Registration Dossier

Administrative data

Description of key information

Skin irritation

The dermal irritation potential of target chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1) was assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical.Based on the available key data and supporting studies,it can be concluded that the testchemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye irritation

The ocular irritation potential of target chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1) was assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical.Based on the available key data and supporting studies,it can be concluded thatchemical is unable to cause eye irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

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:
11-MAY-2004 to 19-AUG-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: Directive 92/69 EEC, B.4. "Acute Toxicity - Skin Irritation", July 31, 1992.
Principles of method if other than guideline:
The purpose of this primary skin irritation study was to assess the possible irritation potential when a single dose of C 010 was placed on the skin of rabbits for approximately four hours.
The test item was administered at 0.5 g/animal, the dose specified in the test guidelines for a solid test item.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride)
- Lot/batch No.of test material: 15
- Expiration date of the lot/batch: 30-DEC-2023
- Purity: 99.3% HPLC

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity:N/A
- Specific activity:N/A
- Locations of the label:N/A
- Expiration date of radiochemical substance:N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator (range of 5 ± 3°C), light protected.
- Stability under test conditions: Stable under storage conditions.
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 0.5 g (per animal) of C 010 was weighed as delivered by the Sponsor and then moistened with approximately 0.1 mL of purified water before application.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material) : No data

OTHER SPECIFICS:
Safety precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.

TEST ITEM PREPARATION
The pH of the test item was measured before the study initiation date. A formulation of a 1 % (w/w) solution was prepared. The pH was found to be 6.53.
Species:
rabbit
Strain:
New Zealand White
Remarks:
SPF
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Scientifique des Dombes F-01400 Chatillon sur Chalaronne / France
- Age at study initiation: 12 weeks (male), 12 weeks (females)
- Weight at study initiation: Given below
- Identification: By unique cage number and corresponding ear number.
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd,Füllinsdorf) and haysticks 4642 (batch no. 80/03, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no.24/04) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: 5 days. Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
- Allocation: Male No. 82 , Female Nos. 83 and 84

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70 %
- Air changes (per hr): Approximately 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The animals were provided with an automatically controlled light cycie of 12 hours light and 12 hours dark. Music was played during the daytime light period.

IN-LIFE DATES: From: 11-MA Y -2004 To: 19-AUG-2004
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.1 mL of purified water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution):No data

POSITIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution): No data
Duration of treatment / exposure:
4 hours
Observation period:
1, 24, 48 and 72 hours, as well as 7 and 10 days.
Number of animals:
3 (Animals of both sexes were used)
Details on study design:
TEST SITE
- Area of exposure: 100 cm2 (10 cm x 10 cm).
- % coverage: No data
- Type of wrap if used: The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The skin was flushed with lukewarm tap water to clean the application site.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : 1, 24, 48 and 72 hours, as well as 7 and 10 days.

SCORING SYSTEM:
- Method of calculation: The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92169/EEC, July 31, 1992 .
Irritation parameter:
erythema score
Basis:
animal: 82,83,84
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: 82,83,84
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal: 82,83,84
Time point:
7 d
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: 82,83,84
Time point:
7 d
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The animals were not sacrificed. No signs of irritation were observed on the treated skin following the application of the test item.
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythemaJeschar grades and for oedema grades, separately.
The mean erythema/eschar score and the mean oedema score was 0.00 for all three animals.
No erythema and no swelling (oedema) was noted in any animal at any time.

CORROSION
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
Other effects:
- Other adverse local effects:
COLORATION
Slight yellow staining produced by the test item of the treated skin was observed in two animals at the 1-and 24-hour reading and persisted in one animal up to 7-days after
treatment.

BODY WEIGHTS
The body weights of all rabbits were considered to be within the normal range of variability.
Body weight in grams
Animal No. Sex First Day of Acclimatization Day of Treatment Last Day of Observation
82 male 2011 2109 2416
83 female 2113 2357 2754
84 female 2094 2247 2533


- Other adverse systemic effects:
VIABILITY/MORTALITY/CLINICAL SIGNS
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

Interpretation of results:
other: Not irritating
Conclusions:
Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) is considered to be "not irritating" to rabbit skin.
Executive summary:

The primary skin irritation potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was investigat ed according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as weil as 7 and 10 days after removal of the dressing.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.

The mean erythema/eschar score and the mean oedema score was 0.00 for all three animals.

