Registration Dossier

Administrative data

Description of key information

Acute oral toxicity: 

Acute oral toxicity dose (LD50) was considered based on experimental study conducted on rats for the given test chemical. The LD50 value was considered to be >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: 

Acute Inhalation toxicity dose (LC50) was considered based on different studies conducted on rats for the test chemical. The LC50 value was considered to be >5.27 mg/L (>527000 mg/m3). The study concluded that the LC50 value is >5 mg/L air. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute Inhalation toxicity.

Acute Dermal toxicity: 

The acute dermal toxicity dose (LD50) was considered based on experimental study conducted on rats for the test chemical. The 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 aim of this study was to assess the toxicity potential of the given test chemical after single oral administration in rats and an observation period of 14 days.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:In-House Bred
- Age at study initiation: Healthy young adult animals were used for the study. 8- 11 weeks at the time of dosing.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation:Minimum: 137 g Maximum: 179 g (Individual body weights were within ± 20% prior to treatment after overnight fasting)
- Fasting period:The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 hours post dosing.
- Housing:The animals were housed individually in polycarbonate cages.
- Bedding: All cages were provided with corn cobs.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed at least twice every week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rodent diet, ad libitum.
- 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 and 4-6 for seven days prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.50°C; Maximum: 22.90°C
- Humidity (%):Minimum: Minimum: 42.90 %; Maximum: 69.10 %
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark

IN-LIFE DATES: From: March 16, 2015 To: April 29, 2015
Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 300 mg/kg and 2000 mg/kg
- Amount of vehicle (if gavage):10 ml
Doses:
G1/ Step I / 300 mg/kg
G1/ Step II / 300 mg/kg
G2/ Step III / 2000 mg/kg
G2/ Step IV / 2000 mg/kg
No. of animals per sex per dose:
G1/ Step I / 300 mg/kg - 3
G1/ Step II / 300 mg/kg - 3
G2/ Step III / 2000 mg/kg - 3
G2/ Step IV / 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 Observation - 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 the animals were observed once a day during the 14 day observation period.
Body weight - All rats were weighed on days 0 (prior to dosing), 7 and 14.
Mortality - All animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period.
- Necropsy of survivors performed: yes, at the end of 14 day observation period, all the 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.
Mortality:
No mortality was observed in the animals treated with 300 and 2000 mg/kg body weight dose throughout the 14 days observation period.
Clinical signs:
At 300 and 2000 mg/kg body weight, animals of G1 (Step I and II) and G2 (Step III and IV) were normal throughout the experimental period.
Body weight:
Mean body weight of all the animals of G1 (Step I and II) and G2 (Step III and IV) treated with 300 and 2000 mg/kg body weight respectively, was observed with gain on day 7 and 14, as compared to day 0.
Gross pathology:
No external and internal gross pathological changes were seen in all the animals of G1 (Step I and II) and G2 (Step III and IV) treated with 300 and 2000 mg/kg body weight respectively, during terminal sacrifice.
Other findings:
not specified

Table 1: Individual Animal Body Weight (g) andBody Weight Changes(%)

Sex: Female

Animal No.

Group/Step/ Dose (mg/kg body weight)

Dose Volume

(ml)

Body Weight (gram)

Body Weight Change (%)

Day 0

Day 7

Day 14

Day

0-7

Day

0-14

1

G1/

Step I/ 300

1.7

169

193

212

14.20

25.44

2

1.6

160

179

197

11.88

23.13

3

1.6

162

194

216

19.75

33.33

4

G1/

Step II/

 300

1.8

179

213

230

18.99

28.49

5

1.7

170

203

214

19.41

25.88

6

1.6

160

187

199

16.88

24.38

7

G2/

Step III/

 2000

1.6

164

182

192

10.98

17.07

8

1.6

157

186

198

18.47

26.11

9

1.8

175

200

218

14.29

24.57

10

G2/

Step IV/

 2000

1.4

140

164

177

17.14

26.43

11

1.4

141

165

177

17.02

25.53

12

1.4

137

156

167

13.87

21.90

*= Dose volume calculated based on day 0 body weight,

 

Table 2: Summary of Animal Body Weight (g) and Body Weight Changes (%)

Sex: Female

Group/Step/Dose (mg/kg body weight)

Rats Body Weight (g)

Body Weight Changes (%)

Day 0

Day 7

Day 14

0-7

0-14

G1/

Step I/ 300

Mean

163.67

188.67

208.33

15.28

27.30

SD

4.73

8.39

10.02

4.05

5.35

N

3

3

3

3

3

G1/

Step II/

 300

Mean

169.67

201.00

214.33

18.43

26.25

SD

9.50

13.11

15.50

1.36

2.08

N

3

3

3

3

3

G2/

Step III/

 2000

Mean

165.33

189.33

202.67

14.58

22.59

SD

9.07

9.45

13.61

3.76

4.84

N

3

3

3

3

3

G2/

Step IV/

 2000

Mean

139.33

161.67

173.67

16.01

24.62

SD

2.08

4.93

5.77

1.86

2.40

N

3

3

3

3

3

Key: SD = Standard Deviation, n = Number of Animals


Table 3: Individual Animal Clinical Signs and Symptoms

 

Sex: Female

Animal No.

