When tested on human cornea model, an irritant potential was shown, however not classification can be made. When tested in ICE Assay, the test item showed irritancy but not sufficient to be considered as Category 1 as Corrosive. With histopathological analysis, the test item showed erosion, vacuolation of the epithelium in this test system. Additional in vivo study showed eye irritation potential of the test substance. Hence, the test item was classified as Category 2A for Eye Irritation by caution.

Key study summaries :

-Loehr, Klimisch 2, 1979, comparable to OECD 405 :

Six rabbits (2 male and 4 female) were used in the study. The 100 mg test substance was applied to the left eye of each rabbit. The test substance was not removed after treatment. The animals were observed for signs of corneal opacity, iris irritation, conjuctivae redness, lid swelling and discharge on Day 1-4 of study. The signs of irritation were observed on Day 1-4 after treatment. Based on above, N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate was found to be irritating when instilled undiluted (without eye rinse) to rabbit’s eye.

-Prinsen, 2016, Klimisch 1, OECD 438, GLP :

N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed. Under experimental conditions of the study, the registered substance N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate caused slight corneal swelling (mean swelling 10%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). Microscopic examination of the corneas revealed very slight (3/3 corneas) erosion and very slight (2/3 corneas; top region) vacuolation of the epithelium. According to the CLP regulation and OECD, the ICE test cannot be used for classify Eye irritation Category 2 due to false negative rate of the test (including alcohol). However, microscopic observation was performed on histopathologic slide of the chicken eye. Hence, by caution, the substance was considered as irritant for thee eyes due to erosion, vacuolation of the epithelium in this test, and classified as Category 2.

-Duschl 2017, GLP, Klimisch 1; OECD 492:

A HCM Test was performed according to OECD 492 Guideline. Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each. Irritating effects were observed following incubation with N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased to 6.3% (threshold for irritancy: < 60%). Since the test item was classified as irritant in the main experiment due to a viability being lower than the cut-off of 60%. In conclusion, it can be stated that in this study and under the experimental conditions reported, N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate (A050) possesses an eye irritating potential.

Application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation. The test substance was classified as non-irritant to rabbit skin. Additionnaly, two in vitro test were performed (irritation and corrosion tests according to OECD TG 431 and 439), none of these test showed irritant properties of the test substance.

-An in vitro skin corrosion test was performed and was quoted as Klimisch 1 (Warren, 2018, GLP):This study was performed according to OECD 431 TG method. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.  The test item was found to have the potential to cause color interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT assay to determine potential skin corrosive properties. The test item when applied on EpiDermTM skin model induced relative cellular viability values at 99.3% and 77.5% respectively for 3 and 60 minutes.

-An in vitro skin irritation test was performed and was quoted as Klimisch 1 (Warren, 2018, GLP) : Triplicate tissues were treated with the test item for an exposure period of 15 minutes.  The test item was found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours.  At the end of the post exposure incubation period each tissue was taken for MTT assay in order to determine the cellular viability. The relative mean viability of the test item treated tissues was 72.4% after the 15 Minute exposure period and 42 Hours post exposure incubation period.

-An in vivo skin irritation was performed (D'Aleo C, 1979, Klimisch 2): 6 rabbits (4 female and 2 male) were used in each treatment group (shaved intact and abraded skin). 500 mg of test substance was applied to the test site of each rabbit. The treated skin site was observed for edema and erythema for 24 h and 72 h after treatment. No signs of irritation were observed in any of the treated animals (shaved intact and abraded skin) during the study period.Based on above, application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation.

Justification for classification or non-classification

According to CLP ( 1272/2008/EC), a classification as Eye Irritation category 2A. should be considered according to the results of the ICE in vitro test and the in vivo test performed with the 100% registered substance. No effect had been observed on the rabbit skin in in vivo study, hence no classification was applied.

Application of undiluted N,N-Bis(2-Hydroxyethyl)-p-Phenylenediamine Sulfate to rabbit’s shaved intact and abraded skin did not produce skin irritation. The test substance was classified as non-irritant to rabbit skin. Additionnaly, two in vitro test were performed (irritation and corrosion tests according to OECD TG 431 and 439), none of these test showed irritant properties of the test substance. Hence, according to CLP criteria, the test substance was not classified for skin irritation.