The application of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) to the skin resulted in no signs of irritation. However slight yellow staining of the treated skin was observed in two animals at the 1- and 24-hour reading and persisted in one animal up to the 7-day examination. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the skin.

Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1 -phenyl-1H- pyrazol- 4-yl)azo] -N,N, N-trimethylanilinium chloride) is considered to be "not irritating" to rabbit skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
equivalent or similar to
Guideline:
other: Standard Operating Procedures (SOP)
Principles of method if other than guideline:
Skin irritation study was performed according to Standard Operating Procedures (SOP) in accordance with OECD guideline TG 439 on Reconstructed human tissue (RhT) using the EpiSkin™in vitro skin irritation test method.
GLP compliance:
not specified
Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
No data available
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
water
Controls:
not specified
Amount / concentration applied:
10 ± 2 mg (26.3 mg/cm2)
Duration of treatment / exposure:
15 – 60 mins
Observation period:
No data available
Number of animals:
3 tissues/run
Details on study design:
TEST SITE
- Area of exposure: Test chemical powder of conc. 26.3 mg/cm2 was applied to the epidermis surface.
- % coverage: No data available
- Type of wrap if used: No data available

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS).
- Time after start of exposure: No data available

SCORING SYSTEM: Result of the study was interpretated based on the cell viability-

If cell viability is ≤ 50% : the chemical was classified as irritant and

If cell viability is > 50% : the chemical was classified as non - irritant
Irritation / corrosion parameter:
other: overall irritation score
Remarks on result:
other: Basis:

Table 1 and 2: Assessment for both OD and HPLC/UPLC-spectrophotometry EpiSkinTMtissue viability quantification from formazan tissue extracts.

Table 1:

Chemical name

Tissue viability OD (%)

Corrected relative viability

Corrected

SD/*Diff.

Tissue 1

Tissue 2

Tissue 3

Final mean viability

(Uncorrected)

Basic yellow 57

111.6

124.7

109.8

115.4 (117.1)

8.14

 

Table 2:

Chemical name

Tissue viability HPLC/UPLC-spectrophotometry(%)

Corrected relative viability

Corrected

SD/*Diff.

Tissue 1

Tissue 2

Tissue 3

Final mean viability

(Uncorrected)

Basic yellow 57

111.7

122.0

106.1

113.3

8.07

 

Where,
*Diff. = Difference between 2 tissue replicates

Interpretation of results:
other: not irritating
Conclusions:
Based on the cell viability of reconstructed human tissue (RhT) after exposure to the test substance, the test chemical was evaluated to be non-irritant.
Executive summary:

Skin irritation study was performed on Reconstructed human tissue (RhT).

 

Standard Operating Procedures (SOP) in accordance with OECD guideline TG 439 was used for the study.

 

Both positive and negative controls were run in parallel with the test items.

 

The test chemical was tested in the EpiSkin™in vitroskin irritation test method in one run. Water was used as a vehicle for the study.

 

The test chemical conc. used for the study was10 ± 2 mg (26.3 mg/cm2) alongwith exposure duration of 15 – 60 mins.Test chemical powder of conc.26.3 mg/cm2was applied to the epidermis surface.Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS).

 

The evaluation of colour interference involved incubation of the test chemical with solvent or water depending on thein vitroRhT test method. If the solution absorbed at 570 nm or became coloured following incubation with the chemical, living tissue adapted controls incubated with medium instead of MTT to define non-specific colour (%NSCliving) were performed.

 

The run was considered as valid for chemicals and all adapted controls if the following criteria were met: difference of viability between the two replicates tissues was≤20% for skin corrosion or Standard Deviation (SD) of viability between 3 replicate tissues was≤18% for skin irritation in accordance with the respective test method SOPs.

 

The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing and also in parallel by HPLC/UPLC-spectrophotometry.

 

Result of the study was interpretated on the basis of cell viability-

 

If cell viability is ≤ 50%: the chemical was classified as irritant and

 

If cell viability is > 50% :the chemical was classified as non - irritant

 

Statistical analysis:Viability was summarized as mean ± SD or mean and the difference. Simple linear regression was used for assessing the agreement between the OD and HPLC/UPLC-spectrophotometry detection methods. The assumption of linearity was verified with a scatter plot of the standardized residuals versus the viability and the normality of the residuals wasverified with a QQ-plot. An alpha level of 0.05 was used as significancelevel. All analyses were performed in R version 3.2.0.