Group/Step/ Dose (mg/kg body weight)

Hours (Day 0)

1/2

1

2

3

4

1

G1/

Step I/ 300

1

1

1

1

1

2

1

1

1

1

1

3

1

1

1

1

1

4

G1/

Step II/

 300

1

1

1

1

1

5

1

1

1

1

1

6

1

1

1

1

1

7

G2/

Step III/

 2000

1

1

1

1

1

8

1

1

1

1

1

9

1

1

1

1

1

10

G2/

Step IV/

 2000

1

1

1

1

1

11

1

1

1

1

1

12

1

1

1

1

1

 

Animal No.

Group/Step/ Dose (mg/kg body weight)

Days post dosing

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

G1/

Step I/ 300

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

G1/

Step II/

 300

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

7

G2/

Step III/

 2000

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

9

1

1

1

1

1

1

1

1

1

1

1

1

1

1

10

G2/

Step IV/

 2000

1

1

1

1

1

1

1

1

1

1

1

1

1

1

11

1

1

1

1

1

1

1

1

1

1

1

1

1

1

12

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Key:1 = Normal


Table 4: Individual Animal Mortality Record

 

Sex: Female

Animal No.

Group/Step/ Dose (mg/kg body weight)

Day of Observation (Day 0 to 14)

Morning Observations

Evening Observations

1

G1/

Step I/ 300

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

G1/

Step II/

 300

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

7

G2/

Step III/

 2000

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

G2/

Step IV/

 2000

No mortality and morbidity

No mortality and morbidity

11

No mortality and morbidity

No mortality and morbidity

12

No mortality and morbidity

No mortality and morbidity

 


Table 5: Gross Necropsy Observation

 

Sex: Female                                                                             Mode of Death: Terminal Sacrifice

Animal No.

Group/Step/ Dose (mg/kg body weight)

Gross Observation

External

Internal

1

G1/

Step I/ 300

No abnormality detected

No abnormality detected

2

No abnormality detected

No abnormality detected

3

No abnormality detected

No abnormality detected

4

G1/

Step II/

 300

No abnormality detected

No abnormality detected

5

No abnormality detected

No abnormality detected

6

No abnormality detected

No abnormality detected

7

G2/

Step III/

 2000

No abnormality detected

No abnormality detected

8

No abnormality detected

No abnormality detected

9

No abnormality detected

No abnormality detected

10

G2/

Step IV/

 2000

No abnormality detected

No abnormality detected

11

No abnormality detected

No abnormality detected

12

No abnormality detected

No abnormality detected

 

Interpretation of results:
other: Not classified
Conclusions:
Under these study conditions, the acute oral toxicity dose (LD50) was considered to be >2000 mg/kg bw, when female Wistar rats were treated with the given test chemical via oral route. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute oral toxicity. CLP Classification "Not classified"

Executive summary:

Acute oral toxicity study of the given test chemical was conducted as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Wistar female rats.

Twelve female Wistar rats were selected for acute oral toxicity study. The animals were fasted for minimum 16-18 hours prior to dosing and for 4 hours post dosing, with feed 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 group G1 (Step I) were dosed with starting dose of 300 mg/kg body weight and no mortality was observed.

Based on the results from G1 (Step I), additional three animals of group G1 (Step II) were dosed with 300 mg/kg body weight. As there was no mortality at group G1 (Step I and II) dose levels, three rats of group G2 (Step III) was dosed with 2000 mg/kg body weight and no mortality was observed so another three rats of group G2 (Step IV) were dosed with 2000 mg/kg body weight. Since there was no mortality in both the groups G1 (Step I and II) and G2 (Step III and IV), further dosing was not required.

Body weights were re­corded on day 0 (prior to dosing) 7 and 14. Mean body weight of all the animals (Step I, Step II, Step III and Step IV) treated with 300 and 2000 mg/kg body weight and was observed with gain on day 7 and 14, as compared to day 0.

At 300 and 2000 mg/kg body weight, animals of G1 (Step I and II) and G2 (Step III and IV) were normal throughout the experimental period.

No external and internal gross pathological changes were seen in all the animals of G1 (Step I and II) and G2 (Step III and IV) treated with 300 and 2000 mg/kg body weight respectively, during terminal sacrifice.

Under these study conditions, the acute oral toxicity dose (LD50) was considered to be >2000 mg/kg bw, when female Wistar rats were treated with the given test chemical via oral route. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute oral toxicity. CLP Classification "Not classified"

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.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
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 403 (Acute Inhalation Toxicity)
Principles of method if other than guideline:
The aim of this study was to determine the LC50 of the given test chemical in Wistar rats, after acute inhalation exposure for approximately 4 hrs and observation period of 14 days.
GLP compliance:
yes
Test type:
other: Acute Inhalation Toxicity
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:In house breed.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks at the time of exposure. Healthy young adult animals were used for the study.
- Weight at study initiation: Male: Maximum: 211 g; Minimum: 199 g
Female:Maximum: 191 g; Minimum: 180 g
- Housing:Group of three animals each were housed in polycarbonate cages.
- Bedding: All cages were provided with corn cobs.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed at least twice every week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rodent diet, ad libitum.
- 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 7 days prior to exposure of the test item.
- Randomization: Animals were selected manually. No computer generated randomization program was used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.70°C; Maximum: 23.20°C
- Humidity (%):Minimum: 43.10 %; Maximum: 67.30 %
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12 h light and 12 h dark

