Registration Dossier

Administrative data

Description of key information

Acute oral toxicity: 

The acute oral toxicity dose (LD50) was considered based on different studies conducted on rats and mice for the test chemical. The LD50 value is >2000 mg/kg bw, for acute oral toxicity. Thus, comparing this value with the criteria of CLP regulation, the given 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) cannot be classified for acute oral toxicity.

 

 

Acute Dermal toxicity:

The acute dermal toxicity dose (LD50) was considered based on different studies conducted on rats for the test chemical. The studies concluded that LD50 value is >2000 mg/kg bw, for acute dermal toxicity. Thus, comparing this value with the criteria of CLP regulation, the given 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) cannot be classified for acute dermal toxicity.

 

 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sustainability Support Services (Europe) AB, Sweden
- Lot/batch No. of test material: D517
- Expiration date of the lot/batch: 15/8/2021
- Purity test date: No data

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity: No data
- Locations of the label: No data
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Test Item was stored at ambient temperature.
- Stability under test conditions: No data
- 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: Test item was suspended in distilled water.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: The formulation was prepared fresh on the day of dosing. The test item was administ ered in the dose volume of 10 ml/kg body weight.
- 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: Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used.
- Weight at study initiation: The weights were within ± 20% of the mean weight of any animal used for dosing. Body weight range was 199.2 to 212.8 grams.
Body weights at the start :
Female
Mean : 205.33 g (= 100 %)
Minimum: 199.2 g (- 2.98 %)
Maximum: 212.8 g (+ 3.64 %)
Total No. of animals : 12
- Fasting period before study: Approximately 16 hours or more.
- Housing: The rats were housed in polycarbonate cages.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 20.8 to 22.7 degree centigrade.
- Humidity (%): Room humidity was maintained at 55.6% to 58.1%.
- Air changes (per hr): The animal room was independently provided with at least ten to fifteen air changes per hour of 100% fresh air that had been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: No data
Route of administration:
oral: gavage
Vehicle:
other: Distilled water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: No data
- Amount of vehicle (if gavage): No data
- Justification for choice of vehicle: No data
- Lot/batch no. (if required): No data
- Purity: No data

MAXIMUM DOSE VOLUME APPLIED: 10 ml per kg of body weight was considered the maximum volume which could be administered to a rat.

DOSAGE PREPARATION (if unusual): No data
CLASS METHOD (if applicable) No data
- Rationale for the selection of the starting dose: No data
Doses:
Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
No. of animals per sex per dose:
Three females were used at each step.
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: yes
- Other examinations performed:
Clinical Observations and General Appearance:
Animals were observed for clinical signs, mortality and morbidity, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.

Body weights:
Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.

Gross Pathology:
Necropsy was performed on all animals at the end of the study period on day 15. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique.
Statistics:
No data
Sex:
female
Dose descriptor:
LD50
Effect level:
2 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals treated at the dose level of 300 mg/kg body weight and 2000mg/kg body weight survived through the study period of 14 days.
Clinical signs:
Group I
Step I :
Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Group I
Step II :
Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Group II
Step I :
Animals treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Group II
Step II :
Animals treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Body weight:
Group I
Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.45% and 13.14% respectively.

Group I
Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.68% and 11.19% respectively.

Group II
Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.13% and 13.00% respectively.

Group II
Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.43% and 11.33% respectively.
Gross pathology:
Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.
Other findings:
No data

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

Group I :   

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs in days

From - to

Mortality

I

300

No clinical signs observed

3

1,2,3

Day 0 - Day 14

0/3

 

 

Group I :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs in days

From - to

Mortality

II

300

No clinical signs observed

3

4,5,6

Day 0 - Day 14

0/3

 

Group II :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs in days

From - to

Mortality

I

2000

No clinical signs observed

3

7,8,9

Day 0 - Day 14

0/3

 

Group II :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs in days

From - to

Mortality

II

2000

No clinical signs observed

3

10,11,

12

Day 0 - Day 14

0/3

 

 

Table No.II

 

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

300

Mean

201.03

209.97

4.45

227.43

8.32

13.14

± SD

2.22

1.66

1.52

2.29

0.37

1.58

 

Group I :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

II

300

Mean

209.83

217.53

3.68

233.30

7.24

11.19

± SD

3.17

2.95

1.29

3.97

0.39

1.46

 

