Registration Dossier

Administrative data

Description of key information

Acute oral toxicity: 

Acute oral toxicity dose (LD50) of the given test chemical was considered based on experimental study conducted on rats, the LD50 value was considered to be >2000 mg/kg bw in rats. 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 the low vapour pressure of the test chemical, which is reported as 0.007 mmHg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver.

Acute Dermal Toxicity:

Acute Dermal toxicity dose (LD50) for the test chemical was considered based on experimental study conducted on rats, the value was considered to be >2000 mg/kg bw in rats. 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:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Principles of method if other than guideline:
The acute oral toxicity study of the given test chemical was performed in rat.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house animals, bred at Animal House, sa-FORD.CPCSEA Registration No. 1256/bc/09/CPCSEA.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Female rats of the age of approximately 8 to 9 weeks old were used.
- Weight at study initiation: Minimum: 129 g Maximum: 143 g (Individual body weights were within ± 4% prior to treatment after overnight fasting)
- Fasting period before study: The food was withheld prior to dosing and 3-4 hours post dosing but drinking water was provided ad libitum.
- Housing: The animals were housed individually in polycarbonate cages.All cages were provided with corn cobs (Sparconn Life Sciences Bangalore) SPAR – 24/ 2013.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rodent diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 400012.
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum via drinking bottles
- Acclimation period:Animal nos. 1-3 were acclimatized for five days, 4-6 for seven days, prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 21.00 to 24.40 degree centigrade.
- Humidity (%): Room humidity was maintained at 36.60% to 57.30%.
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 2000mg/kg bw
- Amount of vehicle (if gavage):10ml
- Justification for choice of vehicle: Test material soluble in corn oil
- Lot/batch no. (if required): MKBD4650

MAXIMUM DOSE VOLUME APPLIED: The maximum dose volume administered was 10 ml/kg body weight.
DOSAGE PREPARATION (if unusual): The dosing solution was prepared fresh, prior to dose administration.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
Dose 2000 mg/kg:6 female
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: once daily
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical Observations and General Appearance: After test item administration, individual animals were frequently observed at 30 minutes, 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all animals were observed once a day during the 14 day observation period.
Mortality: All animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period.
Body weights: All rats were weighed on days 0 (prior to dosing), 7 and 14.
Gross Pathology: At the end of 14 day observation period, all the survived rats were euthanised by overdose of CO2 for external and internal observations.
Statistics:
not specified
Preliminary study:
not specified
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality was observed
Mortality:
No mortality was observed in the animals treated with 2000 mg/kg dose throught out the 14 days observation period
Clinical signs:
At 2000 mg/kg, animal nos. 1, 4, 5 and 6 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and moderate to mild lethargy was observed at 2, 3 and 4 hours post dosing. Animal nos. 2 and 3 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and mild lethargy was observed at 2, 3 and 4 hours post dosing
Body weight:
Body weight gain was observed in all surviving animals treated with 2000 mg/kg body weight, during the 14 day observation period, as compared to day 0
Gross pathology:
No external and internal gross pathological changes were seen in the animals treated with 2000 mg/kg body weight during terminal sacrifice
Other findings:
not specified

Individual Animal Body Weight (g) andBody Weight Changes(%)

Sex:Female

Animal No.

Group/ Dose (mg/kg)

Body Weight Change (%)

Day 0

Day 7

Day 14

Day

0-7

Day

0-14

1

G1/ 2000

143

176

187

23.08

30.77

2

138

170

187

23.19

35.51

3

141

169

185

19.86

31.21

4

129

155

167

20.16

29.46

5

142

155

179

9.15

26.06

6

134

169

174

26.12

29.85

Summary of Animal Body Weight (g) and Body Weight Changes (%)

Sex:Female

Group/ Dose (mg/kg)

Rats Body Weight (g)

Body Weight Changes (%)

Day 0

Day 7

Day 14

0-7

0-14

G1/ 2000

Mean

137.83

165.67

179.83

20.26

30.47

SD

5.42

8.66

8.11

5.90

3.06

n

6

6

6

6

6

Keys:- = Not Applicable, SD = Standard Deviation, n = Number of Animals

Individual Animal Clinical Signs and Symptoms

Sex:Female

Animal No.

