Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Acute oral toxicity: 

The acute oral toxicity dose (LD50) was considered based on different studies conducted on rats and mice for the test chemical. The LD50 value is 300 mg/kg bw. The study concluded that the LD50 value is between >50 - ≤300 mg/kg bw, for acute oral toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical can be classified in “Category 3” 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 4.66E-6 mm Hg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

Acute Dermal toxicity:

The acute dermal toxicity dose (LD50) was considered based on different studies conducted on rats and 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 purpose of this study was to assess the Toxicological profile of test item to a single administration via oral route to Sprague Dawley rats. This study was designed to determine the acute toxicity at fixed dose levels by oral route of the test item.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing.
- Weight at study initiation: Body weight range was 198.6 to 207.2 grams.
Body weights at the start : Female Mean: 202.32 g (= 100 %); Minimum : 198.6 g (- 1.84 %); Maximum : 207.2 g (+ 2.41 %)
- Identification: Each female rat was individually identified by the picric acid marking.
- Fasting period before study: Approximately 16 hours or more.
- Housing: The rats were housed in polycarbonate cages.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 to 22.9 degree centigrade.
- Humidity (%): 56.1% to 60.2%
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 14-06-2017 to 02-07-2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 300 mg/kg, 50 mg/kg and 50 mg/kg
MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight.
Doses:
Dose Group I : 300 mg/kg
Dose Group II : 50 mg/kg
Dose Group II : 50 mg/kg
No. of animals per sex per dose:
Three females were used at each step.
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical Observations and General Appearance: Animals were observed for clinical signs, mortality and morbidity, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

Body weights: Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.

Gross Pathology: Necropsy was performed on all animals, found dead and sacrificed at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15).

Histopathology: Gross pathological examination revealed distended stomach with liquid ingesta and haemorrhages on mucosa, lesion observed due to the localized irritation of test item and no other systemic abnormality observed hence, no organ collected for histopathology.
Statistics:
not specified
Preliminary study:
not specified
Sex:
female
Dose descriptor:
LD50
Effect level:
> 50 - < 300 mg/kg bw
Based on:
test mat.
Remarks on result:
other: LD50 cut off value
Mortality:
Group I Step I : Animals treated at the dose level of 300 mg/kg body weight: Two animals died on day 1 after the dosing.
Group II Step I : Animals treated at the dose level of 50 mg/kg body weight: All animals survived through the study period of 14 days.
Group II Step II : Animals treated at the dose level of 50 mg/kg body weight: All animals survived through the study period of 14 days.
Clinical signs:
Group I Step I : Animals treated at the dose level of 300 mg/kg body weight resulted in diarrhoea, exophthalmos, loss of righting reflex, markedly reduced locomotor activity, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. The only surviving animal was free of signs of toxicity on day 3 after the dosing.
Group II Step I : Animals treated at the dose level of 50 mg/kg body weight resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Group II Step II : Animals treated at the dose level of 50 mg/kg body weight resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Body weight:
Group I Step I (300 mg/kg) – Percent body weight gain after 7 days and 14 days was found to be 3.83% and 9.61% respectively.
Group II Step I (50 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.80% and 12.20% respectively.
Group II Step II (50 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.91% and 12.45% respectively.
Gross pathology:
Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg and 50 mg/kg dose groups. Gross pathological examination revealed distended stomach with liquid ingesta and haemorrhages on mucosa in found dead animals from 300 mg/kg dose group.
Other findings:
not specified

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs in days

From - to

Mortality

I

300

Diarrhoea  

1

2

Day 1

2/3

Exophthalmos

3

1,3

2

30 min. - 1 hr.

1 hr.

Loss of righting reflex 

3

1,3

2

30 min. - 6 hrs.

2 hrs. - Day 1

Markedly Reduced locomotor activity

3

1,2,3

2 hrs. - 6 hrs.

Reduced locomotor activity

3

1,3

2

30 min. - 6 hrs.

30 min. - Day 2

Ataxic gait

3

1,3

2

30 min. - 6 hrs.

30 min. - Day 2

 

Group II :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs

in days

From - to

Mortality

I

50

Reduced locomotor activity 

3

4,5,6

30 min. - 6 hrs.

0/3

Ataxic gait

3

4,5,6

30 min. - 6 hrs.

 

Group II :

Step

No.

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs

in days

From - to

Mortality

II

50

Reduced locomotor activity 

3

7

8,9

1 hr. - 6 hrs.

30 min. - 6 hrs.

0/3

Ataxic gait

3

7

8,9

1 hr. - 6 hrs.