Group II :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

204.43

214.93

5.13

231.03

7.49

13.00

± SD

1.95

4.34

1.14

4.66

0.14

1.21

 

 Group II :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

II

2000

Mean

206.00

213.07

3.43

229.33

7.63

11.33

± SD

2.88

2.35

0.36

3.14

0.48

0.23

 

 

Table No.III

 

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

 Group I :

Step

No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

300

1 - 3

TS

No abnormality detected

 

Group I :

Step

No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

II

300

4 - 6

TS

No abnormality detected

 

Group II :

Step No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

7 - 9

TS

No abnormality detected

                

Group II :

Step No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

II

2000

10 - 12

TS

No abnormality detected

       

 TS = Terminal Sacrifice

 

Interpretation of results:
other: Not Classified
Remarks:
based on EU Criteria
Conclusions:
It was concluded that the acute oral median lethal dose (LD50) of 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride supplied by Sustainability Support Services (Europe) AB, Sweden, when administered to Sprague Dawley rats was determined to be 2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride does not exhibits acute toxicity by the oral route and can be classified under the category “Not Classified”as per the CLP classification criteria.
Executive summary:

The study reported was designed and conducted to determine the acute oral toxicity 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.

Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality after the dosing.

No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality after the dosing. As no mortality were observed at 24 hours after the dosing, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality after the dosing.

Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.

It was concluded that the acute oral median lethal dose (LD50) of 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride supplied by Sustainability Support Services (Europe) AB, Sweden, when administered to Sprague Dawley rats was found to be 2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride does not exhibits acute toxicity by the oral routeand can be classified under the category “Not Classified”as per the CLP classification criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
Report and safety assessment repor

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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 study report.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
This study was designed to determine the dermal LD50 of the test item (up to 2000 mg/kg) or to establish a non-lethal dose level of 2000 milligram of test item per kilogram of body weight.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: Young adult male and female rats aged between 6 – 9 weeks were used.
- Weight at study initiation: The weight ranges of approximately 218.4 to 262.2 grams at initiation of dosing were used.
Body weights at the start : Male Mean: 256.40 g (= 100 %); Minimum : 251.3 g (- 1.99 %); Maximum : 262.2 g (+ 2.26 %)
Female Mean: 224.72 g (= 100 %); Minimum : 218.4 g (- 2.81 %); Maximum : 231.5 g (+ 3.02 %)
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 20.0 to 21.9 degree centigrade.
- Humidity (%): Room humidity was maintained at 56.1% to 58.7%.
- Air changes (per hr): The animal room was independently provided with at least ten to fifteen air changes per hour of 100% fresh air that had been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 08-11-2016 to 23-11-2016
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: Dorsal surface and sides from scapular to pelvic area.
- % coverage: Approximately 10% of the total body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
Duration of exposure:
24 hours
Doses:
A single dose of 2000 mg of the test item per kilogram of body weight was administered to ten rats (five males and five females).
No. of animals per sex per dose:
10 (5/sex).
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical Observations and General Appearance: Animals were observed for clinical signs, mortality, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

Evaluation of Dermal Reaction: Dermal reaction was observed daily for study period of 14 days.

Body weights: Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14.

Gross Pathology: Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15).
Statistics:
not specified
Preliminary study:
not specified
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg: All animals survived through the study period of 14 days.
Sex : Female Group I – Animal treated at the dose level of 2000 mg/kg: All animals survived through the study period of 14 days
Clinical signs:
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Sex : Female Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Body weight:
Sex : Male Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.93% and 20.46% respectively.
Sex : Female Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.18% and 10.21% respectively.
Gross pathology:
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.
Other findings:
- Other observations: Evaluation of Dermal Reaction
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Sex : Female Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Male

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

1 - 5

Day 0 - Day 14

0/5

 

Sex : Female

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

6 - 10

Day 0 - Day 14

0/5

 

Table No. II

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

1 - 5

Day 0 - Day 14

0/5

 

Sex : Female

 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

6 - 10

Day 0 - Day 14

0/5

  

Table No.III

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Male

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

256.40

281.88

9.93

308.94

9.58

20.46

± SD

4.12

6.59

0.83

10.41

1.19

2.16

 

 