 

Based on the cell viability of reconstructed human tissue (RhT) after exposure to the test substance, the test chemical was evaluated to be non-irritant.

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:
03-JUN-2004 to 10-AUG-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental report
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: Directive 92/69 EEC, B.5. "Acute Toxicity - Eye Irritation", July 31, 1992.
Principles of method if other than guideline:
The purpose of this primary eye irritation study was to assess the possible irritation potential when a single dose of C 010 was placed in the conjunctival sac of rabbit eyes.
The test item was applied at 0.1 g/animal, the dose specified in the test guidelines for a solid test item.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride)
- Lot/batch No.of test material: 15
- Expiration date of the lot/batch: 30-DEC-2023
- Purity: 99.3% HPLC

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator (range of 5 ± 3°C), light protected.
- Stability under test conditions: Stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 0.1 g (per animal) of C 010 was weighed and applied undiluted as it was delivered by the Sponsor.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Safety precautions : Routine hygienic procedures were used to ensure the health and safety of the personnel.

TEST ITEM PREPARATION
The pH of a 1 % (w/w) solution of the test item was measured for a previous study (RCC Study number 853983, skin irritation with C 010 in rabbits) and was found to be 6.53.
Species:
rabbit
Strain:
New Zealand White
Remarks:
SPF
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Elevage Scientifique des Dombes
F-01400 Chatillon sur Chalaronne / France
- Age at study initiation: 15 - 16 weeks (male),15 - 16 weeks (females)
- Weight at study initiation: Given below
- Identification: By unique cage number and corresponding ear number.
- Allocation: Male No. 82,Female Nos. 83 and 84
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd,Füllinsdorf) and haysticks 4642 (batch no.80/03, Provimi Kliba AG) were provided for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad Iibitum (batch no.33/04) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water (e.g. ad libitum):Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: 5 days. Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70 %
- Air changes (per hr): approximately 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during
the daytime light period.

IN-LIFE DATES: From:03-JUN-2004 To:18-JUN-2004
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.1 g
- Concentration (if solution): No data

VEHICLE (No vehicle)
- Amount(s) applied (volume or weight with unit): No data
- Concentration (if solution): No data
- Lot/batch no. (if required): No data
- Purity: No data
Duration of treatment / exposure:
24 hours after treatment.
Observation period (in vivo):
1, 24, 48 and 72 hours, as well as 7 and 10 days after instillation.
Number of animals or in vitro replicates:
3 (Animals of both sexes were used)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated and untreated eyes were rinsed with lukewarm tap water.
- Time after start of exposure: 24 hours

SCORING SYSTEM: The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours, as well as 7 and 10 days after instillation.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-Iamp (Roth AG, CH-4153 Reinach/Switzerland) .
Irritation parameter:
cornea opacity score
Basis:
animal: 82,83,84
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 82,83,84
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 82,83
Time point:
24/48/72 h
Score:
1.67
Max. score:
3
Reversibility:
other: Effects were reversible and were no longer evident 10 days after treatment,the end of the observation period for all animals.
Remarks on result:
other: After 24 hours:moderately reddened; moderate swelling with partial eversion of lids. After 48 hours:moderately reddened; slight swelling. After 72 hours:slightly reddened; slight swelling
Irritation parameter:
conjunctivae score
Basis:
animal: 84
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
other: Effects were reversible and were no longer evident 10 days after treatment,the end of the observation period for all animals.
Remarks on result:
other: slightly reddened
Irritation parameter:
chemosis score
Basis:
animal: 82
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
other: Effects were reversible and were no longer evident 10 days after treatment,the end of the observation period for all animals.
Irritation parameter:
chemosis score
Basis:
animal: 83
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
other: Effects were reversible and were no longer evident 10 days after treatment,the end of the observation period for all animals.
Irritation parameter:
chemosis score
Basis:
animal: 84
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal: 82,83,84
Time point:
7 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 82,83,84
Time point:
7 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 82,83
Time point:
7 d
Score:
1
Max. score:
3
Reversibility:
other: Effects were reversible and were no longer evident 10 days after treatment,the end of the observation period for all animals.
Remarks on result:
other:
Remarks:
slightly reddened
Irritation parameter:
conjunctivae score
Basis:
animal: 84
Time point:
7 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: 82,83,84
Time point:
10 d
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
IRRITATION
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 1 .67, 1 .67 and 1.00 for reddening and 1.33,0.67 and 0.00 for chemosis, respectively.
No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals.
Slight to moderate reddening of the conjunctivae was noted in all animals from the 1-hour upto the 48-hour reading. Slight reddening was still present in all animals 72 hours after treatment and persisted in two animals up to the 7-day examination.
Moderate swelling (chemosis) of the conjunctivae with partial eversion of lids to marked swelling with half-closed lids was observed in all animals at the 1-hour reading. Moderate swelling with partial eversion of the lids was still visible in two animals 24 hours after treatment and slight swelling of the conjunctivae was present in one animal at the 48- and 72-hour examination.
The reddening of the sclerae was not assessable in two animals at the 1-hour reading due to swelling of the conjunctivae while moderate reddening was visible in the third animal. Slight to moderate reddening of the sclerae was present in all animals at the 24-hour examination and moderate reddening was observed in two animals 48 hours after treatment. Slight reddening was still evident in two animals at the 72-hour reading.
Discharge with moistening of the lids and hairs just adjacent to the lids was noted in all animals at the 1-hour examination. Slight discharge was still present in two animals 24 hours after treatment.
No abnormal findings were observed in the treated eye of any animal 10 days after treatment, the end of the observation per iod for all animals.