30 min. - 6 hrs.

 

 

 Table No.II 

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

300

Mean

204.47

208.50

3.83

220.10

5.56

9.61

± SD

3.30

-

-

-

-

-

  

Group II :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

50

Mean

201.33

213.00

5.80

225.90

6.05

12.20

± SD

4.14

4.11

0.76

4.65

0.20

0.97

  

Group II :

Step

No.

Dose

(mg/kg body weight)

 

Before Fasting Body weight

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

II

50

Mean

201.17

213.07

5.91

226.23

6.18

12.45

± SD

1.88

3.66

0.95

4.18

0.70

1.03

 

 

 Table No.III 

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

Group I :

 

Step

No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

300

1, 3

FD

Stomach : Distended with liquid ingesta and haemorrhages on mucosa.

2

TS

No abnormality detected

 

Group II :

Step

No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

50

4 - 6

TS

No abnormality detected

 

  Group II :

Step

No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

II

50

7 - 9

TS

No abnormality detected

            

FD = Found dead

TS = Terminal sacrifice   

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Under the condition of the study, the acute oral LD50 value of the given test chemical was considered in between 50-300 mg/kg body weight. 
Thus, it was concluded that the acute toxicity study of the given test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category 3 (50 – ≤ 300)” criteria of CLP.
Executive summary:

The reported study was designed and conducted to determine the acute oral toxicity profile of the given test chemical as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Sprague Dawley rats.

Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item. Administration of the test item at 300 mg/kg resulted in diarrhoea, exophthalmos, loss of righting reflex, markedly reduced locomotor activity, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. Two animals died on day 1 after the dosing. As mortality was observed at 300 mg/kg dose group, additional three female animals were treated with the lower dose of 50 mg/kg of the test item.

Administration of the test item at 50 mg/kg resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three additional female animals were added to the study and treated with the dose of 50 mg/kg of the test item. Administration of the test item at 50 mg/kg resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing and no mortality at 24 hours after the dosing.

One animal from 300 mg/kg dose group and all animals from 50 mg/kg dose group survived through the study period of 14 days. Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg and 50 mg/kg dose groups. Gross pathological examination revealed distended stomach with liquid ingesta and haemorrhages on mucosa in found dead animals from 300 mg/kg dose group.

Under the condition of the study, the acute oral LD50 value of the given test chemical was considered in between 50-300 mg/kg body weight. 

Thus, it was concluded that the acute toxicity study of the given test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category 3 (50 – ≤ 300)” criteria of CLP.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
300 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:
study scientifically not necessary / other information available
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 study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
This study was designed to determine the dermal LD50 of the test item (up to 2000 mg/kg) or to establish a non-lethal dose level of 2000 milligram of test item per kilogram of body weight.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.
- Weight at study initiation: The weight range of approximately 215.4 to 252.7 grams at initiation of dosing.
Body weights at the start : Male Mean: 246.26 g (= 100 %); Minimum : 241.3 g (- 2.01 %); Maximum : 252.7 g (+ 2.62 %)
Female Mean: 219.38 g (= 100 %); Minimum : 215.4 g (- 1.81 %); Maximum : 222.4 g (+ 1.38 %)
- Identification: Each rat was individually identified by the cage number.
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 21.7 degree centigrade.
- Humidity (%): 55.0% to 58.4%
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 14-06-2017 to 07-08-2017
Type of coverage:
occlusive
Vehicle:
water
Remarks:
(Distilled water)
Details on dermal exposure:
TEST SITE
- Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area)
- % coverage: Approximately 10% of the body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- For solids, paste formed: Yes
Duration of exposure:
24 hours
Doses:
A single dose of 2000 mg of the test item per kilogram of body weight was administered to ten rats (five males and five females).
No. of animals per sex per dose:
10 (5/sex).
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical Observations and General Appearance: Animals were observed for clinical signs, mortality, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time. The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

Evaluation of Dermal Reaction: Dermal reaction was observed daily for study period of 14 days.

Body weights: Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14.

Gross Pathology: Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15).

Histopathology: No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.
Statistics:
not specified
Preliminary study:
not specified
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.
Sex : Female Group I - Animal treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.
Clinical signs:
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Sex : Female Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Body weight:
Sex : Male Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.11% and 18.10% respectively.
Sex : Female Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 6.81% and 10.35% respectively.
Gross pathology:
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.
Other findings:
- Other observations: Evaluation of Dermal Reaction
Sex : Male Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Sex : Female Group I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Male

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

1 - 5

Day 0 - Day 14

0/5

 

Sex : Female

Group

 No.

