Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
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 report.

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 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
The study was conducted to find out the mortality, clinical sign of toxicity and histopathological effect of the given test chemical at different dose level in wistar albino rats.
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
yes

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:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Institute for Industrial Research and Toxicology Ghaziabad
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 200±20g
- Fasting period before study: Animals were fasted overnight prior to test and food was offered three hours after dosing.
- Housing: Groups of three animals of same sex in polypropylene cages with stainless steel grill top, facilities for food and water bottle, and bedding of clean paddy husk.
- Identification: By cage tag and corresponding colour body marking
- Diet (e.g. ad libitum): Pelleted feed
- Water (e.g. ad libitum): Fresh and clean water filtered through ‘Aqua Guard on line water filter’, was kept in glass bottles Ad libitum
- Acclimation period: The healthy wistar albino rats selected for study acclimatized to standard laboratory condition for period of one week under close Veterinary supervision.
- Randomization: After acclimation and Veterinary examination all the animals randomly divided into two groups and each group having five male and five female rats.

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

Type of coverage:
occlusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure:back skin of total body surface area
- % coverage: Approximate 10 percent
- Type of wrap if used: The test compound was held in contact with the skin with an impervious dressing secured in place with an adhesive tape. The animals were then housed individually in cages with a collar around the neck in order to avoid the ingestion of the test compound.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the site of application was cleaned with lukewarm water wiping the test compound.
- Time after start of exposure: 24 hours
Duration of exposure:
24 hours
Doses:
Group-I: 2000 mg/kg b.wt (limit test)
Group-II: 2000 mg/kg b.wt (confirmatory test)
No. of animals per sex per dose:
10 (5male & 5 female)
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality - All the animals were observed for mortality at 30 minutes time interval for first six hours 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 thereafter for every 1 hrs interval for 24 hrs after dosing and twice a day for 14 days. All the rats were observed at least twice daily with the purpose of recording any symptoms of ill-health 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 rats to characterize with erythema, hypersensitivity, edema etc.
- Necropsy of survivors performed: yes, necropsy was carried out on all the animals that 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:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
The test compound when applied dermally at the dose level of 2000 mg/kg b.wt. on Wistar albino rats did not produce any mortality during the observation period of 14 days.
Clinical signs:
The Wistar albino rats treated with the test compound were free from any systematic and local clinical signs when observed after 24th hours of patch removal.
Body weight:
The body weight of all the animals was recorded on day 0 (pre treatment) and then 7th and 14th (post treatment) and it has been observed that there was no significant increase and decrease in the body weight. However, normally increase in the body weight was recorded on day 14th.
Gross pathology:
NECROPSY FINDING
EXTERNAL
i.Skin- Skin and hair coat was observed wet.
ii.All external orifices- Normal
B. INTERNAL
i. Subcutaneous- No changes were 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- No changes were recorded.
iii.Digestive system- No gross changes were observed in stomach and intestine.
iv.Liver and biliary ducts- No gross pathological changes were observed
v.Excretory system- No gross pathological changes were observed.
vi.Adrenal- Observed normal.
vii.Male/female genital organs – Showed normal colour, consistency and no inflammatory changes.
2. THORACIC CAVITY
i.Opening and general examination- Thoracic cavity was found to be normal without any fluid, mucous or blood etc.
ii.Lungs- No changes were recorded.
iii.Heart- No changes were observed in color and consistency. Heart found normal.
iv.Thyroid- Normal in shape, size and surface.
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

Animal ID

Day 0

Day 7

% Gain/loss

Day 14

% Gain/loss

Group-I

2000 mg/kg b. wt

 

 

 

20164-1

203.15

206.12

1.46

210.90

3.80

20164-2

201.91

204.95

1.50

208.99

3.50

20164-3

201.12

213.47

2.08

217.50

4.0

20164-4

211.12

215.91

2.26

219.98

3.82

20164-5

200.19

204.93

2.36

208.13

3.96

20164-6

198.99

203.01

2.02

207.0

4.02

20164-7

203.40

206.50

2.15

210.75

3.61

20164-8

207.6

212.12

1.96

215.36

4.00

20164-9

204.91

207.98

1.47

21.03

3.47

20164-10

207.11

210.19

1.48

214.50

3.56

Group-II

2000 mg/kg b. wt

20164-11

200.32

203.94

1.80

207.92

3.79

20164-12

201.91

204.99

1.52

208.62

3.32

20164-13

207.21

211.29

1.96

215.92

4.20

20164-14

205.14

209.31

2.03

213.34

3.99

20164-15

211.20

215.93

2.23

219.56

3.99

20164-16

212.12

216.18

1.91

220.38

3.89

20164-17

203.12

207.45

2.13

211.76

4.25

20164-18

200.19

204.91

2.35

218.87

4.33

20164-19

199.80

209.99

2.59

208.77

4.48

20164-20

208.11

212.09

1.91

215.74

3.68

TABLE – 3

CLINICAL SIGNS AND MORTALITY

Parameters

Incidence of Clinical Signs Observed after Dosing on

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

0/10

0

Clinical Signs- Local

 