IN-LIFE DATES: From: March 20, 2015 To: April 13, 2015
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: Dimethyl sulfoxide (DMSO)
Mass median aerodynamic diameter (MMAD):
3.02 µm
Geometric standard deviation (GSD):
2.45
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the nose only inhalation chamber
- Exposure chamber volume: 22.52 liters
- Method of holding animals in test chamber: Each rat was accommodated in the portholes of inhalation chamber with the help of rat exposure tube. It was made up of transparent polycarbonate with adjustable unit and all rats were kept for four hour continuous exposure in inhalation chamber.
- Source and rate of air: Air compressor is used for supply of air to the inhalation unit. The applied air flow rate was 8 liter per minute
- Method of conditioning air: Air was passed into nebulizer at 30 psi pressure and 18 liter per minute air was drawn from the chamber to ensure slight negative pressure inside the chamber to prevent leakage of test item into the surrounding area
- System of generating particulates/aerosols: 70% formulated test item in Dimethyl sulfoxide (DMSO) was loaded into a 50 ml syringe and kept in infusion syringe pump. The infusion pump flow rate was 40 ml/hour. The test item was infused into nebulizer; aerosol was formed and distributed in the inhalation chamber.
- Method of particle size determination: The seven stage cascade impactor was used to determine the particle size.
- Treatment of exhaust air: The outgoing air from the chamber passes through conical flask containing 1% sodium hydroxide and moisture trap (cotton) by using the suction pump.
- Temperature, humidity, pressure in air chamber: The mean chamber temperature, relative humidity, oxygen and carbon dioxide concentration were 22.40°C, 54.03%, 21.08% and 0.09%, respectively; which were measured four times during 4 hour exposure period.

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes, the breathing zone concentration and particle size were measured by using open face filter holder and seven stage cascade impactor four times during four hour exposure period.

VEHICLE
- Concentration of test material in vehicle (if applicable): 70% formulated test item in Dimethyl sulfoxide (DMSO)
- Lot/batch no. (if required): MMBB3970

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The MMAD was calculated according to the particle size at which the line across the 50% Mark whereas GSD was calculated according to the particle size at which the line across the 84.1% Mark divided by 50% Mark
Analytical verification of test atmosphere concentrations:
not specified
Duration of exposure:
4 h
Concentrations:
5.27 mg/l of air
No. of animals per sex per dose:
Three male and three female wistar rats
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical Observation - During inhalation exposure, individual animals were frequently observed at 1, 2, 3 and 4 hours and 30 minutes and one hour post exposure at day 0 (day of exposure). Subsequently, all animals were observed once a day during the 14 day observation period, after exposure.
Mortality - Animals were observed twice daily for mortality/morbidity during experimental period.
Body weight - All rats weighed on test days 0 (prior to application), 1, 3, 7 and 14 and found dead.
- Necropsy of survivors performed: yes, at the end of 14 day observation period, all surviving rats were euthanised by over dose of thiopentone sodium by intraperitoneal injection and subjected to gross pathology examination of external and internal observations. All animals showed no evidence of gross pathology hence, microscopic examination was not performed.
Statistics:
not specified
Preliminary study:
not specified
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.27 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
One mortality was observed at the breathing zone concentration of 5.27 mg/liter of air of test item during the 14 day observation period.
Clinical signs:
other: Clinical signs like lethargy and tremors were observed after exposure. Animal nos. 1, 2 and 4 were found normal from day 5 till terminal sacrifice whereas animal nos. 3 and 5 were found normal from day 6 till terminal sacrifice. Animal no. 6 was found dea
Body weight:
Decrease in mean body weight was observed on days 1 and 3 while increase in mean body weight was observed on days 7 and 14 in male rats whereas in female rats decrease in mean body weight was observed on day 1, 3, 7 and 14 when compared with day 0 mean body weight.
Gross pathology:
All the animals were subjected to gross pathological examination.
Other findings:
External Observation : External observation of found dead animal and terminally sacrificed animals did not show any pathological abnormality.
Internal Observation : Internal observation of all terminally sacrificed animals did not show any pathological abnormality.
In found dead animal following changes were observed in organs like: Lungs: Red discoloration was observed in Animal no. 6.

TABLE 1

Inhalation Chamber – Temperature, Relative Humidity, Oxygen, Carbon dioxide Concentration and Air Flow Rate During Exposure

Dose (mg/liter)

Time (Hours)

1

2

3

4

Mean

SD

5.27

Temperature (°C)

22.5

22.3

22.6

22.2

22.40

0.18

Relative Humidity (%)

53.9

54.2

53.5

54.5

54.03

0.43

Oxygen Concentration (%)

21.2

21.1

20.9

21.1

21.08

0.13

Carbon Dioxide Concentration (%)

0.05

0.09

0.13

0.08

0.09

0.03

In let Air Flow Rate              (liter per Minute)

8.00

8.00

8.00

8.00

8.00

0.00

Key :SD = StandardDeviation. 


TABLE 2

Concentration Measurement of Inhalation Chamber by Gravimetric Analysis

No.

Pre Weight (mg)

Post Weight (mg)

Difference in Weight (mg)

Air Suction Rate (LPM)

Time (minute)

Breathing Zone Concentration* (mg/l air)

Mean   ±

SD

1

26.44

36.98

10.54

2

1

5.27

5.27      ±             0.02

2

26.39

36.87

10.48

2

1

5.24

3

26.28

36.86

10.58

2

1

5.29

4

26.72

37.24

10.52

2

1

5.26

Keys :No. = Number, mg = Milligram,SD = Standard Deviation, LPM = Liter per Minute, l = liter

Differencein Weight (mg)

*BreathingZone Concentration                     =   ______________________________________

Air Suction Rate (liter/minute) X Time (minute)

 

 


TABLE 3

Nominal Concentration Details

 

Density of 70% formulated Test Item in Dimethyl Sulfoxide (DMSO)

 

No.