Group/ Dose (mg/kg)

Hours (Day 0)

1/2

1

2

3

4

1

G1/ 2000

1

1

99++

99++

99+

2

1

1

99+

99+

99+

3

1

1

99+

99+

99+

4

1

1

99++

99+

99+

5

1

1

99++

99+

99+

6

1

1

99++

99+

99+

Animal No.

Group/ Dose (mg/kg)

Days post dosing

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

G1/ 2000

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

1

1

1

1

1

1

4

1

1

1

1

1

1

1

1

1

1

1

1

1

1

5

1

1

1

1

1

1

1

1

1

1

1

1

1

1

6

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Keys:1 = Normal, 99 = Lethargy, + = Mild, ++ = Moderate

Individual Animal Mortality Record

Sex:Female

Animal No.

Group/ Dose (mg/kg)

Day of Observation (Day 0 to 14)

Morning Observations

Evening Observations

1

G1/ 2000

No mortality and morbidity

No mortality and morbidity

2

No mortality and morbidity

No mortality and morbidity

3

No mortality and morbidity

No mortality and morbidity

4

No mortality and morbidity

No mortality and morbidity

5

No mortality and morbidity

No mortality and morbidity

6

No mortality and morbidity

No mortality and morbidity

Gross Necropsy Observation

 Sex:Female

Animal No.

Group/ Dose (mg/kg)

Mode of Death

Gross Observation

External

Internal

1

G1/ 2000

Terminal sacrifice

No abnormality detected

No abnormality detected

2

Terminal sacrifice

No abnormality detected

No abnormality detected

3

Terminal sacrifice

No abnormality detected

No abnormality detected

4

Terminal sacrifice

No abnormality detected

No abnormality detected

5

Terminal sacrifice

No abnormality detected

No abnormality detected

6

Terminal sacrifice

No abnormality detected

No abnormality detected


Interpretation of results:
other: Not classified
Conclusions:
The LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical orally.
Executive summary:

The acute oral toxicity study of the given test chemical was performed as per OECD 423 guideline in 6 female wistar rats. The test material dissolved in corn oil. The volume was made up to 10 ml to achieve the appropriate concentrations. The dosing solution was prepared fresh, prior to dose administration and given in dose concentration 2000mg/kg bw by oral gavage route. The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 hours post dosing, with food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of first group were dosed with starting dose of 2000 mg/kg body weight and the animals did not show any mortality so another three animals of the same group were dosed with 2000 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped. Body weights were re­corded on day 0 (prior to dosing) 7 and 14. Body weight gain was observed in all surviving animals treated with 2000 mg/kg body weight, during the 14 day observation period, as compared to day 0. At 2000 mg/kg, animal nos. 1, 4, 5 and 6 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and moderate to mild lethargy was observed at 2, 3 and 4 hours post dosing. Animal nos. 2 and 3 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and mild lethargy was observed at 2, 3 and 4 hours post dosing. No external and internal gross pathological changes were seen in the animals treated with 2000 mg/kg body weight during terminal sacrifice. Hence, the LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical orally.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is Klimicsh 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 experimental report.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
Acute dermal toxicity study of the given test chemical was performed in rats.
GLP compliance:
yes
Test type:
other: not specified
Limit test:
yes
Specific details on test material used for the study:
- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house animals, bred at Animal House, sa-FORD.CPCSEA Registration No. 1256/bc/09/CPCSEA.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Weight at study initiation: Male:Minimum: 229 g and Maximum: 262 g
(Prior to Treatment) Female:Minimum: 203 g and Maximum: 241 g
- Fasting period before study: The food was withheld prior to dosing and 3-4 hours post dosing but drinking water was provided ad libitum.
- Housing: The animals were housed individually in polycarbonate cages.All cages were provided with corn cobs (Sparconn Life Sciences,Bangalore) SPAR – 24/ 2013.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rodent diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 400012.
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum via drinking bottles
- Acclimation period:All animals were acclimatized to the test conditions for 5 days prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 21.10 to 24.40 degree centigrade.
- Humidity (%): Room humidity was maintained at 36.60% to 57.30%.
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: over clipped dorsal area of rat skin. Approx. 10% of body surface area of rat
- % coverage: porous gauze dressing
- Type of wrap if used: The test site was further covered in a suitable manner to retain the gauze dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, At the end of the exposure period, residual test item was removed by using distilled water.
- Time after start of exposure:24hr