Redness

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Pain

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Swelling

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Systemic signs

 

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Group: I Limit test                                                                        Dose: 2000 mg/kg b.wt

                                                                                                                                                          

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE – 3 Contd…………….

CLINICAL SIGNS AND MORTALITY

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

                                                                                                                                    

 

Parameters

Incidence of Clinical Signs Observed after Dosing on

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

0/10

0

Clinical Signs- Local

 

Redness

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Pain

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Swelling

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Systemic signs

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE – 4

SUMMARY OF NECROPSY FINDINGS

S. No.

 

Fate

 

Wistar albino rats

Dose (mg/kg b. wt)

2000

(limit test)

2000

(confirmatory test)

1

Terminal sacrifice

10/10

10/10

2

Found Dead

0/10

0/10

3

Abnormalities detected

0/10

0/10

TABLE – 5

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

Group-I (limit test)                                                                         2000 mg/kg b.wt.

Animal ID

Fate

Time

Gross Findings

20164-1

TS

Day 14

NAD

20164-2

TS

Day 14

NAD

20164-3

TS

Day 14

NAD

20164-4

TS

Day 14

NAD

20164-5

TS

Day 14

NAD

20164-6

TS

Day 14

NAD

20164-7

TS

Day 14

NAD

20164-8

TS

Day 14

NAD

20164-9

TS

Day 14

NAD

20164-10

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: II(confirmatory test)                                                     Dose: 2000 mg/kg b.wt.                                                                                

Animal ID

Fate

Time

Gross Findings

20164-11

TS

Day 14

NAD

20164-12

TS

Day 14

NAD

20164-13

TS

Day 14

NAD

20164-14

TS

Day 14

NAD

20164-15

TS

Day 14

NAD

20164-16

TS

Day 14

NAD

20164-17

TS

Day 14

NAD

20164-18

TS

Day 14

NAD

20164-19

TS

Day 14

NAD

20164-20

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 acute dermal LD50 value was considered to be >2000 mg/kg bw, when male/female Wistar albino rats were treated with the given test chemical by dermal application. Thus, it can be concluded that the test compound is non toxic to Wistar albino rats as per the criteria of CLP regulation.
Executive summary:

The acute dermal toxicity study was conducted according to OECD guideline 402 for testing of chemicals on Wistar albino rats. The summary of the study was as follows -

LIMIT TEST (2000 mg/kg b.wt): Ten healthy wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test compound was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. 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 body weight of each rat was observed on day 0 (pre treatment), 7th and 14th (post treatment). The necropsy was performed on all animals at termination of the study.

The test compound applied at the dose level of 2000 mg/kg b.wt in Wistar albino rats did not show any mortality as well as clinical signs of toxicity throughout the observation period of 14 days.  Necropsy finding did not reveal any gross pathological changes under test condition.

CONFIRMATORY TEST: After 72 hrs, a confirmatory test was conducted in same species of animals to confirm the results obtained from limit test (OECD-402 guidelines). Ten healthy Wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test chemical was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. 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 body weight of each rat was observed on day 0 (pre treatment), 7th and 14th (post treatment). The necropsy was performed on all animals which was died during the study or were sacrificed at termination of the study.

No mortality was recorded in Wistar albino rats after administration of test compound at the dose level of 2000 mg/kg b.wt throughout the period of observation (14 days). The test compound did not elicit any clinical signs of toxicity during the observation period. No skin reaction was observed after 24th hrs of patch removal. The body weight of each animal recorded on day 7th and 14th showed normal increase as compared to day 0 (pre treatment).

From the results obtained from present investigation, the acute dermal LD50 value was considered to be >2000 mg/kg bw, when male/female Wistar albino rats were treated with the given test chemical by dermal application. Thus, it can be concluded that the test compound is non toxic to Wistar albino rats as per the criteria of CLP regulation.