Amount of Sample (ml)

Weight of Sample (mg)

Mean±SD

1

1

1015.29

1015.71±0.41

2

1

1015.73

3

1

1016.11

 

Calculationof Nominal Concentration of Test Item

 

                                                                       Infusion Flow Rate (ml/ hour) X Density (mg/ml)

Nominal concentration (mg/l air)       =      _______________________________________       

                                                                                   Air Flow Rate per Hour (LPH)

 

Density (mg/ml)

Infusion Flow Rate (ml/hour)

Air flow rate (LPM))

Air flow Rate (LPH)

Nominal Concentration

(mg/liter)

1015.71

40

8

480

84.64

 

Keys :LPM = Liter per Minute, LPH = Liter per Hour,SD = Standard Deviation.


TABLE 4

Particle Size Distribution

Dose :5.27 mg/l of air

Stage

Effective Cutoff Diameter

Initial Weight (gm)

Final Weight (gm)

Difference in weight (gm)

% of Total Particles captured (by weight)

Cumulative (%)*

Sample 1

1

10.40

1657.35

1657.41

0.06

2.94

97.06

2

6.45

1660.63

1660.82

0.19

9.31

87.75

3

3.96

1647.35

1647.77

0.42

20.59

67.16

4

2.33

1638.78

1639.53

0.75

36.76

30.39

5

1.52

1661.30

1661.81

0.51

25.00

5.39

6

0.94

1659.64

1659.67

0.03

1.47

3.92

7

0.56

1653.56

1653.62

0.06

2.94

0.98

F

0.00

242.94

242.96

0.02

0.98

0.00

Sample 2

1

10.40

1657.35

1657.42

0.07

3.38

96.62

2

6.45

1660.64

1660.84

0.20

9.66

86.96

3

3.96

1647.35

1647.78

0.43

20.77

66.18

4

2.33

1638.79

1639.57

0.78

37.68

28.50

5

1.52

1661.31

1661.80

0.49

23.67

4.83

6

0.94

1659.64

1659.66

0.02

0.97

3.86

7

0.56

1653.56

1653.61

0.05

2.42

1.45

F

0.00

242.94

242.97

0.03

1.45

0.00

Keys: *= Percent of Particles smaller than corresponding effective cutoff diameter, F= filter
TABLE 4Continued...

Dose :5.27 mg/l of air

Stage

Effective Cutoff Diameter

Initial Weight (gm)

Final Weight (gm)

Difference in weight (gm)

% of Total Particles captured (by weight)

Cumulative (%)*

Sample 3

1

10.40

1657.35

1657.40

0.05

2.42

97.58

2

6.45

1660.63

1660.83

0.20

9.66

87.92

3

3.96

1647.35

1647.80

0.45

21.74

66.18

4

2.33

1638.79

1639.55

0.76

36.71

29.47

5

1.52

1661.31

1661.81

0.50

24.15

5.31

6

0.94

1659.64

1659.67

0.03

1.45

3.86

7

0.56

1653.56

1653.61

0.05

2.42

1.45

F

0.00

242.93

242.96

0.03

1.45

0.00

Sample 4

1

10.40

1657.35

1657.41

0.06

2.91

97.09

2

6.45

1660.63

1660.82

0.19

9.22

87.86

3

3.96

1647.35

1647.78

0.43

20.87

66.99

4

2.33

1638.78

1639.53

0.75

36.41

30.58

5

1.52

1661.31

1661.83

0.52

25.24

5.34

6

0.94

1659.64

1659.66

0.02

0.97

4.37

7

0.56

1653.56

1653.62

0.06

2.91

1.46

F

0.00

242.94

242.97

0.03

1.46

0.00

Keys: *= Percent of Particles smaller than corresponding effective cutoff diameter, F = Filter


TABLE 4Continued...

Summary of Particle Size Distribution

Collection Time :1 minute                                                  Air Flow Rate :2 liter per minute

 

Time

1

2

3

4

Mean±SD

MMAD

3.01

3.06

3.02

3.00

3.02 ± 0.03

GSD

2.45

2.47

2.44

2.45

2.45 ± 0.01

Keys:MMAD = Mass Median Aerodynamic Diameter,GSD = Geometric Standard Deviation, SD = Standard Deviation.


Interpretation of results:
other: Not classified
Conclusions:
Under the conditions of this study, the median lethal concentration (LC50) of 70% of the given test chemical diluted in Dimethyl Sulfoxide (DMSO) in male and female Wistar rats was estimated as being greater than the breathing zone concentration of 5.27 mg/l air after four hour inhalation exposure and classified as “Not classified” as per CLP classification.
Executive summary:

Acute inhalation toxicity study of the given test chemical was conducted  as per OECD Guideline No. 403 in Wistar Rats.

Three male and three female healthy young adult rats were randomly selected and used for conducting acute inhalation toxicity limit study. Rats free from injuries were selected for the study. The study was carried out in dynamic nose only inhalation chamber. The dynamic inhalation chamber has three main parts namely inlet, exposure and outlet chambers. Total capacity of chamber is 22.52 liters. Each rat was restrained in a single transparent polycarbonate exposure tube with adjustable unit.