CORROSION
No corrosion of the cornea was observed at any of the reading times.
Other effects:
NECROPSY : No necropsy was performed on the animals sacrificed at termination of observation.
All rabbits were sacrificed by an intravenous injection of Vetanarcol into the ear vein at a dose of at least 1 mL/kg body weight (equivalent to 162 mg sodium pentobarbitone/kg body weight) and discarded.

VIABILlTY/MORTALITY AND CLINICAL SIGNS
No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

COLORATION
No staining of the treated eyes produced by the test item was observed.

BODY WEIGHTS
The body weights of all rabbits were considered to be within the normal range of variability.
Body weight in grams
Animal No. Sex First Day of Acclimatization Day of Treatment Last Day of Observation
82 male 2591 2783 2950
83 female 3036 3277 3543
84 female 2720 2832 2999
Interpretation of results:
other: Not irritating
Remarks:
based on CLP criteria
Conclusions:
Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) is considered to be "not irritating" to the rabbit eye.

Executive summary:

The primary eye irritation potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. The treated and untreated eyes were rinsed with lukewarm tap water 24 hours after instillation. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7 and 10 days after test item instillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 1.67, 1.67 and 1.00 for reddening and 1.33, 0.67 and 0.00 for chemosis, respectively.

The instillation of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride) into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 10 days after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H- pyrazol-4-yl)azo] -N,N,N-trimethylanilinium chloride) is considered to be "not irritating" to the rabbit eye.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
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)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Specific details on test material used for the study:

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST:solid
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien

The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek,In Vitro Life Science Lab. (Bratislava, Slovakia).The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of solid test chemical
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles, and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA)
for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles and controls at approximately 37°C, 5% CO2 in a humidified incubator.
After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls.Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.
- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette.2 tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid test articles and controls.

- Justification for the use of a different negative control than ultrapure H2O (Not applicable)
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
0
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of OD :0.00;irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

The analysis contains the results of so called corrected adsorbance since the colors themselves disturbed the MTT salt. Thus, a test with only the colored chemicals were conducted without adding the MTT salt during the MTT analysis. Hence, the results presented here are the corrected result (i.e. the results from the assay with MTT minus the results from the assay without MTT) and shows the true, corrected MTT analysis without the effect of the chemical absorbance included for test chemical.

Interpretation of results:
other: irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean of OD for test chemical was determined to be 0.00. The mean % tissue viability of test chemical was determined to be 0.0%. Thus, test chemical was considered to be irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.00.The mean % tissue viability of test chemical was determined to be 0.0%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

Various studieshas been investigated for the test chemical3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in-vitro and in-vivo experiments conducted for target chemicalwhich have beensummarized as below;

 

The primary skin irritation potential of test chemical3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)was investigated according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7 and 10 days after removal of the dressing. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The mean erythema/eschar score and the mean oedema score was 0.00 for all three animals. The application of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) to the skin resulted in no signs of irritation. However slight yellow staining of the treated skin was observed in two animals at the 1- and 24-hour reading and persisted in one animal up to the 7-day examination. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed. Thus, the test item did not induce significant or irreversible damage to the skin. Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1 -phenyl-1H- pyrazol- 4-yl)azo] -N,N, N-trimethylanilinium chloride) is considered to be "not irritating" to rabbit skin.

