Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Acute oral toxicity: 

The acute oral toxicity dose (LD50) was considered based on different studies conducted on rats for the given test chemical. The studies concluded that 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 cannot be classified for acute oral toxicity.

Acute Inhalation Toxicity:

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 6.79E-42 mm Hg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

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 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
experimental data from various test chemicals
Justification for type of information:
Data is summarized based on the available information from various test chemicals.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on 3 acute oral toxicity studies as - WoE 2, WoE 3 and WoE-4.
Acute oral toxicity test was carried out to study the effects of the test chemicals on rodents.
GLP compliance:
not specified
Test type:
other: not specified
Limit test:
no
Species:
other: 2. mouse 3. rat 4. rat
Strain:
other: 2. DDY 3. Sprague-Dawley 4. Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
2. TEST ANIMALS
Source: Japan SLC Co., Shizuoka, Japan
Age at study initiation: 7 weeks
Weight at study initiation: no data
Fasting period before study: no data
Housing: no data
Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan), ad libitum
Water (e.g. ad libitum): Tap Water, ad libitum
Acclimation period: 1 week of acclimatization

ENVIRONMENTAL CONDITIONS
Temperature (°C): The animal room was kept at 20–24°C.
Humidity (%): No data
Air changes (per hr): No data
Photoperiod (hrs dark / hrs light): 12 h Light – dark cycle.
3. 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 191.5 to 205.7 grams.
Body weights at the start : Female Mean : 199.26 g (= 100 %); Minimum: 191.5 g (- 3.89 %); Maximum: 205.7 g (+ 3.23 %) 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 20.1 to 22.7 degree centigrade.
- Humidity (%): Room humidity was maintained at 55.1% to 61.2%.
- 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.
4. 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.
- Weight at study initiation: Body weight range was 199.1 to 219.9 grams. Body weights at the start : Female Mean : 206.81 g (= 100 %); Minimum : 199.1 g (- 3.73 %); Maximum : 219.9 g (+ 6.33 %) 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): 20.6 to 23.2 degree centigrade.
- Humidity (%): 55.1% to 58.6%.
- 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: 26-09-2016 to 15-10-2016
Route of administration:
oral: gavage
Vehicle:
other: 2. physiological saline 3. Distilled water 4. Distilled water
Details on oral exposure:
2. MAXIMUM DOSE VOLUME APPLIED: 0.5 × LD50 or the limit dose of 2000 mg/kg.
3. MAXIMUM DOSE VOLUME APPLIED: 10 ml per kg of body weight was considered the maximum volume which could be administered to a rat.
4. VEHICLE
- Concentration in vehicle: 300 mg/kg, 300 mg/kg, 2000 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 per kg of body weight.
Doses:
2. 0.5 × LD50 or the limit dose of 2000 mg/kg.
3. Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
4. Dose Group I : 300 mg/kg
Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
Dose Group II : 2000 mg/kg
No. of animals per sex per dose:
2. 4 - 5 male mice
3. Three females were used at each step.
4. Three females were used at each step.
Control animals:
not specified
Details on study design:
2. no data
3. - 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.
4. - 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 therea fter 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.
Histopathology: No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.
Statistics:
No data
Preliminary study:
No data
Sex:
male
Dose descriptor:
LD50
Remarks:
2
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality was observed
Sex:
female
Dose descriptor:
LD50
Remarks:
3
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality was observed
Sex:
female
Dose descriptor:
LD50
Remarks:
4
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality was noted
Mortality:
2. no mortality observed at dose 2000 mg/kg bw.
3. 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.
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: 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 : All animals survived through the study period of 14 days.
4. 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:
2. no data
3. 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.
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.
Group II Step I : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (reddish colour stools) in all animals 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 on day 1 after the dosing.
Group II Step II : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (reddish colour stools) in all animals 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 on day 1 after the dosing.
Staining of the stool is attributed to the reddish colour of the test item.
4. 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.
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.
Group II Step I : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Group II Step II : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals 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 on day 1 after the dosing.
Staining of the stool is attributed to the black colour of the test item.
Body weight:
2. no data
3. Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.26% and 12.80% respectively.
Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.49% and 13.33% respectively.
Group II Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.42% and 14.33% respectively.
Group II Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.85% and 12.91% respectively.
4. Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.45% and 12.76% respectively.
Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.56% and 12.49% respectively.
Group II Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.06% and 10.98% respectively.
Group II Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 2.17% and 9.86% respectively.
Gross pathology:
2. no data
3. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.
4. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.
Other findings:
No data

2.

