Registration Dossier

Administrative data

Description of key information

Acute toxicity: oral
Under the condition of the study, the acute oral LD50 (Cut-off value) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) was 1000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Acute toxicity: inhalation
The study need not be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Acute toxicity: dermal
It was concluded that the acute dermal median lethal dose (LD50) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) does not classify as an acute dermal toxicant. CLP Classification: “Unclassified”.

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
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Principles of method if other than guideline:
The purpose of this study was to assess the Toxicological profile of test item to a single administration via oral route to Sprague Dawley rats. This study was designed to determine the acute toxicity at fixed dose levels by oral route of the test item.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Item: 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5)
- Source of test material: Sustainability Support Services (Europe) AB, Sweden
- Batch No. of test material: KCP/FS/43/17
- Manufacturing Date: January; 2017
- Expiration date of the lot/batch: December; 2017
- Purity test date: No data
- Consistency: Solid, powder

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: Test Item and prepared formulation(s) were stored at ambient temperature.
- 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: Test item was suspended in Polyethylene Glycol - 400. The formulation was prepared fresh on the day of dosing.
- 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 (if different from that of starting material): No data available

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 nulliparous and non-pregnant: yes
- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing.
- Weight at study initiation: Body weight range was 197.2 to 208.2 grams.
Body weights at the start :
Female
Mean : 201.32 g (= 100 %)
Minimum : 197.2 g (- 2.05 %)
Maximum : 208.2 g (+ 3.42 %)
Total No. of animals : 9
- Identification: Each female rat was individually identified by the picric acid marking.
- 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): 20.1 to 22.3 degree centigrade.
- Humidity (%): 55.1% to 58.4%
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 12-06-2017 to 30-06-2017
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene Glycol - 400
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 300 mg/kg, 300 mg/kg and 2000 mg/kg
- 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/kg body weight.

DOSAGE PREPARATION (if unusual): No data available

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 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:
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 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.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

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, found dead and sacrificed 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).

Histopathology:
Gross pathological examination revealed distended stomach with test item coloured ingesta and small and large intestine with liquid test item coloured ingesta the localized colouration imparted to the abdominal organs is of the test item and no gross abnormality observed except colouration hence, no organ collected for histopathology.
Statistics:
No data
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
1 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: LD50 cut off value
Mortality:
Group I
Step I :
Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days and were free of signs of toxicity at 6 hours after the dosing.

Group I
Step II :
Animals treated at the dose level of 300 mg/kg body weight: 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: Two animals died on day 1 after the dosing.
Clinical signs:
Group I
Step I :
Animals treated at the dose level of 300 mg/kg body weight resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity at 6 hours after the dosing.

Group I
Step II :
Animals treated at the dose level of 300 mg/kg body weight resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity at 6 hours after the dosing.

Group II
Step I :
Animals treated at the dose level of 2000 mg/kg body weight resulted in polyurea, diarrhoea, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. The only surviving animal was free of signs of toxicity on day 7 after the dosing.
Body weight:
Group I
Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 1.97% and 9.21% respectively.

Group I
Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 2.02% and 9.58% respectively.

Group II
Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 1.93% and 10.09% respectively.
Gross pathology:
Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg and 2000 mg/kg dose groups.
Gross pathological examination revealed distended stomach with test item coloured ingesta and small and large intestine with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group.

Other findings:
No data available

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

Reduced locomotor activity

1

2

4 hrs.

0/3

Ataxic gait 

1

2

4 hrs.

 

  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

Reduced locomotor activity

1

4

4 hrs.

0/3

Ataxic gait 

1

4

4 hrs.

 

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

Polyurea

1

9

Day 1 - Day 6

2/3

Diarrhoea

3

7,8,9

4 hrs. - 6 hrs.

Reduced locomotor activity

3

7,8

9

30 min. - 6 hrs.

30 min. - 4 hrs.

Ataxic gait 

3

7,8

9

1 hr. - 6 hrs.

1 hr. - Day 6

 

 

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

205.13

209.17

1.97

224.00

7.10

9.21

± SD

2.91

3.23

0.61

1.28

1.28

1.42

 

 

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

200.37

204.40

2.02

219.57

7.42

9.58

± SD

2.06

2.35

1.14

3.25

1.45

0.50

 

 

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

198.47

201.00

1.93

217.10

8.01

10.09

± SD

2.11

-

-

-

-

-

 

 

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, 8

FD

Stomach : Distended with test item coloured ingesta.