Continuous aerosol generation from as such (undiluted) test item was not possible hence test item was diluted in Dimethyl sulfoxide (DMSO) at the concentration of 70% (v/v) based on the solubility test. Formulated test item was loaded into a 50 ml syringe which was positioned to continuous infusion syringe pump at a flow rate of 40 ml/hour that was selected for main study based on pre test results. The rats were exposed to inhalation chamber exposure part. At a flow rate of 8 liter per minute, air was passed into nebulizer at 30 psi pressure and 18 liter per minute air was drawn from the chamber to ensure slight negative pressure inside the chamber to prevent leakage of test item into the surrounding area. Minimum 15 air changes per hour were maintained in inhalation chamber. The rats were exposed for continuous 4 hours after 20 minute equilibration period at the breathing zone concentration of 5.27 mg/liter air in Dimethyl Sulfoxide (DMSO). The mean chamber temperature, relative humidity, oxygen and carbon dioxide concentration were 22.40°C, 54.03%, 21.08% and 0.09%, respectively; which were measured four times during 4 hour exposure period. The mass median aerodynamic diameter of test item was 3.02 µm with geometric standard deviation of 2.45.

The animals were observed daily during the acclimatization period and mortality/morbidity and clinical signs were recorded. All animals were observed for clinical signs at 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0. Mortality/Morbidity was recorded during 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0 (in common with the clinical signs) and twice daily during days 1-14 (at least once on the day of sacrifice). Body weights were re­corded on day 0 (prior to exposure) and on days 1, 3, 7 and 14 and found dead. All animals were necropsied and examined macroscopically.

Clinical signs like lethargy and tremors were observed after exposure. Animal nos. 1, 2 and 4 were found normal from day 5 till terminal sacrifice whereas animal nos. 3 and 5 were found normal from day 6 till terminal sacrifice. Animal no. 6 was found dead on day 2 post exposure.

Decrease in mean body weight was observed on days 1 and 3 while increase in mean body weight was observed on days 7 and 14 in male rats whereas in female rats decrease in mean body weight was observed on day 1, 3, 7 and 14 when compared with day 0 mean body weight. ll the animals were subjected to gross pathological examination. External observation of found dead animal and terminally sacrificed animals did not show any pathological abnormality. Internal observation of all terminally sacrificed animals did not show any pathological abnormality. In found dead animal following changes were observed in organs like: Lungs: Red discoloration was observed in Animal no. 6.

Under the conditions of this study, the median lethal concentration (LC50) of 70% of the given test chemical diluted  in Dimethyl Sulfoxide (DMSO) in male and female Wistar rats was estimated as being greater than the breathing zone concentration of 5.27 mg/l air after four hour inhalation exposure and classified as “Not classified” as per CLP classification.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
527 000 mg/m³
Quality of whole database:
Data is Klimisch 1 and from experimental study report.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
The study was conducted to find out the LD50 value, clinical sign and histopathological effect of the given test chemical at different dose level in wistar albino rats.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Institute for Industrial Research and Toxicology Ghaziabad,
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 200±20g
- Housing: Groups of three animals of same sex in polypropylene cages with stainless steel grill top, facilities for food and water bottle, and bedding of clean paddy husk.
- Diet (e.g. ad libitum): Pelleted feed
- Water (e.g. ad libitum): Fresh and clean water filtered through ‘Aqua Guard on line water filter’, was kept in glass bottles Ad libitum
- Acclimation period:The healthy wistar albino rats selected for study acclimatized to standard laboratory condition for period of one week under close Veterinary supervision.
- Randomization:After acclimation and Veterinary examination all the animals randomly divided into two groups and each group having five male and five female rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 22-25°C
- Humidity (%): 40-60%
- Air changes (per hr): Air conditioned rooms with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.
Type of coverage:
open
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: back skin of total body surface area
- % coverage: Approximate 10 percent
- Type of wrap if used: The test compound was held in contact with the skin with an impervious dressing secured in place with an adhesive tape.The animals were then housed individually in cages with a collar around the neck in order to avoid the ingestion of the test compound.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the site of application was cleaned with lukewarm water wiping the test compound.
- Time after start of exposure:24 hours
Duration of exposure:
24 hours
Doses:
No. of dose groups
Group-I: 2000 mg/kg b.wt (limit test)
Group-II: 2000 mg/kg b.wt (confirmatory test)
No. of animals per sex per dose:
10 (5male & 5 female)
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The treated animals were closely observed for clinical signs of intoxication, first 4 hours and thereafter for every 1 hrs interval for 24 hrs after dosing and twice a day for 14 days. All the rats were observed at least twice daily with the purpose of recording any symptoms of ill-health or behavioral changes. These observations included changes in skin and fur in the eyes and mucous membranes, respiratory, circulatory, central nervous and autonomous systems, somatomotor activity and behavior changes. The following clinical signs were observed in female rats to characterize with erythema, hypersensitivity, edema etc.
Mortality - All the animals were observed for mortality at 30 minutes time interval for first six hours on the day of test compound administration and thereafter twice a day for 14 days.
Body weight: The body weight of all the animals was observed weekly on day 0 (pre treatment), 7th and 14th (post treatment).
- Necropsy of survivors performed: yes, necropsy was carried out on all the animals that died during the study or surviving animals were sacrificed at the end of the study to observe any gross pathological changes.
- Other examinations performed: The organ which showed gross pathological change during necropsy subjected for histopathological study.
Statistics:
not specified
Preliminary study:
not specified
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:
The test compound did not produce any mortality throughout the observation period of 14 days
Clinical signs:
The test compound did not elicit any clinical signs at the dose level of 2000 mg/kg b.wt. in entire observation period.
Body weight:
All the animals treated with the test compound at the dose level of 2000 mg/kg b.wt did not show any significant loss or gain in their weight when observed on day 0th (pre treatment) and then 7th and 14th (post treatment).
Gross pathology:
EXTERNAL
i. Skin- Skin and hair coat was observed wet.
ii. All external orifices- Normal
B. INTERNAL
i. Subcutaneous- No changes was observed.
ii. Superficial and deep lymph nodes- No change in mesenteric lymph node.
ABDOMINAL CAVITY
i. Opening and general examination- In the abdominal cavity all the organs were present in normal position.
ii. Spleen- No changes were recorded.
iii. Digestive system- No gross changes were observed in stomach and intestine.
iv. Liver and biliary ducts- No gross pathological changes were observed
v. Excretory system- No gross pathological changes were observed.
vi. Adrenal- Observed normal.
vii. Male/female genital organs – Showed normal colour, consistency and no inflammatory changes.
2. THORACIC CAVITY
i. Opening and general examination- Thoracic cavity was found to be normal without any fluid, mucous or blood etc.
ii. Lungs- No changes were recorded.
iii. Heart- No changes were observed in color and consistency. Heart found normal.
iv. Thyroid- Normal in shape, size and surface.
3. CRANIAL CAVITY
Brain- Normal in shape and size.
Other findings:
not specified