Table 1: Acute Oral Toxicity study results

 

Test chemical

Vehicle

Source*

LD50**

mg/kg

TEST CHEMICAL

Saline

T

>2000

 

*T = Tokyo Kasei Kogyo Industry Ltd., Tokyo, Japan;

** In order to set appropriate doses for the assay, we determined approximate LD50 by simple acute toxicity experiments on four–five animals. When no death was observed at 2000 mg/kg, the LD50 was defined as >2000 mg/kg.

3.

Individual Animal - Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

 Group I :   

 

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

I

300

3

No clinical signs observed

1

Day 0 - Day 14

0

No clinical signs observed

2

Day 0 - Day 14

0

No clinical signs observed

3

Day 0 - Day 14

0

 

  Group I :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

II

300

3

No clinical signs observed

4

Day 0 - Day 14

0

No clinical signs observed

5

Day 0 - Day 14

0

No clinical signs observed

6

Day 0 - Day 14

0

 

  Group II :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

I

2000

3

Diarrhoea (Reddish colour stools)

7

4 hrs. - 6 hrs.

0

Diarrhoea (Reddish colour stools)

8

4 hrs. - 6 hrs.

0

Diarrhoea (Reddish colour stools)

9

4 hrs. - 6 hrs.

0

 

  Group II :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

II

2000

3

Diarrhoea (Reddish colour stools)

10

4 hrs. - 6 hrs.

0

Diarrhoea (Reddish colour stools)

11

4 hrs. - 6 hrs.

0

Diarrhoea (Reddish colour stools)

12

4 hrs. - 6 hrs.

0

 

Staining of the stool is attributed to the reddish colour of the test item.

 

  

Individual Animal - Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group : I                       Step I :                                 Dose  : 300 mg/kg body weight

Animal No.

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

1

193.2

202.5

4.81

219.9

8.59

13.82

2

197.4

203.7

3.19

222.5

9.23

12.72

3

201.5

205.1

1.79

225.4

9.90

11.86

 

Group : I                       Step II :                                 Dose  : 300 mg/kg body weight

Animal No.

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

4

203.9

210.2

3.09

228.6

8.75

12.11

5

198.5

206.2

3.88

224.5

8.87

13.10

6

196.4

203.3

3.51

225.4

10.87

14.77

 

Group : II                      Step I :                                 Dose  : 2000 mg/kg body weight

Animal No.

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

7

202.1

210.2

4.01

229.6

9.23

13.61

8

198.2

206.3

4.09

225.1

9.11

13.57

9

191.5

201.4

5.17

221.8

10.13

15.82

 

Group : II                      Step II :                                 Dose  : 2000 mg/kg body weight

Animal No.

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

10

204.0

210.2

3.04

227.6

8.28

11.57

11

205.7

214.3

4.18

232.2

8.35

12.88

12

198.7

207.3

4.33

227.1

9.55

14.29

 

 

Appendix No.III

 

Individual Animal - Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

Group : I

Step I :

Dose  : 300 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

1

TS

No abnormality detected

2

TS

No abnormality detected

3

TS

No abnormality detected

 

Group : I

Step II :

Dose  : 300 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

4

TS

No abnormality detected

5

TS

No abnormality detected

6

TS

No abnormality detected

 

Group : II

Step I :

Dose  : 2000 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

7

TS

No abnormality detected

8

TS

No abnormality detected

9

TS

No abnormality detected

 

Group : II

Step II :

Dose  : 2000 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

10

TS

No abnormality detected

11

TS

No abnormality detected

12

TS

No abnormality detected

 

TS = Terminal sacrifice

4.

