Registration Dossier

Administrative data

Description of key information

Acute oral toxicity:

Acute oral toxicity study was conducted in order assess the toxicological profile of the test item on wistar rats. Based on the results, The LD50 of test chemical was determined to be 5000 mg/kg ,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 4.55E-07 Pa. 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 rabbits 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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Principles of method if other than guideline:
The objective of this acute oral toxicity study was to assess the toxicological profile of the test item when administered to rats by a single oral gavage.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
- Purity as per Certificate of Analysis:97%
- Lot No.:20120720
- Manufactured date:DEC-2013
- Retest date:20 June 2027
- pH:4.69 at 26.8°C
- Density:1.176 g/cm3 at 27°C
- Storage conditions:Ambient (+15 to +25°C)
- SAFETY PRECAUTIONS: Gloves, cap and face mask were used in addition to protective body garments and shoes, to ensure adequate personal health and safety and to avoid inhalation and skin contact with the test item.
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd. Bengaluru
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 12 Weeks
- Weight at study initiation: 177.51 g to 202.61 g
- Identification:By rat accession number. Identification of individual rats was by cage card and turmeric colour body marking. The rat accession number was allotted during the course of the study. The temporary body marking during acclimatization period was done with crystal violet.
- Fasting period before study: rats were fasted for approximately 16 to 18 hours
- Housing:Three rats/cage were housed (except during exposure period and up to observation period of 48 hours wherein housed individually) in the standard polysulfone cages (Size: 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 at least once a week.
- Diet (e.g. ad libitum):Hypro Rat & Mice pellet feed
- 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, Mumbai, ad libitum
- Acclimation period: The animals were acclimatized six days for G1-FTS, eight days for G1-STS and twelve days for G2-FTS before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 65 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: 03 July 2018 To: 09 July 2018
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 300 & 2000 mg/kg body weight.
- Amount of vehicle (if gavage):dose volume was 20 mL/kg
Doses:
G1 (FTS) - 300 mg/kg
G1 (STS) - 300 mg/kg
G2 (FTS) - 2000 mg/kg
G2 (STS) - 2000 mg/kg
G2 (FTS) - 2000 mg/kg
No. of animals per sex per dose:
G1 (FTS) - 300 mg/kg - 3
G1 (STS) - 300 mg/kg - 3
G2 (FTS) - 2000 mg/kg - 3
G2 (STS) - 2000 mg/kg - 3
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical signs and pre-terminal deaths - At each step, the animals were observed five times on test day 1 (day of administration) i.e. at 30 minutes and four times at hourly intervals and once daily during days 2 to 15 post administration. Observations included 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 the observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma and all observed clinical signs were recorded. Body weights - The body weights were recorded on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration).
- Necropsy of survivors performed: yes, the rats surviving to the end of the observation period were euthanised by using isoflurane anaesthesia and subjected to detailed necropsy.
- Other examinations performed: Gross pathological findings were recorded and reported. Microscopic examination was not carried out as no gross pathological changes were observed.
Statistics:
not specified
Preliminary study:
not specified
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
5 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no other details available
Mortality:
G1 (FTS) - 300 mg/kg - There were no pre-terminal deaths observed.
G1 (STS) - 300 mg/kg - There were no pre-terminal deaths observed.
G2 (FTS) - 2000 mg/kg - No deaths
G2 (STS) - 2000 mg/kg - No deaths
Clinical signs:
G1 - [300 mg/kg body weight - Treatment (FTS and STS)]: There were no clinical sings and pre-terminal deaths.
G2 - [2000 mg/kg body weight - Treatment (FTS and STS)]: There were no clinical signs observed in any of the rats.
Body weight:
G1 - [300 mg/kg body weight - Treatment (FTS and STS)]: The body weights of all the rats increased throughout the observation period.
G2 - [2000 mg/kg body weight - Treatment (FTS and STS)]: The body weights of all the rats increased throughout the observation period.
Gross pathology:
There were no gross pathological changes at necropsy.
Other findings:
not specified

TABLE 1.      Body weight, body weight change and pre-terminal deaths

Group and

Dose

(mg/kg

body weight)

Rat

No.

Sex

Body weight (g)

Day of Death

 (Time of Death)

No. dead/

 No. tested

Pre-terminal deaths

(%)

Initial

(Day 1)

8thday

Weight change

(day 8 – Initial)

15thday

Weight change          

(day 15 – Initial)

At Death

G1

(FTS)

300

 

Rw271

F

183.00

189.24

6.24

196.41

13.41

NA

NA

 

0/3

 

0

Rw272

F

191.08

198.16

7.08

203.67

12.59

NA

NA

Rw273

F

192.26

201.49

9.23

208.12

15.86

NA

NA

G1

(STS)

300

 

Rw274

F

194.47

201.67

7.2

208.38

13.91

NA

NA

 

0/3

 

0

Rw275

F

202.61

209.28

6.67

215.83

13.22

NA

NA

Rw276

F

191.21

198.96

7.75

204.71

13.50

NA

NA

Group and

Dose

(mg/kg

body weight)

Rat

No.