 

 

Another study was designed and conducted to determine the dermal reaction profile of 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride in Sprague Dawley rats. The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.  Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. From the study, it can be concluded that the test substance ’3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substance 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride can be classified under nonirritant category.

 

 

Further, in-vitro skin irritation study was performed on reconstructed human tissue (RhT). Standard Operating Procedures (SOP) in accordance with OECD guideline TG 439 was used for the study. Both positive and negative controls were run in parallel with the test items. The test chemical was tested in the EpiSkin™in vitroskin irritation test method in one run. Water was used as a vehicle for the study. The test chemical conc. used for the study was10 ± 2 mg (26.3 mg/cm2) alongwith exposure duration of 15 – 60 mins. Test chemical powder of conc.26.3 mg/cm2was applied to the epidermis surface. Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS). The evaluation of colour interference involved incubation of the test chemical with solvent or water depending on thein vitroRhT test method. If the solution absorbed at 570 nm or became coloured following incubation with the chemical, living tissue adapted controls incubated with medium instead of MTT to define non-specific colour (%NSCliving) were performed. The run was considered as valid for chemicals and all adapted controls if the following criteria were met: difference of viability between the two replicates tissues was≤20% for skin corrosion or Standard Deviation (SD) of viability between 3 replicate tissues was≤18% for skin irritation in accordance with the respective test method SOPs. The resulting formazan tissue extracts were analyzed by photometry (OD) in accordance with the SOP for each test method after testing and also in parallel by HPLC/UPLC-spectrophotometry. Result of the study was interpreted on the basis of cell viability-If cell viability is ≤ 50%: the chemical was classified as irritant and If cell viability is > 50% : the chemical was classified as non – irritant. Statistical analysis: Viability was summarized as mean ± SD or mean and the difference. Simple linear regression was used for assessing the agreement between the OD and HPLC/UPLC-spectrophotometry detection methods. The assumption of linearity was verified with a scatter plot of the standardized residuals versus the viability and the normality of the residuals was verified with a QQ-plot. An alpha level of 0.05 was used as significance level. All analyses were performed in R version 3.2.0. Based on the cell viability of reconstructed human tissue (RhT) after exposure to the test substance, the test chemical was evaluated to be non-irritant.

 

 

Next, skin irritation study was carried out on New Zealand White rabbits according to OECD 404 Guidelines to determine the irritation potency of Basic Yellow 57. Approximately 24 hours prior to the treatment, the dorsal fur was shaved, to expose an area of about 100 cm². 0.5 g of the moistened test substance was applied under a 4 cm x 4 cm semi-occlusive patch to the intact shaved back skin of each animal. The patch was removed 4 hours after contact. Animals were examined for signs of erythema, eschar and oedema formation. The skin reactions were assessed approx. 1 hour, 24, 48 and 72 hours, 7 and 10 days after exposure. Slight yellow staining of the treated skin was observed in two animals at the 1- and 24-hour reading and persisted in one animal up to the 7-day examination. There was no sign of irritation at any observation point. Under the conditions of the study, Basic Yellow 57 was not irritating to rabbit skin.

 

 

Another skin irritation study was carried out on New Zealand White rabbits to determine the irritation potency of Basic Yellow 57. 0.5 g of the test material was dampened with 0.5 ml distilled water and applied to an area of 1 in2on the backs of 3 rabbits each with shorn intact or scarified skin. The sample was covered by an impervious material and left in place for 24 hours. Skin reactions were recorded after 24 and 72 hours. There were no observable reactions to the dye. Hence it was reported that Basic Yellow 57 is non irritating to rabbit skin.

 

 

 

Above results were further supported by dermal irritation study carried out on New Zealand White rabbits to determine the irritation potency of Basic Yellow 57. Undiluted Basic Yellow 57 was applied at the level of 0.5 g/in2 to the backs of 3 rabbits of each sex with shorn intact or scarified skin. The sample was occlusively covered and left in place for 24 hours. Readings were made according to Draize upon removal of the test material anddaily for 14 days post administration. There were no observable reactions to the dye. Hence it was reported that Basic Yellow 57 is non irritating to rabbit skin.