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 - 3

0 to 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 - 6

0 to 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

Diarrhoea

(Black colour stools)

3

7,9

8

4 hrs. - 6 hrs.

2 hrs. - 6 hrs.

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

Diarrhoea

(Black colour stools)

3

10,11,12

4 hrs. - 6 hrs.

0/3

 

Staining of the stool is attributed to the black colour of the test item.

 

 

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

201.47

210.43

4.45

227.17

7.95

12.76

± SD

2.63

2.15

0.39

2.19

0.25

0.68

 

 

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

203.23

210.47

3.56

228.60

8.62

12.49

± SD

1.35

1.86

0.76

1.71

0.87

1.33

 

 

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

207.00

213.33

3.06

229.73

7.69

10.98

± SD

2.52

2.51

0.67

3.33

0.29

0.81

 

 

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

215.53

220.20

2.17

236.77

7.52

9.86

± SD

4.45

3.22

0.67

3.50

0.29

0.91

 

 

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

Appendix No.I

 

Individual Animal - Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

 Group I :   

 

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

I

300

3

No clinical signs observed

1

Day 0 - Day 14

0

No clinical signs observed

2

Day 0 - Day 14

0

No clinical signs observed

3

Day 0 - Day 14

0

 

  Group I :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

II

300

3

No clinical signs observed

4

Day 0 - Day 14

0

No clinical signs observed

5

Day 0 - Day 14

0

No clinical signs observed

6

Day 0 - Day 14

0

 

  Group II :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

I

2000

3

Diarrhoea (Black colour stools)

7

4 hrs. - 6 hrs.

0

Diarrhoea (Black colour stools)

8

2 hrs. - 6 hrs.

0

Diarrhoea (Black colour stools)

9

4 hrs. - 6 hrs.

0

 

  Group II :

Step

No.

Dose

mg/kg

Total Number of

Animals

Observed Signs

Animal Nos.

Period of signs

in days

From - to

Mortality

II

2000

3

Diarrhoea (Black colour stools)

10

4 hrs. - 6 hrs.

0

Diarrhoea (Black colour stools)

11

4 hrs. - 6 hrs.

0

Diarrhoea (Black colour stools)

12

4 hrs. - 6 hrs.

0

 

Staining of the stool is attributed to the black colour of the test item.

 

 

Appendix No.II

 

Individual Animal - Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group : I                       Step I :                                 Dose  : 300 mg/kg body weight

Animal No.

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

1

201.0

210.6

4.78

227.9

8.21

13.38

2

199.1

208.2

4.57

224.7

7.93

12.86

3

204.3

212.5

4.01

228.9

7.72

12.04

 

Group : I                       Step II :                                 Dose  : 300 mg/kg body weight

Animal No.

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

4

201.9

210.3

4.16

230.2

9.46

14.02

5

203.2

208.7

2.71

226.8

8.67

11.61

6

204.6

212.4

3.81

228.8

7.72

11.83

 

Group : II                      Step I :                                 Dose  : 2000 mg/kg body weight

Animal No.

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

7

209.8

215.3

2.62

232.4

7.94

10.77

8

206.3

214.2

3.83

230.8

7.75

11.88

9

204.9

210.5

2.73

226.0

7.36

10.30

 

Group : II                      Step II :                                 Dose  : 2000 mg/kg body weight

Animal No.

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

10

219.9

223.6

1.68

240.7

7.65

9.46

11

215.7

219.8

1.90

235.6

7.19

9.23

12

211.0

217.2

2.94

234.0

7.73

10.90

 

 

Appendix No.III

 

Individual Animal - Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

Group : I

Step I :

Dose  : 300 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

1

TS

No abnormality detected

2

TS

No abnormality detected

3

TS

No abnormality detected

 

Group : I

Step II :

Dose  : 300 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

4

TS

No abnormality detected

5

TS

No abnormality detected

6

TS

No abnormality detected

 

Group : II

Step I :

Dose  : 2000 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

7

TS

No abnormality detected

8

TS

No abnormality detected

9

TS

No abnormality detected

 

Group : II

Step II :

Dose  : 2000 mg/kg body weight

Animal No.

