Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Data is from experimental study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Principles of method if other than guideline:
The study was conducted to findout mortality, clinical signs, necropsy finding and histopathological changes in wistar albino rats by using the given test chemical.
GLP compliance:
not specified
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylenediamine, salt with phosphoric acid
EC Number:
238-914-9
EC Name:
Ethylenediamine, salt with phosphoric acid
Cas Number:
14852-17-6
Molecular formula:
C2H8N2.xH3O4P
IUPAC Name:
ethane-1,2-diamine; phosphoric acid
Test material form:
solid: particulate/powder
Details on test material:
Name of the test chemical: ethylenediamine, salt with phosphoric acid
Molecular Formula: C2H11N2O4P
Molecular Weight: 158.094 g/mol
Physical State: Solid, White powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Institute for Industrial Research & Toxicology
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 200±20g
- Identification:By cage tag and corresponding colour body marking
- Fasting period before study: Fasted overnight prior to treatment. Food was offered three hours after dosing.
- Housing: Groups of three animals of similar sex in polypropylene cages with stainless steel grill top, facilities for food and water bottle, and bedding of clean paddy husk.
- Diet (e.g. ad libitum): Pelleted feed
- Water (e.g. ad libitum): Aqua Guard filter water was kept in PVC bottles, ad libitum
- Acclimation period: One week in experimental room after veterinary examination.
- Randomization:After acclimation and veterinary examination randomly selected in groups of three females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature between 22-25°C
- Humidity (%): relative humidity 40-60%
- Air changes (per hr): Air conditioned rooms with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 2000 mg/kg bw
- Amount of vehicle (if gavage): 10ml/kg bw

DOSAGE PREPARATION (if unusual): Dose preparation of the test article was done freshly, few minutes prior to dosing. Test substance was dissolved in distilled water to obtain final concentration of 200 mg/ml.
Doses:
Two + one vehicle control
Group I: Dist. water, 10ml/kg body wt.
Group II : 2000 mg/kg body wt.
Group III : 2000 mg/kg body wt.
No. of animals per sex per dose:
Three (3 females)/step
Control animals:
yes
Remarks:
Group I: Dist. water, 10ml/kg body wt.
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality: The test compound was administered at different dose level in wistar albino rats observed for mortality at the time interval of 30 minutes, 1hr, 2hr, 4 hr, and 6hr time interval on the day of test compound administration and thereafter twice a day for 14 days.
Body weight: The body weight of all the animals was observed weekly on day 0 (pre treatment), 7th and 14th (post treatment).
Clinical Signs: The treated animals were closely observed for clinical signs of intoxication, first 4 hours and every 1 hrs interval for 24 hrs after dosing and thereafter twice a day for 14 days. All the rats were observed at least twice daily to observe any clinical signs or behavioral changes. These observations included changes in skin and fur, in the eyes and mucous membranes, respiratory, circulatory, central nervous and autonomic nervous systems, somatomotor activity and behavioral changes. The following clinical signs were observed in female mice to characterize the various systemic studies: Salivation, lacrimation, pale mucous membrane, diarrheal feaces, hunched posture, scratching, polyuria, hypoactivity etc.
- Necropsy of survivors performed: yes, necropsy was carried out on all the animals which died during the study or surviving animals were sacrificed at the end of the study to observe any gross pathological changes.
Statistics:
not specified

Results and discussion

Preliminary study:
not specified
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality recorded in any of the Wistar albino rats after administration of the test compound during the entire observation period (14 days).
Clinical signs:
The test compound did not produce any clinical signs of toxicity at the tested dose level throughout the period of observation.
Body weight:
Wistar albino rats treated with the test compound at the dose level of 2000 mg/kg b.wt. showed significant normal gain in body weight on day 7th and 14th (post treatment) as compared to control group.
Gross pathology:
NECROPSY FINDING
EXTERNAL
i.Skin- Skin and hair coat was observed wet.
ii.All external orifices- Normal
B. INTERNAL
i. Subcutaneous- No change was observed.
ii. Superficial and deep lymph nodes- No change in mesenteric lymph node.
ABDOMINAL CAVITY
i.Opening and general examination- In the abdominal cavity all the organs were present in normal position.
ii.Spleen- Normal upto highest tested dose level 2000 mg/kg b.wt.
iii.Digestive system- No gross changes were observed in stomach and intestine upto highest tested dose level 2000 mg/kg b.wt.
iv.Liver and biliary ducts- No gross pathological changes were observed
v.Excretory system- No gross pathological changes were observed upto highest tested dose level 2000 mg/kg b.wt.
vi.Adrenal- Observed normal.
vii.Male/female genital organs – Showed normal colour, consistency and no inflammatory changes upto highest tested dose level 2000 mg/kg b.wt.
2. THORACIC CAVITY
i.Opening and general examination- Thoracic cavity was found to be normal without any fluid, mucous or blood etc.
ii.Lungs- observed normal.
iii.Heart- No changes were observed in color and consistency. Heart found normal upto highest tested dose level 2000 mg/kg b.wt.
iv.Thyroid- Normal in shape, size and surface upto highest tested dose level 2000 mg/kg b.wt.
3. CRANIAL CAVITY
Brain- Normal in shape and size.
Other findings:
not specified

Any other information on results incl. tables

TABLE – 2

SUMMARY OF BODY WEIGHT (GM)

Group

Day 0

Day 7

% Gain/loss

Day 14

% Gain/loss

Group-I distilled water 10ml/kg

202.40

208.90

3.21

217.40

7.41

Group-II

2000 mg/kg b. wt

203.70

209.2

2.70

219.9

7.95

Group-III

2000 mg/kg b. wt

201.90

208.30

3.16

216.70

7.33


TABLE – 3

CLINICAL SIGNS AND MORTALITY

Group: I (Vehicle Control)                                                           Dose: 10 ml/kg b.wt

                                           

FEMALE RATS 

Parameters

Incidence of clinical signs observed after dosing

Mortality

Day 0

DAY

Min

Hour

30

1

2

4

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total*

Mortality (total)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

0/3

 

Clinical Signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

0         =   No clinical sign (Normal)

+         =   Clinical Sign

 


TABLE – 3 (Contd.)