TEST MATERIAL
- Concentration (if solution): 2000mg/kgbw
- Constant volume or concentration used: yes
Duration of exposure:
24hr
Doses:
2000mg/kg bw
No. of animals per sex per dose:
Total : 10
male : 5
female: 5
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs:After test item administration, individual animals were frequently observed at 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all animals were observed once a day during the 14 day observation period.
Local Signs/Skin Reactions: All animals were observed once daily during days 1-14 (in common with clinical signs).
body weight:All rats were weighed on days 0 (prior to dosing), 7 and 14.
Mortality:Animals were observed twice daily for any mortality during the experimental period
Pathology:At the end of 14 day observation period, all the surviving rats were euthanised by overdose of CO2 and subjected to gross pathology examination, for external and internal observations.
Statistics:
No statistical analysis was performed since the study was terminated with limit test
Preliminary study:
not specified
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality was observed
Mortality:
No mortality was observed at limit dose of 2000 mg/kg body weight of test item during the 14 day observation period
Clinical signs:
No systemic or local signs of toxicity were observed at limit dose of 2000 mg/kg body weight of test item during the experimental period
Body weight:
The male and female animals were observed with body weight gain compared to day 0 throughout the experiment
Gross pathology:
The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality
Other findings:
not specified

Individual Animal Body Weight (g) andBody Weight Changes(%)

 

Dose:2000 mg/ kg bodyweight                                                                             

 Density:0.9407

Animal No.

Sex

Dose (ml) Applied*

Body Weight (gram)

Body Weight Change (%)

Day 0

Day 7

Day 14

Day 0-7

Day 0-14

1

Male

0.49

229

247

256

7.86

11.79

2

0.50

235

256

278

8.94

18.30

3

0.49

230

245

272

6.52

18.26

4

0.53

247

262

296

6.07

19.84

5

0.56

262

289

310

10.31

18.32

6

Female

0.51

241

240

258

-0.41

7.05

7

0.43

203

205

211

0.99

3.94

8

0.43

203

206

213

1.48

4.93

9

0.50

236

241

245

2.12

3.81

10

0.48

226

231

232

2.21

2.65

Key:* = Based on density of test item and day 0 body weight taken prior to dose application.

Individual Animal Clinical Signs and Symptoms

 

Dose:2000 mg/kg body weight

Animal No.

Sex

Hour(s) - Day 0

Day

1

2

3

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

Male

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

4

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

5

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

6

Female

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

7

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

8

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

9

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

10

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Key: 1 = Normal

Individual Animal Mortality Record

 

Dose:2000 mg/kg body weight

       Animal No.

Sex

Days of Observation (0 to 14)

Morning Observations

Evening Observations

1

Male

No mortality and morbidity

No mortality and morbidity

2

No mortality and morbidity

No mortality and morbidity

3

No mortality and morbidity

No mortality and morbidity

4

No mortality and morbidity

No mortality and morbidity

5

No mortality and morbidity

No mortality and morbidity

6

Female

No mortality and morbidity

No mortality and morbidity

7

No mortality and morbidity

No mortality and morbidity

8

No mortality and morbidity

No mortality and morbidity

9

No mortality and morbidity

No mortality and morbidity

10

No mortality and morbidity

No mortality and morbidity

Summaryof Animal Body Weight (g) and Body Weight Changes (%)

 

Dose:2000 mg/kg body weight

Sex

Body Weight (gram)

Body Weight Changes (%)

Day 0

Day 7

Day 14

Day 0-7

Day 0-14

Male

Mean

231.20

242.20

257.10

7.94

17.30

SD

18.12

25.01

33.03

1.74

3.15

n

10

10

10

5

5

Female

Mean

221.80

224.60

231.80

1.28

4.48

SD

17.99

17.87

20.29

1.07

1.65

n

5

5

5

5

5

Keys:SD= Standard deviation, n = Number of animals

Gross Necropsy Observation

 

 Dose:2000 mg/kg body weight                                               

Mode of Death:Terminal Sacrifice


Animal No.