Fate

Gross Pathological Findings

10

TS

No abnormality detected

11

TS

No abnormality detected

12

TS

No abnormality detected

 

TS = Terminal sacrifice

Interpretation of results:
other: not classified
Conclusions:
The test chemical cannot be classified for acute oral toxicity, as the LD50 value is >2000 mg/kg bw according to CLP regulation.
Executive summary:

In different studies, the given test chemical 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 the given test chemical. The studies are summarized as below –

 

Acute oral toxicity experiment was conducted prior to Comet assay in male DDY mice. Male ddY mice were obtained from Japan SLC Co., Shizuoka, Japan, at 7 weeks of age and used after 1 week of acclimatization. They were fed commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) and tap water ad libitum throughout the acclimatization period and the experiment. The animal room was kept at 20–24°C with a 12 h light – dark cycle. In order to set appropriate doses for the assay, the approximate LD50 was determined by simple acute toxicity experiments on four–five animals. When no death was observed at 2000 mg/kg, the LD50 was defined as >2000 mg/kg. The acute oral LD50 in male DDY mice was determined to be greater than 2000 mg/kg after dosing with test chemical.

 

The above study is supported with another acute oral toxicity study designed and conducted to determine LD5 value of the test chemical in Sprague Dawley rats. The study was conducted as per OECD 423 Guidelines. 12 Female Sprague Dawley rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing and its weight were within ± 20% of the mean weight of any animal used for dosing. Body weight range was 191.5 to 205.7 grams. Females were nulliparous and non- pregnant. 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 resulted in diarrhoea (reddish colour stools) in all animals with onset at 4 hours and no 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 resulted in diarrhoea (reddish colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool was attributed to the reddish colour of the test item. 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 LD50 of the test chemical was greater than 2000mg/kg. It was classified under the category "Not Classified".

 

Both the above studies are further supported with the reported study designed and conducted to determine the acute oral toxicity profile of test chemical 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 resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours and no 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 resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool is attributed to the black colour of the test item. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. Under the condition of the study, the acute oral LD50 value was considered o be >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of the given test chemical, when administered via oral route in Sprague Dawley rats falls into the Category "Not classified” criteria of CLP.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that 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 cannot be classified for acute oral toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is Klimisch 2 and from handbook or collection of data.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
exposure considerations
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
Quality of whole database:
Waiver

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
experimental data from various test chemicals
Justification for type of information:
Data is summarized based on the available information from various test chemicals.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on 2 acute dermal toxicity studies as - WoE 2 and WoE 3.
Acute dermal toxicity test was carried out to study the effects of the test chemicals on rodents.
GLP compliance:
not specified
Test type:
other: not specified
Limit test:
no
Species:
rat
Strain:
other: 2. Wistar 3. Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
2. TEST ANIMALS
- Source: Vivo Bio Tech Ltd. Telangana
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 10 Weeks
- Weight at study initiation: 159.10 g to 169.08 g
- Fasting period before study: Rats were fasted overnight
- Identification:By rat accession number. Identification of individual rats was by cage card and turmeric colour body markings. The rat accession number was allotted during the course of the study. The temporary body marking during acclimatization period was done with crystal violet.
- Housing:Rats were housed individually in standard polysulfone cages
(Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage once a week
- Diet (e.g. ad libitum): Rat & Mice pellet feed, manufactured by Krishna Valley Agro Tech LLP, MIDC Kupwad block, Sangli, Maharashtra, was provided to animals
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes
- Acclimation period:After physical examination for good health and suitability for experiment, the animals were acclimatized five days for G1-FTS and ten days for G1-STS before treatment. Animals were observed once daily during acclimatization period. Females were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 59 to 67%,
- Air changes (per hr): air conditioned with adequate fresh air supply (12.4 air changes/hour)
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From
3. TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females (if applicable) nulliparous and non-pregnant: [no]
- 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 201.4 to 232.7 grams at initiation of dosing were used.
Body weights at the start : Male Mean : 222.62 g (= 100 %); Minimum : 216.7 g (- 2.66 %); Maximum : 232.7 g (+ 4.53 %) Total No. of animals : 5
Female Mean : 207.88 g (= 100 %); Minimum : 201.4 g (- 3.12 %); Maximum : 218.1 g (+ 4.92 %) Total No. of animals : 5
- Fasting period before study: No data
- 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 20.1 to 21.9 degree centigrade.
- Humidity (%): Room humidity was maintained at 55.5% to 59.2%.
- 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
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
2. TEST SITE
- Area of exposure: dorsolateral thoracic surface of the skin
- % coverage: 10% of the body surface of the rat
- Type of wrap if used: The applied area was covered with cotton gauze

