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Acute Toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
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).

Test animals

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

Administration / exposure

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

Results and discussion

Effect levels
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.