Sex

Body weight (g)

Day of Death

 (Time of Death)

No. dead/

 No. tested

Pre-terminal deaths

(%)

Initial

(Day 1)

8thday

Weight change

(day 8 – Initial)

15thday

Weight change          

(day 15 – Initial)

At Death

G2

(FTS)

2000

 

Rw277

F

190.07

198.12

8.05

204.96

14.89

NA

NA

 

0/3

 

0

Rw278

F

189.94

199.61

9.67

207.21

17.27

NA

NA

Rw279

F

177.51

186.32

8.81

194.68

17.17

NA

NA

G2

(STS)

2000

 

Rw280

F

187.43

195.82

8.39

203.78

16.35

NA

NA

 

0/3

 

0

Rw281

F

186.19

197.13

10.94

206.18

19.99

NA

NA

Rw282

F

180.49

189.78

9.29

198.19

17.70

NA

NA

F: Female        FTS: First Treatment Step            STS: Second Treatment Step             NA: Not Applicable           

 

APPENDIX 1.      Individual clinical signs, dose administration and necropsy findings

Group and

Dose

(mg/kg

body weight)

Date and

time of administration

Rat

No.

Sex

Body

weight

(Day 1)

(g)

Total volume administered

(mL)

Day of observation

Day 1

30 min

1hour

2 hours

3 hours

4 hours

G1

(FTS)

300

 

 

03 July 2018 and

11:30 AM

to

11:33 AM

Rw271

F

183.00

3.66

N

N

N

N

N

Rw272

F

191.08

3.82

N

N

N

N

N

Rw273

F

192.26

3.85

N

N

N

N

N

Group and

Dose

(mg/kg

body weight)

Rat

No.

Sex

Day of observation

Necropsy

Findings

2

3

4

5

6

7

8

9

10

11

12

13

14

15

G1

(FTS)

300

 

Rw271

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw272

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw273

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

F: Female            FTS: First treatment step              N: Normal            min: minutes         mg: milligrams            kg: kilograms       

mL: millilitre         055 (b): Recumbent;            043 (1): Ataxia – slight;              NAD: No Abnormality Detected 

 

APPENDIX 2 contd. Individual clinical signs, dose administration and necropsy findings               

Group and

Dose

(mg/kg

body weight)

Date and

time of administration

Rat

No.

Sex

Body

weight

(Day 1)

(g)

Total volume administered

(mL)

Day of observation

Day 1

30 min

1hour

2 hours

3 hours

4 hours

G1

(STS)

300

 

 

05 July 2018 and

11:48 AM

to

11:50 AM

Rw274

F

194.47

3.89

N

N

N

N

N

Rw275

F

202.61

4.05

N

N

N

N

N

Rw276

F

191.21

3.82

N

N

N

N

N

  

 

Group and

Dose

(mg/kg

body weight)

Rat

No.

Sex

Day of observation

Necropsy

Findings

2

3

4

5

6

7

8

9

10

11

12

13

14

15

G1

(STS)

300

 

Rw274

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw275

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw276

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

F: Female        STS: Second treatment step               N: Normal              min: minutes       mg: milligrams                      kg: kilograms             

mL: millilitre         043 (1): Ataxia – slight;             NAD: No Abnormality Detected           

 

APPENDIX 2 contd. Individual clinical signs, dose administration and necropsy findings         

Group and

Dose

(mg/kg

body weight)

Date and

time of administration

Rat

No.

Sex

Body

weight

(Day 1)

(g)

Total volume administered

(mL)

Day of observation

Day 1

30 min

1hour

2 hours

3 hours

4 hours

G2

(FTS)

2000

 

 

10 July 2018 and

11:33 AM

to

11:35 AM

Rw277

F

190.07

3.80

N

N

N

N

N

Rw278

F

189.94

3.80

N

N

N

N

N

Rw279

F

177.51

3.55

N

N

N

N

N

        

 

Group and

Dose

(mg/kg

body weight)

Rat

No.