TABLE – 2

SUMMARY OF BODY WEIGHT (gm)

Group

Animal ID

Day 0

Day 7

% Gain/loss

Day 14

% Gain/loss

Group-I

2000 mg/kg b. wt

 

 

 

20174-1

198.2

203.5

2.67

209.3

5.60

20174-2

201.4

205.6

2.08

211.5

5.01

20174-3

200.6

204.5

1.94

213.2

6.28

20174-4

205.4

211.4

2.92

214.4

4.38

20174-5

204.2

210.3

2.98

215.3

5.43

20174-6

203.1

209.4

3.10

215.2

5.95

20174-7

204.7

211.1

3.12

216.7

5.86

20174-8

200.5

205.5

2.49

214.2

6.83

20174-9

202.6

208.5

2.91

214.1

5.67

20174-10

202.5

207.2

2.32

215.3

6.32

Group-II

2000 mg/kg b. wt

20174-11

197.5

203.1

2.83

208.6

5.62

20174-12

206.2

211.4

2.52

217.3

5.38

20174-13

203.4

208.2

2.35

213.4

4.91

20174-14

202.7

207.4

2.31

215.3

6.21

20174-15

205.3

210.3

2.43

216.7

5.55

20174-16

199.7

204.8

2.55

212.4

6.35

20174-17

204.9

209.6

2.29

217.5

6.14

20174-18

202.2

208.4

3.06

215.6

6.62

20174-19

201.4

206.2

2.38

211.4

4.96

20174-20

200.8

205.2

2.19

213.4

6.27

TABLE – 3

CLINICAL SIGNS AND MORTALITY

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

 

Parameters

Incidence of Clinical Signs Observed after Dosing on

Mortality

Day 0

DAY

Min

Hour

30

1

2

4

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total

%

Mortality (total)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0/10

0

Clinical Signs- Local

 

Redness

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Pain

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Swelling

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Systemic signs

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe


TABLE – 3 Contd…………….

CLINICAL SIGNS AND MORTALITY

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

                                                                                                                                      

 

Parameters

Incidence of Clinical Signs Observed after Dosing on

Mortality

Day 0

DAY

Min

Hour

30

1

2

4

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total

%

Mortality (total)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0/10

0

Clinical Signs- Local

 

Redness

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Pain

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Swelling

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Systemic signs

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE – 4

SUMMARY OF NECROPSY FINDINGS

S. No.

 

Fate

 

Wistar albino rats

Dose (mg/kg b. wt)

2000

(limit test)

2000

(confirmatory test)

1

Terminal sacrifice

10/10

10/10

2

Found Dead

0/10

0/10

3

Abnormalities detected

0/10

0/10

TABLE – 5

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

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

Animal ID

Fate

Time

Gross Findings

20174-1

TS

Day 14

NAD

20174-2

TS

Day 14

NAD

20174-3

TS

Day 14

NAD

20174-4

TS

Day 14

NAD

20174-5

TS

Day 14

NAD

20174-6

TS

Day 14

NAD

20174-7

TS

Day 14

NAD

20174-8

TS

Day 14

NAD

20174-9

TS

Day 14

NAD

20174-10

TS

Day 14

NAD

Day 0 is the day of dose administration.

TS- Terminal Sacrifice

NAD- No abnormality Detected

FD-Found dead

TABLE-5 Contd………..

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

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

Animal ID

Fate

Time

Gross Findings

20174-11

TS

Day 14

NAD

20174-12

TS

Day 14

NAD

20174-13

TS

Day 14

NAD

20174-14

TS

Day 14

NAD

20174-15

TS

Day 14

NAD

20174-16

TS

Day 14

NAD

20174-17

TS

Day 14

NAD

20174-18

TS

Day 14

NAD

20174-19

TS

Day 14

NAD

20174-20

TS

Day 14

NAD

 

Day 0 is the day of dose administration.