Sex

Gross Observation

External

Internal

1

Male

No abnormalities detected

No abnormalities detected

2

No abnormalities detected

No abnormalities detected

3

No abnormalities detected

No abnormalities detected

4

No abnormalities detected

No abnormalities detected

5

No abnormalities detected

No abnormalities detected

6

Female

No abnormalities detected

No abnormalities detected

7

No abnormalities detected

No abnormalities detected

8

No abnormalities detected

No abnormalities detected

9

No abnormalities detected

No abnormalities detected

10

No abnormalities detected

No abnormalities detected


Interpretation of results:
other: Not classified
Conclusions:
The LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical by dermal application.
Executive summary:

Acute dermal toxicity study of the given test chemical was performed as per OECD No.402 in Wistar Rats. Five male and five female healthy young adult rats were randomly selected. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item was applied by single dermal application and observed for 14 days after treatment. On test day 0, as such amount of test item, calculated based on density (0.9407) and body weight was applied directly on the intact skin of clipped area of rats; the surgical gauze patch was put on to the intact skin of clipped area. This gauze patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non-irritating adhesive tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically. No mortality was observed in any animal till the end of the experimental period. No clinical signs and any skin reaction were observed throughout the experimental period in all treated animals. The male and female animals were observed with body weight gain compared to day 0 throughout the experiment. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Hence the LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical by dermal application.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is Klimicsh 1 and from experimental study report.

Additional information

Acute oral toxicity:

In different experimental 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 test chemical. The studies are summarized as below –

The acute oral toxicity study of the given test chemical was performed as per OECD 423 guideline in 6 female wistar rats. The test material dissolved in corn oil. The volume was made up to 10 ml to achieve the appropriate concentrations. The dosing solution was prepared fresh, prior to dose administration and given in dose concentration 2000mg/kg bw by oral gavage route. The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 hours post dosing, with food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of first group were dosed with starting dose of 2000 mg/kg body weight and the animals did not show any mortality so another three animals of the same group were dosed with 2000 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped. Body weights were re­corded on day 0 (prior to dosing) 7 and 14. Body weight gain was observed in all surviving animals treated with 2000 mg/kg body weight, during the 14 day observation period, as compared to day 0. At 2000 mg/kg, animal nos. 1, 4, 5 and 6 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and moderate to mild lethargy was observed at 2, 3 and 4 hours post dosing. Animal nos. 2 and 3 were observed normal at 30 minutes, 1 hour, on day 1 to 14 post dosing and mild lethargy was observed at 2, 3 and 4 hours post dosing. No external and internal gross pathological changes were seen in the animals treated with 2000 mg/kg body weight during terminal sacrifice. Hence, the LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical orally.