Sex : Female

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

224.72

236.38

5.18

247.66

4.78

10.21

± SD

5.10

6.72

1.47

6.41

0.57

1.15

  

Table No.IV

 Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

 Sex : Male

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

1 - 5

TS

No abnormality detected

 

Sex : Female

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

6 - 10

TS

No abnormality detected

  TS = Terminal Sacrifice

Interpretation of results:
other: Not classified
Conclusions:
It was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibits acute toxicity by the dermal route. CLP Classification: “Not classified”.
Executive summary:

The reported study was designed and conducted to determine the acute dermal toxicity profile of the given test chemical as per OECD Guideline 402 (Acute Dermal Toxicity) 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.

Hence, it was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibits acute toxicity by the dermal route. CLP Classification: “Not classified”.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
Study report

Additional information

Acute oral toxicity: 

In different studies, the given 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 acute oral toxicity to a greater or lesser extent. Often are the studies based on in-vivo experiments in rodents, i.e. most commonly in rats and mice for test chemical. The studies are summarized as below –

 

The study reported was designed and conducted to determine the acute oral toxicity 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. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality after the dosing. As no mortality were observed at 24 hours after the dosing, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality after the dosing. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. It was concluded that the acute oral median lethal dose (LD50) of 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride supplied by Sustainability Support Services (Europe) AB, Sweden, when administered to Sprague Dawley rats was found to be 2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride it infers that 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethyl anilinium chloride does not exhibits acute toxicity by the oral routeand can be classified under the category “Not Classified” as per the CLP classification criteria.

 

Another acute oral study was carried on Sprague Dawley rats to determine the acute oral toxicity of Basic Yellow 57. In this study, groups of 5 male and 5 female rats received a single oral dose of 2000 mg/kg bw. The animals were observed daily for 14 days for mortality and clinical abnormalities. Body weights and macroscopic observations were recorded, but histological examinations were not performed. No mortalities were reported. Clinical signs of reaction to treatment observed shortly after dosing in all rats were piloerection, abnormal body carriage (hunched posture), abnormal gait (waddling) and increased salivation. The bodyweights were reported to be normal for the strain, but there was no control group for comparison. The LD50 was reported to be greater than 2000 mg/kg bw.

 

The above results were supported by next acute oral study performed on CF1 male mice to determine the acute oral toxicity of Basic Yellow 57. Basic Yellow 57 was administered to groups of 10 male CF1 mice at dose levels of 631,1000, 2510 and 5010 mg/kg bw Animals were observed for a period of 7 days. Signs of reaction to treatment were an increased respiratory rate, and tremors. The general condition of the mice deteriorated at the higher dose levels. The LD50 was reported to be 2350 mg/kg bw.

 

In the last study, the acute oral test was carried out on CFY rats consist of groups of 2 male and 2 female rats. Each rat received a single oral dose of 100, 1000, 2000 and 4000 mg/kg bw. Control animals received 1% aqueous methylcellulose in a volume of 40 ml/kg. The animals were observed daily for 14 days for clinical abnormalities and mortality. Body weights and macroscopic observations were recorded, but histological examinations were not performed. One male and two female rats died after a dose of 2.0 g/kg bw. All animals treated at 4.0 g/kg bw died within one week of dosing. There were no mortalities at lower doses. Signs of reaction to treatment, observed shortly after dosing, included piloerection and abnormal body carriage (hunched posture), which were accompanied by lethargy and diarrhoea in rats treated with doses greater than 100 mg/kg, and decreased respiratory rate, pallor of the extremities and ptosis at 1000 mg/kg and by increased salivation and diuresis in rats treated with 2000 mg/kg. Therefore the LD50 was reported to be between 1000 and 2000 mg/kg bw.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that LD50 value of test chemical is >2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, 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) cannot be classified for acute oral toxicity.

 

Acute Dermal Toxicity:

The reported study was designed and conducted to determine the acute dermal toxicity profile of the given test chemical as per OECD Guideline 402 (Acute Dermal Toxicity) 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. 

Hence, it was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibits acute toxicity by the dermal route. CLP Classification: “Not classified”.

Justification for classification or non-classification

Based on the above studies on 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), it can be concluded that LD50 value is >2000 mg/kg bw, for acute oral and acute dermal toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral and acute dermal toxicity.