 

 

The overall result was further supported by another in-vitro study performed according to ECVAM Standard Operating Procedures (SOP) on human tissue using the EpiSkin™in vitro skin irritation test method. Both positive and negative controls were used for the study. Test chemical was applied to the epidermis surface of the skin of human for 15 mins.Exposure of test animal to the test substance was terminated by rinsing with phosphate buffered saline (PBS) (using a cotton bud if necessary).After the termination of exposure, further incubation of the epidermis at 37°C for 42 ± 1 hrs (37°C, 5% CO2, 95% humidity) and assessment of cell viability was done by incubating the tissues for 3 hours with MTT solution, after whichthe precipitated formazan is extracted and quantified spectrophotometrically at 570 nm. For each treated tissue the viability is expressed as a percentage of the mean of the negative control tissues’ values. For each test material, three independent tests with three different batches of Episkin™ samples have been carried out. In case viability of the tissues is > 50% after treatment, a second endpoint, namely interleukin-1α(IL-1α) release into the culture medium, is measured. To that end, samples are taken after the 42 hours of incubation and frozen until subjected to an ELISA (enzyme linked immunosorbant assay) test. Result of the study was interpreted on the basis of cell viability and/or IL-1α release. Based on the cell viability of human tissue > 50% (i.e. 105 and 108 % of sample 1 & 2) andIL - 1α release of < 50 pg/ml (i.e. 5 pg/ml of both sample 1 & 2) after exposure to the test substance, the test chemical Basic yellow 57 was evaluated to be non-irritant.

 

Based on the available data for key and supporting studies, it can be concluded that test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye irritation:

In different studies, the test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo and in-vitro experiments conducted for target chemical that have been summarized as below;

 

The primary eye irritation potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. The treated and untreated eyes were rinsed with lukewarm tap water 24 hours after instillation. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7 and 10 days after test item instillation. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 1.67, 1.67 and 1.00 for reddening and 1.33, 0.67 and 0.00 for chemosis, respectively. The instillation of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride) into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 10 days after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed. Thus, the test item did not induce significant or irreversible damage to the rabbit eye. Based on these results and according to the CLP classification criteria: the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H- pyrazol-4-yl)azo] -N,N,N-trimethylanilinium chloride) is considered to be "not irritating" to the rabbit eye.

 

 

The ocular irritation potential of test article was determined in an in-vitro study performed according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean of OD for test chemical was determined to be 0.00.The mean % tissue viability of test chemical was determined to be 0.0%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

 

Another in-vivo eye irritation study was carried out on New Zealand White rabbits according to determine the irritation potency of Basic Yellow 57. 0.1 ml of 0.5% solution Basic Yellow 57 was instilled into the conjunctival sac of the left eye of three rabbits. The right eye was treated with 0.1 ml of the vehicle and served as a control. Eye reactions were recorded at 30 and 60 minutes and 1 and 2 days following and evaluated by the Draize method. The treatment provoked no effects on the cornea or iris in any of the test animals, however, there was a discoloration of the conjunctivae. This test was conducted at a concentration below the intended in-use maximum of 2%. A mild reaction could have been masked by the discoloration caused by the dye. Hence it was reported that Basic Yellow 57 is non irritating to rabbit eyes.

 

 

The overall results were further supported by an eye irritation study performed on New Zealand White rabbits according to OECD 405 Guidelines for test chemical Basic Yellow 57. 0.1 g of Basic Yellow 57 was placed into the conjunctival sac of one eye of the test animals. The substance remained in permanent contact with the eyes until rinsing with warm tap water, 24 hours after instillation. The other eyes served as controls. The eye irritation reactions were scored approx. 1 hour, 24, 48 and 72 hours, 7 and 10 days after instillation of the test solution. Basic Yellow 57 caused mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclera and chemosis. These effects were reversible and had resolved at 10 days in the three animals. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No staining of the treated eyes by the test item was observed. Since the observed effects were fully reversible within 10 days, the test chemical was considered as not irritating to the skin under the tested conditions.

 

Based on the available data for key and supporting studies, it can be concluded that test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)is unable to cause ocular irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Justification for classification or non-classification

The skin and eye irritation potential of test chemical were observed in various studies. The results obtained from these studies indicates that the chemical is unlikely to cause skin and eye damage. Hence the test chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride / Basic Yellow 57 (CAS No. 68391-31-1)can be classified under the category “Not Classified” for skin and eye as per CLP.