In another study, acute oral toxicity dose was determined for the test chemical.The acute oral range-finding toxicity study of the given test chemical was conducted on mice. Oral intubation 4-5 weeks old white mice. Undiluted test concentrations were used in the study. Mice was given the dosages of 9300.0, 4650.0, 1860.0 mg/kg bw (equivalent to 10.0, 5.0, and 2.0 ml/kg bw). Animals were observed for up to 7 days after intubation. All animals dying were autopsied. All survivors were killed and examined post mortem after 7 days. Animals were checked for clinical signs, body weight, and mortality and for gross pathology. One out of two animal died at 9300.0 mg/kg bw (10.0 ml/kg bw) whereas no animals died at 4650.0 mg/kg bw and 1860.0 mg/kg bw (5.0 ml/kg bw and 2.0 ml/kg bw). Mice dosed at 5.0 and 10.0 ml/kg were showing signs of stress and exhibiting laboured breathing and unco-ordinated movement within 30 mins to 1 hour after treatment. The female mouse dosed at 2.0 ml/kg was also exhibiting an unusual 'swimming' action when moving within 1 hour after intubation. The mice dosed at 5.0 and 10.0 ml/kg were hypothermic within 2 hours after treatment and the mice dosed at 2.0 ml/kg had their eyes closed, with evidence of lacrimation. Within 18 hours the female mouse dosed at 10.0 ml/kg died and the male mouse had a bloated stomach. The mice dosed at 2.0 and 5.0 ml/kg recovered within 42 hours after treatment. Autopsy of the animal that died revealed gross gaseous distension of the stomach and intestines and a pale mottled liver. Surviving animals gained weight during the 7 day observation period and autopsy revealed slight thickening of the cardiac region of the stomach and areas of bright red tissue in the lungs of the survivors dosed at 5.0 and 10.0 ml/kg. Hence the approximate LD50 value was considered to be about 9440.0 mg/kg bw (10.0 ml/kg body weight), when mice were treated with the given test chemical orally.

Above studies are supported with the data mentioned in peer reviewed journal and conducted in rats. In acute oral toxicity study of the given test chemical groups of 10 young adult Osborne-Mendel rats evenly divided by sex were fasted for approximately 18 hr prior to treatment. The undiluted test material in dose concentration upto 5400 mg/kg bw were administered by oral gavage route. All animals were maintained under close observation for recording toxic signs and time of death. Such observation was continued until animals appeared normal and showed weight gain. The usual observation period was 2 weeks. LD50's were computed by the method of Litchfield & Wilcoxon (1949).50% mortality was observed at dose concentration 4590 mg/kg bw in 4hr-4days. Clinical signs like Depression, tremors were observed in treated rats. Hence, LD50 value was considered to be 4590 mg/kg bw (slope function1.4 (1.3 – 1.5) when Osborne-Mendel rats were treated with the given test chemical orally.

All the above experimental studies are further supported with the data mentioned in peer reviewed journal and conducted on mice. Acute oral toxicity study was conducted by using the given test chemical in male and female mice. Animals were divided into three groups as1 male and 1 female in the 10000 mg/kg bw group, 3 males and 3 females in the 5000 mg/kg bw group; 1 male and 1 female in the 2000 mg/kg bw group. The test material was administered in dose concentration 10000, 5000 and 2000 mg/kg bw by oral gavage route. Mortality and toxicity signs were observed for up to 7 days. No deaths were observed at 2000 and 5000 mg/kg; one animal (1/2) died at 10000 mg/kg. Clinical signs included stress, laboured breathing, uncoordinated movement, hypothermia, lacrimation and bloated stomach. Necropsy of the animal that died revealed gross gaseous distension of the stomach and intestines and a pale mottled liver. Necropsy of the surviving animals revealed slight thickening of the cardiac region of the stomach and areas of bright red tissue in the lungs of animals at 5000 and 10000 mg/kg. Hence the LD50 value was considered to be 10000 mg/kg bw when male and female mice were treated with the given test chemical orally.

Thus, based on the above studies on test chemical, it can be concluded that LD50 value is >2000 mg/kg bw. 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 the low vapour pressure of the test chemical, which is reported as 0.007 mmHg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver.

Acute Dermal toxicity:

Acute dermal toxicity study of the given test chemical was performed as per OECD No.402 in Wistar Rats. Five male and five female healthy young adult rats were randomly selected. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item was applied by single dermal application and observed for 14 days after treatment. On test day 0, as such amount of test item, calculated based on density (0.9407) and body weight was applied directly on the intact skin of clipped area of rats; the surgical gauze patch was put on to the intact skin of clipped area. This gauze patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non-irritating adhesive tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically. No mortality was observed in any animal till the end of the experimental period. No clinical signs and any skin reaction were observed throughout the experimental period in all treated animals. The male and female animals were observed with body weight gain compared to day 0 throughout the experiment. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Hence the LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical by dermal application.

Justification for classification or non-classification

Based on the above experimental studies on 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 were added so, not possible to classify.