Sex

Day of observation

Necropsy

Findings

2

3

4

5

6

7

8

9

10

11

12

13

14

15

G2

(FTS)

2000

 

Rw277

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw278

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rw279

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

F: Female         FTS: First treatment step                 min: minutes   mg: milligrams                       kg: kilograms     mL: millilitre      

055 (b): Recumbent;    016: Dead;              NAD: No Abnormality Detected

 


Interpretation of results:
other: Not classified
Conclusions:
Acute oral toxicity study was conducted in order assess the toxicological profile of the test item on wistar rats. Based on the results, The LD50 of test chemical was determined to be 5000 mg/kg ,Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity.
Executive summary:

The acute oral toxicity study with test chemical in Wistar rats was conducted to assess the toxicological profile of the test item. The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (16 to 18 hours) three female rats (G1-FTS) at the dose of 300 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths observed. As all the rats survived at this step, the test was confirmed with three additional female animals with the same dose of 300 mg/kg body weight (G1-STS). There were no clinical signs of toxicity and pre-terminal deaths observed at this step.Based on the scheme - Annex 2c of the guideline OECD 423, the test was repeated at the dose of 2000 mg/kg body weight (G2-FTS). There were no clinical signs of toxicity observed. Hence, the test was confirmed with three additional female rats at the same dose of 2000 mg/kg body weight (G2-STS) as per the scheme - Annex 2c of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths observed at this confirmatory test. Hence, the further dosing was stopped.The rats were observed for mortality and clinical signs for 14 days post treatment. Body weights were recorded prior to dosing on day 1 and again on days 8 and 15. Gross necropsy was performed for all the rats at termination. The body weight growth of all rats was unaffected by the test item. There were no gross pathological changes at necropsy, hence histopathology was not performed.Based on the results of the present study, the LD50of test item is 5000 mg/kg body weight or infinity as per LD50cut-off value,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:
5 000 mg/kg bw
Quality of whole database:
Data is Klimisch 1 and from experimental study report.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Quality of whole database:
Waiver

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
The objective of this acute dermal toxicity study was to assess the toxicological profile of the test item on application as a single semi-occlusive dermal application to rats.
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
no
Specific details on test material used for the study:
- Purity as per Certificate of Analysis:97%
- Lot No.:20120720
- Manufactured date:DEC-2013
- pH:4.69 at 26.8°C
- Density:1.176 g/cm3 at 27°C
- Storage conditions:Ambient (+15 to +25°C)
- SAFETY PRECAUTIONS: Gloves, cap and face mask were used in addition to protective body garments and shoes, to ensure adequate personal health and safety and to avoid inhalation and skin contact with the test item.
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd. Bengaluru
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: Females: 235.18 to 238.33 g
- Identification:By rat accession number. Identification of individual rats is by cage card and crystal violet colour body markings. The temporary body marking during acclimatization period was done with crystal violet. The rat accession numbers were allotted during the course of the study and was included in raw data and reported.
- Housing: Animals were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill. Additionally, polycarbonate rat huts were placed inside the cage as enrichment objects and were changed along with the cage once a week. Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.
- Diet (e.g. ad libitum): Hypro Rat & Mice pellet feed, ad libitum
- 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, ad libitum
- Acclimation period: The rats were acclimatized for six, eight, twelve and fourteen days before treatment for dose range finding and main study respectively under standard laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 65 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: 03 July 2018 To: 09 July 2018
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: clipped skin of dorsolateral thoracic surface
- % coverage: 10% of the body surface
- Type of wrap if used: The applied area was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso.

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

-Preparation of test site for application
Approximately 24 hours before treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 10 x 8 cm) with an electric clipper Care was taken to avoid abrading the skin, which could alter its permeability
Duration of exposure:
24 hours
Doses:
DRF G1 - 200 mg/kg
DRF G2 - 1000 mg/kg
DRF G3 - 2000 mg/kg
Main G3 - 2000 mg/kg
No. of animals per sex per dose:
DRF G1 - 200 mg/kg - 1
DRF G2 - 1000 mg/kg - 1
DRF G3 - 2000 mg/kg - 1
Main G3 - 2000 mg/kg - 2
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical examination and pre-terminal deaths - 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. In addition, the treatment site was observed for skin reactions at 24, 48 and 72 hours after removal of test chemical using the Draize criteria. 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).
- Necropsy of survivors performed: yes, 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.
- Other examinations performed: Microscopic examination was not carried out as no gross pathological changes were observed.
Statistics:
not specified
Preliminary study:
not specified
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no other details available
Mortality:
There were no pre-terminal deaths (mortality) observed during the study.
Clinical signs:
There were no clinical signs observed during the study.
There were no skin reactions at the site of application at 24 and 48 hours after test patch removal (as per draize method).
Body weight:
All rats gained body weight throughout the observation period
Gross pathology:
No abnormality was detected at necropsy.
Other findings:
not specified

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

200

DRF

Rw283

F

235.18

242.68

7.50

248.19

13.01

0

G2 and

1000

DRF

Rw284

F

238.33

245.45

7.12

251.67

13.34

0

G3 and

2000

DRF

Rw285

F

238.16

244.73

6.57

249.14

10.98

0

G3 and

2000

Main study

Rw286

F

230.34

236.59

6.25

241.08

10.74

0

Rw287

F

234.62

242.93

8.31

247.63

13.01

0

 DRF: Dose Range Finding   F: Female

APPENDIX 1.      Individual test item application, clinical signs, skin reactionand 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