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the applied area was washed with deionized water and wiped dry using clean towel.
- Time after start of exposure:24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000 (1.99 mL/kg body weight) was applied
3. 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
- Constant volume or concentration used: No data
- For solids, paste formed: No data
Duration of exposure:
2. 24 hours
3. 24 hours
Doses:
2. DRF G1 - 2000 mg/kg
Main G2- 2000 mg/kg
3. 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:
2. 3 [ 1 female/group for dose range finding study and 2 female / main study group (1 for dose range finding study and 2 for main study)
3. 10 (5/sex).
Control animals:
not specified
Details on study design:
2. - Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs and pre-terminal deaths (mortality) once during first 30 minutes after application, and at hourly intervals for 6 hours after application on the day of treatment (day 1) and once daily during Days 2 to 15.
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical examination and pre-terminal deaths: All rats were observed for changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Body weights: Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application), and 15 (14 days post application).
Gross Pathology: At the end of the observation period, all rats were euthanised and exsanguinated under isoflurane anesthesia and subjected to detailed necropsy by an experienced prosector and the findings were recorded. Microscopic examination was not carried out as no gross pathological changes were observed.
3. - 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:
2. Dose range finding study - Selection of dose level: An initial starting dose of 200 mg/kg body weight was tested with 1 female rat (dose range finding study). As there was no mortality at this dose range finding study the dose range finding study was continued with 1 female rat (dose range finding study) at the dose of 1000 mg/kg body weight. There was no mortality, hence the dose range finding study was continued with 1 female rat (dose range finding study) at the dose of 2000 mg/kg body weight.
3. No data
Sex:
female
Dose descriptor:
LD50
Remarks:
2
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality was observed
Sex:
male/female
Dose descriptor:
LD50
Remarks:
3
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality observed
Mortality:
2. There were no pre-terminal deaths (mortality) observed during the study.
3. 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:
2. There were no clinical signs observed during the study.
3. 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:
2. All rats gained body weight throughout the observation period.
3. Sex : Male Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 10.60% and 21.82% respectively.
Sex : Female Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 6.04% and 11.76% respectively.
Gross pathology:
2. No abnormality was detected at necropsy.
3. Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.
Other findings:
2. not specified
3. - 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.

2.

TABLE 1.            Individual body weight, body weight changes and pre-terminal deaths

Group and

Dose

(mg/kg body weight)

Rat

No.

S

e

x

Body weight (g)

Pre-terminal deaths

Initial

(Day 1 - at treatment)

8th  

day

Weight change

(day 8 – Initial)

15th

day

Weight change

(day 15 – Initial)

G1 and

2000

DRF

Rm8779

F

229.80

244.45

14.65

232.63

2.83

0

G1 and

2000

Main study

Rm8780

F

237.85

239.22

1.37

248.70

10.85

0

Rm8781

F

240.16

242.81

2.65

249.49

9.33

0

 DRF: Dose Range Finding   F: Female

 

APPENDIX 2.    contd. Individual test item application, clinical signs, skin reaction and necropsy findings