TS- Terminal Sacrifice

NAD- No abnormality Detected

FD-Found dead

Interpretation of results:
other: Not classified
Conclusions:
From the results obtained from present investigation, it can be concluded that the test chemical is non toxic to Wistar albino rats at the dose level of 2000 mg/kg body weight and the LD50 of this compound is >2000 mg/kg body weight.
Executive summary:

The acute dermal toxicity study of the given test chemical was conducted on wistar albino rats under OECD guideline-402 Guideline for Testing of Chemicals.

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

The test chemical applied at the dose level of 2000 mg/kg b.wt in Wistar albino rats did not show any clinical signs of toxicity throughout the observation period of 14 days.   Furthermore, no mortality was observed throughout the period of observation (14 days). After 14 days necropsy was conducted on all the animals which did not reveal any significant gross pathological changes.

CONFIRMATORY TEST:  After 72 hrs. a confirmatory test was conducted in same species of animals to confirm the limit test of test compound (OECD-402 guidelines).

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

There was no mortality recorded throughout the observation period after application of the test compound at the dose level of 2000 mg/kg body weight. The test compound did not elicit any clinical signs of toxicity during the entire observation period. The body weight of each animal treated with test compound observed on day 0th(pre treatment) and then 7thand 14th(post treatment) did not show any significant increase or decrease in their body weight. Necropsy was conducted on day 15th(end of study) on all the animals which did not reveal any significant gross pathological changes related to compound toxicity.

From the results obtained from present investigation, it can be concluded that the test chemical is non toxic to Wistar albino rats at the dose level of 2000 mg/kg body weight and the LD50 of this compound is >2000 mg/kg body weight.

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 study report.

Additional information

Acute oral toxicity:

Acute oral toxicity study of the given test chemical was conducted as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Wistar female rats.

Twelve female Wistar rats were selected for acute oral toxicity study. The animals were fasted for minimum 16-18 hours prior to dosing and for 4 hours post dosing, with feed 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 group G1 (Step I) were dosed with starting dose of 300 mg/kg body weight and no mortality was observed.

Based on the results from G1 (Step I), additional three animals of group G1 (Step II) were dosed with 300 mg/kg body weight. As there was no mortality at group G1 (Step I and II) dose levels, three rats of group G2 (Step III) was dosed with 2000 mg/kg body weight and no mortality was observed so another three rats of group G2 (Step IV) were dosed with 2000 mg/kg body weight. Since there was no mortality in both the groups G1 (Step I and II) and G2 (Step III and IV), further dosing was not required.

Body weights were re­corded on day 0 (prior to dosing) 7 and 14. Mean body weight of all the animals (Step I, Step II, Step III and Step IV) treated with 300 and 2000 mg/kg body weight and was observed with gain on day 7 and 14, as compared to day 0.

At 300 and 2000 mg/kg body weight, animals of G1 (Step I and II) and G2 (Step III and IV) were normal throughout the experimental period.

No external and internal gross pathological changes were seen in all the animals of G1 (Step I and II) and G2 (Step III and IV) treated with 300 and 2000 mg/kg body weight respectively, during terminal sacrifice.

Under these study conditions, the acute oral toxicity dose (LD50) was considered to be >2000 mg/kg bw, when female Wistar rats were treated with the given test chemical via oral route. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute oral toxicity. CLP Classification "Not classified"

Acute Inhalation Toxicity:

In different studies, the given test chemical has been investigated for acute inhalation 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:

 

Acute inhalation toxicity study of the given test chemical was conducted as per OECD Guideline No. 403 in Wistar Rats.

Three male and three female healthy young adult rats were randomly selected and used for conducting acute inhalation toxicity limit study. Rats free from injuries were selected for the study. The study was carried out in dynamic nose only inhalation chamber. The dynamic inhalation chamber has three main parts namely inlet, exposure and outlet chambers. Total capacity of chamber is 22.52 liters. Each rat was restrained in a single transparent polycarbonate exposure tube with adjustable unit.

Continuous aerosol generation from as such (undiluted) test item was not possible hence test item was diluted in Dimethyl sulfoxide (DMSO) at the concentration of 70% (v/v) based on the solubility test. Formulated test item was loaded into a 50 ml syringe which was positioned to continuous infusion syringe pump at a flow rate of 40 ml/hour that was selected for main study based on pre test results. The rats were exposed to inhalation chamber exposure part. At a flow rate of 8 litres per minute, air was passed into nebulizer at 30 psi pressure and 18 litres per minute air was drawn from the chamber to ensure slight negative pressure inside the chamber to prevent leakage of test item into the surrounding area. Minimum 15 air changes per hour were maintained in inhalation chamber. The rats were exposed for continuous 4 hours after 20 minute equilibration period at the breathing zone concentration of 5.27 mg/liter air in Dimethyl Sulfoxide (DMSO). The mean chamber temperature, relative humidity, oxygen and carbon dioxide concentration were 22.40°C, 54.03%, 21.08% and 0.09%, respectively; which were measured four times during 4 hour exposure period. The mass median aerodynamic diameter of test item was 3.02 µm with geometric standard deviation of 2.45.