Small and large intestine with liquid test item coloured ingesta.

9

TS

No abnormality detected

                

FD = Found dead

TS = Terminal Sacrifice

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the condition of the study, the acute oral LD50 (Cut-off value) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) was 1000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.
Executive summary:

The study now reported was designed and conducted to determine the acute oral toxicity profile of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) 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 resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing and no 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 resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing and no 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 resulted in polyurea, diarrhoea, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. Two animals died on day 1 after the dosing. 

All animals from 300 mg/kg dose group survived and exhibited normal body weight gain through the study period of 14 days.

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

Gross pathological examination revealed distended stomach with test item coloured ingesta and small and large intestine with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group.

The acute oral LD50 (Cut-off value) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) was 1000 mg/kg body weight.  

Thus, it was concluded that the acute toxicity study of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
300 mg/kg bw
Quality of whole database:
Data is of K1 reliability and is obtained from experimental study report.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
the study does not need to be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size
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 experimental 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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Item: 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5)
- Source of test material: Sustainability Support Services (Europe) AB
- Batch No.of test material: KCP/FS/43/17
- Manufacturing Date: January; 2017
- Expiration date of the lot/batch: December; 2017
- Purity test date: No data available
- Consistency: Solid, powder

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: Ambient Temperature
- 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: Test item was grounded to fine powder prior to application. The particulates were moistened
with distilled water before application.
- 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: Paste

OTHER SPECIFICS:
Safety Precautions : Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females nulliparous and non-pregnant: No data available
- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.
- Weight at study initiation: The weight range of approximately 213.4 to 252.2 grams at initiation of dosing.
Body weights at the start :
Male
Mean : 245.26 g (= 100 %)
Minimum : 239.8 g (- 2.23 %)
Maximum : 252.2 g (+ 2.83 %)
Total No. of animals : 5
Female
Mean : 218.46 g (= 100 %)
Minimum : 213.4 g (- 2.32 %)
Maximum : 222.7 g (+ 1.94 %)
Total No. of animals : 5
- Identification: Each rat was individually identified by the cage number.
- Fasting period before study: No data available
- 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): 19.6 to 21.6 degree centigrade.
- Humidity (%): 55.0% to 58.4%.
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 12-06-2017 to 27-06-2017
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
(Distilled water)
Details on dermal exposure:
TEST SITE
- Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area)
- % coverage: Approximately 10% of the 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
- Constant volume or concentration used: No data available
- For solids, paste formed: Yes

VEHICLE
- Amount(s) applied (volume or weight with unit): No data available
- Concentration (if solution): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
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).

Histopathology:
No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.




Statistics:
No data
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Sex : Male
Group I -
All animals survived through the study period of 14 days.

Sex : Female
Group I -
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.

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.
Body weight:
Sex : Male
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.03% and 18.46% respectively.

Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.69% and 9.55% 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

245.26

267.38

9.03

290.50

8.65

18.46

± SD

5.06

4.63

0.64

4.65

0.40

0.95

 

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

218.46

230.88

5.69

239.32

3.66

9.55

± SD

3.57

3.95

0.59

4.16

0.59

0.71

 

   

  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 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) does not classify as an acute dermal toxicant.  
CLP Classification: “Unclassified”.
Executive summary:

The study now reported was designed and conducted to determine the acute dermal toxicity profile of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5)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. It was concluded that the acute dermal median lethal dose (LD50) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) does not classify as an acute dermal toxicant.  CLP Classification: “Unclassified”.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is of K1 reliability and is obtained from experimental study report.

Additional information

Acute toxicity: oral
Different experimental studies were reviewed for acute oral toxicity endpoint for the test substance 4-(phenyldiazenyl)benzene-1,3-diamine and are presented below as key and supporting studies:

The study now reported was designed and conducted to determine the acute oral toxicity profile of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) 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 resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing and no 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 resulted in reduced locomotor activity and ataxic gait with onset at 4 hours after the dosing and no 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 resulted in polyurea, diarrhoea, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. Two animals died on day 1 after the dosing.  All animals from 300 mg/kg dose group survived and exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg and 2000 mg/kg dose groups. Gross pathological examination revealed distended stomach with test item coloured ingesta and small and large intestine with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group. The acute oral LD50 (Cut-off value) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) was 1000 mg/kg body weight.  Thus, it was concluded that the acute toxicity study of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Supporting data were obtained from Initial submission report from National Technical Report library as follows:

Acute oral toxicity study was conducted on young adult Sprague Dawley rats using the test compound Solvent orange 3/ Calco Chrysoidine Y Base. Further to dose range finding study and the trial study, the dose range selected for the main study was 500, 813, 1323, 2152 and 3500 mg/Kg bw. Cage side observations, clinical signs and mortality was checked. On the basis of observations made,Solvent orange 3/ Calco Chrysoidine Y Baseshows an acute oral LD50 for male rats to be 2212 mg/kg bw with a 95% confidence interval of 1558 to 3859 mg/kg. The LD50 for females is to be 1037 mg/kg bw with a 95% confidence interval of 698 to 1540 mg/kg and the combined response (males and females) is 1532 mg/kg with a 95% confidence interval of 1192 to 1993 mg/kg.

Range finding acute oral toxicity study was conducted on young adult Sprague Dawley rats using the test compound Solvent orange 3/ Calco Chrysoidine Y Base. In the dose range finding study, the dosage level used was 500, 813, 1581, 2812 and 5000 mg/Kg. LD50 value was found out on the basis of mortality observed. On the basis of observations made,Solvent orange 3/ Calco Chrysoidine Y Bases hows an acute oral LD50 for male/female rats to be 1581 mg/kg bw.

Acute oral toxicity study was conducted on young adult Sprague Dawley rats using the test compound Solvent orange 3/ Calco® Chrysoidine Y Base. A trial study was conducted at a dosage level of 5000 mg/Kg bw.Cage side observations, clinical signs and mortality was checked. On the basis of observations made, Solvent orange 3/ Calco® Chrysoidine Y Base shows an acute oral LD100 for male/female rats to be 5000 mg/kg.

Based on the values from the above studies it can be concluded that the substance can be classified in acute category IV as per the criteria of CLP regulation.

Further supporting above experimental data was based on the QSAR prediction done using the Danish (Q)SAR Database, the LD50 was estimated to be 1100 mg/kg/d on rat for substance m-Phenylenediamine, 4(phenylazo)-. Thus based on this value it can be concluded that the substance can be classified in acute category IV as per the criteria of CLP regulation.

Data obtained from RTECS database also indicates the acute oral LD50 value of m-Phenylenediamine, 4(phenylazo)- as 1650 mg/kg in rat.

Thus, considering the key and various supporting studies for the substance 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), it was concluded that the substance is toxic via oral route and hence it can be classified in acute category IV as per the criteria of CLP regulation.

Acute toxicity: inhalation
The study need not be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Acute toxicity: dermal
Experimental data for the test substance 4-(phenylazo)benzene-1,3-diamine (CAS 495-54-5) and its structurally related substance 4-Aminodiphenylamine (CAS 101-54-2) were reviewed for classification of the substance in acute dermal category and are presented below:

The study now reported was designed and conducted to determine the acute dermal toxicity profile of4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5)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. It was concluded that the acute dermal median lethal dose (LD50) of 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) does not classify as an acute dermal toxicant.  CLP Classification: “Unclassified”.

24 hrs acute toxicity test was performed on rabbits to measure the toxicity potential of the structurally related substance 4-Aminodiphenylamine (CAS 101-54-2) via dermal route of application (as cited in OECD SIDS assessment report). No deaths occurred after a single 24-hour occlusive dermal application of 5000 mg of test substance/kg bw (applied as a paste, moistened with saline) to the clipped, intact skin of 5 male and 5 female rabbits. During the 14 day post-exposure observation period only a few animals showed decreased food consumption, and nasal and ocular discharge. Single occurrences of red and/or swollen eyes were observed. Necropsy findings were not different from controls. The dermal LD50 in rabbits is greater than 5000 mg/kg bw.

Thus, considering the key study for target and supporting study for structurally related substance. it can be concluded that the target substance 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) is not toxic via dermal and hence is considered as Not classified as per the criteria of CLP regulation.

Justification for classification or non-classification

Considering the key studies, it can be concluded that the substance 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5) is toxic via oral route and non toxic via dermal route and hence is classified in Acute category IV for oral route and Not classified for dermal route as per the criteria of CLP regulation.