Dose range finding study

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mg)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

 min

1 h

2 h

3 h 

4 h

5 h

6 h

*

Er

@

Ed

@

*

Er

@

Ed

@

*

Er

@

Ed

@

G1 and

2000

DRF

 

05 April 2018

and

10.27 AM

Rm8779

F

229.80

460

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and

2000

DRF

Rm8779

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                      NAD: No abnormality detected      Er: Erythema                      Ed: Edema  

Score 0: No Erythema / Edema       

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal


APPENDIX 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

main study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mg)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G1 and

2000

Limit test

Main

 

10 April 2018

and

10.14AM and 10.15 AM

Rm8780

F

237.85

476

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Rm8781

F

240.16

480

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observations

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G2 and

2000

Limit test

Main

 

Rm8780

F

N

N

N

N

N

N

N

N

N

N

NAD

Rm8781

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

3.

Individual Animal - 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

Day 0 - Day 14

0

2

Day 0 - Day 14

0

3

Day 0 - Day 14

0

4

Day 0 - Day 14

0

5

Day 0 - Day 14

0

 

 

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

Day 0 - Day 14

0

7

Day 0 - Day 14

0

8

Day 0 - Day 14

0

9

Day 0 - Day 14

0

10

Day 0 - Day 14

0

 

 

Individual Animal - Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

 

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

5

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Sex : Female  

Group : I

Dose  : 2000 mg/kg body weight

Animal

Dermal

D A Y S

 

No.

Reaction

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

6

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

8

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

9

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

10

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Individual Animal - Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex    : Male

Group : I

Dose  : 2000 mg/kg body weight

 

Animal No.

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

1

216.7

239.0

10.29

264.5

10.67

22.06

2

217.9

241.2

10.69

268.1

11.15

23.04

3

222.4

244.9

10.12

264.2

7.88

18.79

4

223.4

247.2

10.65

273.7

10.72

22.52

5

232.7

258.9

11.26

285.5

10.27

22.69

 

 

Sex    : Female

Group : I

Dose  : 2000 mg/kg body weight

 

Animal No.

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

6

201.4

213.4

5.96

224.5

5.20

11.47

7

202.0

216.7

7.28

228.7

5.54

13.22

8

206.6

218.4

5.71

231.4

5.95

12.00

9

211.3

223.4

5.73

235.7

5.51

11.55

10

218.1

230.2

5.55

241.1

4.74

10.55

 

 

Individual Animal - Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex    : Male

Group : I

Dose  : 2000 mg/kg body weight

 

Animal No.

Fate

Gross Pathological Findings

1

TS

No abnormality detected

2

TS

No abnormality detected

3

TS

No abnormality detected

4

TS

No abnormality detected

5

TS

No abnormality detected

 

 

Sex    : Female

Group : I

Dose  : 2000 mg/kg body weight

 

Animal No.

Fate

Gross Pathological Findings

6

TS

No abnormality detected

7

TS

No abnormality detected

8

TS

No abnormality detected

9

TS

No abnormality detected

10

TS

No abnormality detected

 

TS = Terminal sacrifice


Interpretation of results:
other: not classified
Conclusions:
According to CLP regulation, the test chemical cannot be classified for acute dermal toxicity, as the LD50 value is >2000 mg/kg bw.
Executive summary:

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

 

The acute dermal toxicity of the given test chemical was tested with 2000 mg/kg with 1 female for the dose range finding study, followed by additional 2 females for main study at the dose of 2000 mg/kg body weight in Wistar rats. Based on the individual body weight, the test item at the dose of 2000 mg/kg body weight was weighed on an aluminium foil and made as a paste in Milli-Q water and applied directly to the clipped skin (Clipping was done approximately 24 hour prior to treatment) of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wrapped around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weight was measured on days 1, 8 and 15 and all rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy. Based on the present study results, the acute dermal LD50of the given test chemical is more than 2000 mg/kg body weight in female Wistar rats.