The animals were observed daily during the acclimatization period and mortality/morbidity and clinical signs were recorded. All animals were observed for clinical signs at 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0. Mortality/Morbidity was recorded during 1, 2, 3 and 4 hours during exposure period and 30 minutes and one hour after exposure period on day 0 (in common with the clinical signs) and twice daily during days 1-14 (at least once on the day of sacrifice). Body weights were re­corded on day 0 (prior to exposure) and on days 1, 3, 7 and 14 and found dead. All animals were necropsied and examined macroscopically.

Clinical signs like lethargy and tremors were observed after exposure. Animal nos. 1, 2 and 4 were found normal from day 5 till terminal sacrifice whereas animal nos. 3 and 5 were found normal from day 6 till terminal sacrifice. Animal no. 6 was found dead on day 2 post exposure.

Decrease in mean body weight was observed on days 1 and 3 while increase in mean body weight was observed on days 7 and 14 in male rats whereas in female rats decrease in mean body weight was observed on day 1, 3, 7 and 14 when compared with day 0 mean body weight. ll the animals were subjected to gross pathological examination. External observation of found dead animal and terminally sacrificed animals did not show any pathological abnormality. Internal observation of all terminally sacrificed animals did not show any pathological abnormality. In found dead animal following changes were observed in organs like: Lungs: Red discoloration was observed in Animal no. 6.

Under the conditions of this study, the median lethal concentration (LC50) of 70% of the given test chemical diluted  in Dimethyl Sulfoxide (DMSO) in male and female Wistar rats was estimated as being greater than the breathing zone concentration of 5.27 mg/l air after four hour inhalation exposure and classified as “Not classified” as per CLP classification.

 

The above study is further supported with the study mentioned in another study report for the test chemical. The acute inhalation study of the test chemical was conducted in albino rats according to OECD-Guideline -403 for testing of chemicals.

LIMIT TEST: Ten healthy Wistar albino rats of both sexes (5 male and 5 female) of body weight 200±20 gm were selected for study after acclimatization. The test compound was dissolved in 0.1% tween 80 in distilled water. The test group of animals was exposed to aerosol at the concentration of 5 mg/L for period of 4 hrs. After exposure all the animals were closely observed for clinical signs of toxicity at various intervals such as 1 hr, 2 hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day throughout the experimentation period of 14 days. The necropsy was performed on all the animals at termination of experiment.

All the albino rats exposed to aerosol of the test chemical at the concentration of 5 mg/L did not show any clinical signs of intoxication. Furthermore, no mortality was observed throughout the period of observation. The necropsy was performed on all the animals at the termination of study did not show any gross pathological changes.

CONFIRMATORY TEST: After 72 hrs, the result obtained from limit test was confirmed in another 10 animal of both sex at similar concentration following same guideline.

Ten healthy Wistar albino rats of both sexes (body weight 200±20 gm) were selected for study after acclimatization. The test group animals were exposed to aerosol of the test compound at the concentration of 5 mg/L for period of 4 hrs. After exposure all the animals were closely observed for any clinical signs of toxicity at various intervals such as 1 hr, 2 hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day throughout the experimentation period of 14 days. The necropsy was performed on all the animals at termination of experiment.

All the albino rats exposed to aerosol at the concentration of 5 mg/L did not show any clinical signs of intoxication. Again there was no mortality recorded during the entire observation period. The body weight of animals exposed to test compound, observed on day 0th (pre treatment) and day 7th (post treatment) did not differ significantly as compared to day 0th. Whereas, body weight of animals observed on day 14th showed normal increase as compared to day 0th. The necropsy was performed on all the animals at the termination of study did not show any gross pathological changes.

The result obtained from present investigation can be concluded that the test chemical is acutely non toxic to Wistar albino rats at the exposure concentration of 5 mg/l. The acute lethal Concentration (LC50) of test chemical was considered to be >5 mg/L (>5000 mg/m3).

 

Thus, based on the above summarised studies on the test chemical, it can be concluded that LC50 value is >5 mg/L air. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute inhalation toxicity.

 

Acute Dermal toxicity:

The acute dermal toxicity study of the given test chemical was conducted on wistar albino rats under OECD guideline-402 Guideline for Testing of Chemicals.

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

The test chemical applied at the dose level of 2000 mg/kg b.wt in Wistar albino rats did not show any clinical signs of toxicity throughout the observation period of 14 days.   Furthermore, no mortality was observed throughout the period of observation (14 days). After 14 days necropsy was conducted on all the animals which did not reveal any significant gross pathological changes.

CONFIRMATORY TEST:  After 72 hrs. a confirmatory test was conducted in same species of animals to confirm the limit test of test compound (OECD-402 guidelines).

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

There was no mortality recorded throughout the observation period after application of the test compound at the dose level of 2000 mg/kg body weight. The test compound did not elicit any clinical signs of toxicity during the entire observation period. The body weight of each animal treated with test compound observed on day 0th (pre treatment) and then 7thand 14th (post treatment) did not show any significant increase or decrease in their body weight. Necropsy was conducted on day 15th (end of study) on all the animals which did not reveal any significant gross pathological changes related to compound toxicity.

From the results obtained from present investigation, it can be concluded that the test chemical is non toxic to Wistar albino rats at the dose level of 2000 mg/kg body weight and the LD50 of this compound is >2000 mg/kg body weight.

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, for acute oral toxicity; LC50 value is >5 mg/L air, for acute inhalation toxicity; and LD50 value is >2000 mg/kg bw, for acute dermal toxicity. Thus, comparing these values with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral, acute inhalation and acute dermal toxicity.