(mL)

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

200

DRF

11 April 2018

and

10.55 AM

Rm8907

F

223.58

0.04

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

Observation

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and

200

DRF

Rm8907

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

 

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

30

 min

1 h

2 h

3 h 

4 h

5 h

6 h

*

Er

@

Ed

@

*

Er

@

Ed@

G1- DRF

 and

200

 

03 July 2018

and

11.36 AM

Rw283

F

235.18

47

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G1- DRF

 and

200

 

Rw283

F

N

0

0

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

 

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

30

 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er

@

Ed

@

*

Er

@

Ed

@

G2- DRF

 and

1000

 

05 July 2018

and

11.56 AM

Rw284

F

238.33

238

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G2- DRF

 and

1000

 

Rw284

F

N

0

0

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

30

min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

G3- Main

and

2000

 

12 July 2018

and

11.41 AM to 11.42 AM

Rw286

F

230.34

461

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Rw287

F

234.62

469

N

N

N

N

N

N

N

N

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G3- Main

 and

2000

 

Rw286

F

N

0

0

N

N

N

N

N

N

N

N

N

N

NAD

Rw287

F

N

0

0

N

N

N

N

N

N

N

N

N

N

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

 


Interpretation of results:
other: Not classified
Conclusions:
Acute dermal toxicity study was conducted in order assess the toxicological profile of the test item on wistar rats. Based on the results, The LD50 of test chemical was determined to be >2000 mg/kg ,Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute dermal toxicity.
Executive summary:

The acute dermal toxicity of test chemical in Wistar rats was tested with 200 (G1-DRF), 1000 (G2-DRF) and 2000 (G3-DRF) mg/kg with 1 female each for the dose range finding study and 2 female for main study (G3).Based on the individual body weight, the test item at the dose of 200, 1000 and 2000 mg/kg body weight was weighed on an aluminum foil and made as a paste in Milli-Q water and applied directly to the clipped skin 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 wound 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 the site of application of test item. Body weights were measured on Days 1, 8 and 15 and growth was unaffected by test item. At the end of observation period (Day 15), all animals were euthanized and subjected to necropsy, and there were no gross lesions noted at the necropsy.Based on the present study results, the acute dermal LD50of test chemical is greater than 2000 mg/kg body weight in female Wistar rats.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 1 and from experimental study report.

Additional information

Acute oral toxicity:

The acute oral toxicity study with test chemical in Wistar rats was conducted to assess the toxicological profile of the test item. The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (16 to 18 hours) three female rats (G1-FTS) at the dose of 300 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths observed. As all the rats survived at this step, the test was confirmed with three additional female animals with the same dose of 300 mg/kg body weight (G1-STS). There were no clinical signs of toxicity and pre-terminal deaths observed at this step.Based on the scheme - Annex 2c of the guideline OECD 423, the test was repeated at the dose of 2000 mg/kg body weight (G2-FTS). There were no clinical signs of toxicity observed. Hence, the test was confirmed with three additional female rats at the same dose of 2000 mg/kg body weight (G2-STS) as per the scheme - Annex 2c of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths observed at this confirmatory test. Hence, the further dosing was stopped.The rats were observed for mortality and clinical signs for 14 days post treatment. Body weights were recorded prior to dosing on day 1 and again on days 8 and 15. Gross necropsy was performed for all the rats at termination. The body weight growth of all rats was unaffected by the test item. There were no gross pathological changes at necropsy, hence histopathology was not performed.Based on the results of the present study, the LD50of test item is 5000 mg/kg body weight or infinity as per LD50cut-off value,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 4.55E-07 Pa. 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 of test chemical in Wistar rats was tested with 200 (G1-DRF), 1000 (G2-DRF) and 2000 (G3-DRF) mg/kg with 1 female each for the dose range finding study and 2 female for main study (G3).Based on the individual body weight, the test item at the dose of 200, 1000 and 2000 mg/kg body weight was weighed on an aluminum foil and made as a paste in Milli-Q water and applied directly to the clipped skin 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 wound 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 the site of application of test item. Body weights were measured on Days 1, 8 and 15 and growth was unaffected by test item. At the end of observation period (Day 15), all animals were euthanized and subjected to necropsy, and there were no gross lesions noted at the necropsy.Based on the present study results, the acute dermal LD50of test chemical is greater than 2000 mg/kg body weight in female Wistar rats.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 on test chemical, it can be concluded that LD50 value is >2000 mg/kg bw and 5000 mg/kg bw for acute dermal and acute oral toxicity respectively. 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.