 

The above study is supported with another study designed and conducted to determine the acute dermal toxicity profile of the test chemical in Sprague Dawley rats. The study was performed according to OECD 402 Guidelines. A limit test with 5 male and 5 female Sprague Dawley rats were carried out at the dose of 2000 mg/kg body weight. The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. 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 the test chemical when exposed to Sprague Dawley rats was greater than 2000 mg/kg. Hence the test chemical cannot be classified for acute dermal toxicity.

 

Thus, based on the above summarised studies for test chemical, it can be 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 cannot be classified for acute dermal toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is Klimisch 2 and from handbook or collection of data.

Additional information

Acute oral toxicity:

In different studies, the given test chemical 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 the given test chemical. The studies are summarized as below –

 

Acute oral toxicity experiment was conducted prior to Comet assay in male DDY mice. Male ddY mice were obtained from Japan SLC Co., Shizuoka, Japan, at 7 weeks of age and used after 1 week of acclimatization. They were fed commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) and tap water ad libitum throughout the acclimatization period and the experiment. The animal room was kept at 20–24°C with a 12 h light – dark cycle. In order to set appropriate doses for the assay, the approximate LD50 was determined by simple acute toxicity experiments on four–five animals. When no death was observed at 2000 mg/kg, the LD50 was defined as >2000 mg/kg. The acute oral LD50 in male DDY mice was determined to be greater than 2000 mg/kg after dosing with test chemical.

 

The above study is supported with another acute oral toxicity study designed and conducted to determine LD5 value of the test chemical in Sprague Dawley rats. The study was conducted as per OECD 423 Guidelines. 12 Female Sprague Dawley rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing and its weight were within ± 20% of the mean weight of any animal used for dosing. Body weight range was 191.5 to 205.7 grams. Females were nulliparous and non- pregnant. 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 resulted in diarrhoea (reddish colour stools) in all animals with onset at 4 hours and no 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 resulted in diarrhoea (reddish colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool was attributed to the reddish colour of the test item. 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 LD50 of the test chemical was greater than 2000mg/kg. It was classified under the category "Not Classified".

 

Both the above studies are further supported with the reported study designed and conducted to determine the acute oral toxicity profile of test chemical 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 resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours and no 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 resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool is attributed to the black colour of the test item. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. Under the condition of the study, the acute oral LD50 value was considered o be >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of the given test chemical, when administered via oral route in Sprague Dawley rats falls into the Category "Not classified” criteria of CLP.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that 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 cannot be classified for acute oral toxicity.

 

Acute Inhalation Toxicity:

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 6.79E-42 mm Hg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

Acute Dermal Toxicity:

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

 

The acute dermal toxicity of the given test chemical was tested with 2000 mg/kg with 1 female for the dose range finding study, followed by additional 2 females for main study at the dose of 2000 mg/kg body weight in Wistar rats. Based on the individual body weight, the test item at the dose of 2000 mg/kg body weight was weighed on an aluminium foil and made as a paste in Milli-Q water and applied directly to the clipped skin (Clipping was done approximately 24 hour prior to treatment) of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wrapped around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weight was measured on days 1, 8 and 15 and all rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy. Based on the present study results, the acute dermal LD50of the given test chemical is more than 2000 mg/kg body weight in female Wistar rats.

 

The above study is supported with another study designed and conducted to determine the acute dermal toxicity profile of the test chemical in Sprague Dawley rats. The study was performed according to OECD 402 Guidelines. A limit test with 5 male and 5 female Sprague Dawley rats were carried out at the dose of 2000 mg/kg body weight. The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. 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 the test chemical when exposed to Sprague Dawley rats was greater than 2000 mg/kg. Hence the test chemical cannot be classified for acute dermal toxicity.

 

Thus, based on the above summarised studies for test chemical, it can be 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 cannot be classified for acute dermal toxicity.

 

 

Justification for classification or non-classification

Based on the above studies for the test chemical, 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. For acute inhalation toxicity wavier was added so, not possible to classify.