CLINICAL SIGNS AND MORTALITY

Group: II                                                                                           Dose: 2000 mg/kg b. wt.

 

FEMALE RATS

 

Parameters

Incidence of clinical signs observed after dosing

Mortality

Day 0

DAY

Min

Hour

30

1

2

4

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total*

Mortality (total)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

0/3

Clinical Signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

  0           =   Normal

+            =    Mild

++           =   Moderate

+++         =   High

 ++++     =   Severe


TABLE – 3 (Contd.)

CLINICAL SIGNS AND MORTALITY

Group: III                                                                                          Dose: 2000 mg/kg b.wt

                                            

FEMALE RATS 

 

Parameters

Incidence of clinical signs observed after dosing

Mortality

Day 0

DAY

Min

Hour

30

1

2

4

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total

Mortality (total)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

0/3

Clinical Signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 0            =   Normal

+            =    Mild

++           =   Moderate

+++         =   High

 ++++     =   Severe


TABLE – 4

SUMMARY OF NECROPSY FINDINGS

S. No.

 

Fate

 

Wistar albino rats

Dose (mg/kg b. wt)

Distilled water (10 ml/kg)

 

2000

 

 

2000

1

Terminal sacrifice

3/3

3/3

3/3

2

Found Dead

0/3

0/3

0/3

3

Abnormalities detected

NAD

NAD

NAD

NAD - No abnormality recorded


TABLE - 5

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

Group: I (Vehicle Control)                                                           Dose: 10ml/kg b.wt

                                                                                                                    

FEMALE RATS

 

Animal ID

Fate

Time

Gross Findings

20164-1

TS

Day 14

NAD

20164-2

TS

Day 14

NAD

20164-3

TS

Day 14

NAD

 

Day 0 is the day of dose administration.

TS- Terminal Sacrifice

NAD- No abnormality Detected

 

 

TABLE – 5 Contd……..

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

Group: II                                                                                            Dose: 2000 mg/kg b.wt.                  

FEMALE RATS

 

Animal ID

Fate

Time

Gross Findings

20164-4

TS

Day 14

NAD

20164-5

TS

Day 14

NAD

20164-6

TS

Day 14

NAD

 

Day 0 is the day of dose administration.

TS- Terminal Sacrifice

NAD- No abnormality Detected

FD- Found Dead

 

TABLE – 5 contd……….

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

Group: III                                                                                          Dose: 2000 mg/kg b.wt.      

                                                     

                                                           FEMALE RATS 

Animal ID

Fate

Time

Gross Findings

20164-7

TS

Day 14

NAD

20164-8

TS

Day 14

NAD

20164-9

TS

Day 14

NAD

 

Day 0 is the day of dose administration.

TS- Terminal Sacrifice

NAD- No abnormality Detected

FD- Found Dead 

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Conclusions:
From the results obtained from present investigation, the LD50 value was considered to be >2000 mg/kg bw. Thus, it can be concluded that the test chemical is non toxic to Wistar albino rats. CLP classification "Not classified"
Executive summary:

The acute oral toxicity study was conducted under the OECD Guideline-423 for testing of chemicals in wistar albino rats. The healthy wistar albino rats of body weight 200±20 gm were selected for study after acclimatization to standard laboratory condition and divided into test compound and vehicle control group each having three animals. The study was conducted stepwise as follow:

Starting dose 2000 mg/kg body weight: Step-I -  The test compound was mixed with distilled water and administered orally at the dose level of 2000 mg/kg body weight (dose volume 10ml/kg) to three female rats. However; vehicle control group treated with the distilled water at the dose level of 10 ml/kg b.wt.  The treated animals were closely observed for clinical signs of intoxication during first four hours of test compound administration. Thereafter, all the animals were observed periodically at one hour interval for 24 hrs and twice daily for a period of 14 days.  The necropsy was performed on all animals at the termination of the study.

The test compound did not elicit any clinical signs of intoxication throughout the period of observation at the tested dose level 2000 mg/kg body weight. Furthermore, no mortality was recorded at test dose level. Vehicle control group of animals were also free from any mortality and clinical signs. The necropsy finding did not reveal any gross pathological changes at the tested dose level. Furthermore, no gross pathological change was observed in vehicle control group.

Step -II: After 72 hrs, the result of step-I was confirmed by administration of same dose level (2000 mg/kg. b.wt) of test compound in additional three animals of same sex (OECD-423 guidelines) under same test condition.

The test compound administered at the dose level of 2000 mg/kg b.wt did not produce any mortality and clinical sign of intoxication throughout the observation period of 14 days. Body weight of each rat was also recorded on day 7th and 14th showed normal gain as compared to control group. Necropsy was conducted at the end of the study (15th day) on all the animals which did not reveal any gross pathological changes.

From the results obtained from present investigation, the LD50 value was considered to be >2000 mg/kg bw. Thus,  it can be concluded that the test chemical is non toxic to Wistar albino rats. CLP classification "Not classified"