Dose mg/kg

                            Observed Signs

Total Number of

Animals

 

Animal Nos.

Period of signs in days

 From - to

 

Mortality

I

2000

No clinical signs observed

5

6 - 10

Day 0 - Day 14

0/5

 

 

 

 Table No. II

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs

in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

1 - 5

Day 0 - Day 14

0/5

 

Sex : Female

Group

 No.

Dose mg/kg

                          

Dermal Reaction

Total Number of

Animals

 

Animal Nos.

Period of signs

in days

 From - to

 

Mortality

I

2000

No dermal reaction observed

5

6 - 10

Day 0 - Day 14

0/5

 

 

Table No.III

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Male

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

246.26

268.68

9.11

290.82

8.24

18.10

± SD

4.71

4.11

0.47

4.22

0.32

0.64

 

  

Sex : Female

Group No.

Dose

(mg/kg body weight)

 

Body weight Day 0

Body weight Day 7

% body weight gain

day 0-7

Body weight Day 14

% body weight gain

day 7- 14

% body weight gain

day 0- 14

I

2000

Mean

219.38

234.32

6.81

242.08

3.31

10.35

± SD

2.70

3.45

0.55

3.46

0.25

0.47

 

 

Table No.IV

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Male

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

1 - 5

TS

No abnormality detected

 

Sex : Female

Group No.

Dose

mg/kg

Animal Numbers

Animal Fate

Gross Pathological Findings

I

2000

6 - 10

TS

No abnormality detected

 TS = Terminal Sacrifice

Interpretation of results:
other: Not Classified
Conclusions:
It was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not classify as an acute dermal toxicant.  
CLP Classification: “Not classified”.
Executive summary:

The reported study was designed and conducted to determine the acute dermal toxicity profile of the given test chemical as per OECD Guideline 402 (Acute Dermal Toxicity) in Sprague Dawley rats.

The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.   

Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. 

It was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not classify as an acute dermal toxicant.  

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.

Additional information

Acute oral toxicity:

In different studies, the given test chemical has been investigated for acute oral toxicity to a greater or lesser extent. Often are the studies based on in-vivo experiments in rodents,

i.e. most commonly in rats and mice for test chemical. The studies are summarized as below –

 

The reported key study was designed and conducted to determine the acute oral toxicity profile of the given test chemical as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Sprague Dawley rats.

Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item. Administration of the test item at 300 mg/kg resulted in diarrhoea, exophthalmos, and loss of righting reflex, markedly reduced locomotor activity, reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing. Two animals died on day 1 after the dosing. As mortality was observed at 300 mg/kg dose group, additional three female animals were treated with the lower dose of 50 mg/kg of the test item.

Administration of the test item at 50 mg/kg resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three additional female animals were added to the study and treated with the dose of 50 mg/kg of the test item. Administration of the test item at 50 mg/kg resulted in reduced locomotor activity and ataxic gait with onset at 30 minutes to day 1 after the dosing and no mortality at 24 hours after the dosing.

One animal from 300 mg/kg dose group and all animals from 50 mg/kg dose group survived through the study period of 14 days. Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg and 50 mg/kg dose groups. Gross pathological examination revealed distended stomach with liquid ingesta and haemorrhages on mucosa in found dead animals from 300 mg/kg dose group.

Under the condition of the study, the acute oral LD50 value of the given test chemical was considered in between 50-300 mg/kg body weight. Thus, it was concluded that the acute toxicity study of the given test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category 3 (50 – ≤ 300)” criteria of CLP.

 

This study is supported with another study mentioned in peer-reviewed journal and authoritative databases for the given test chemical. In acute oral toxicity study, Wistar male and female rats were treated with the given test chemical orally by gavage in corn oil.

In the first stage test, 3 rats per group were treated with 10, 100 and 1000 mg/kg. No mortality was observed with treatment of 10 and 100 mg/kg. 2 male rats and all three female rats died within 32 hour when treated with 1000 mg/kg. The remaining one male rat died within 72 hour. Treatment with 10 mg/kg did not induce any acute toxic signs. At 100 mg/kg, decreases in spontaneous movement, ataxic gait, eyelid closure, and lacrimation were observed within 1 hour of treatment. Treatment with 1000 mg/kg immediately caused paralytic gait, prone position with coma, lacrimation and hypothermia. Based on the results of the mortality in the first stage test, the acute toxicity of 140, 225, 370 and 600 mg/kg was examined using two rats of each sex/dose group as a second stage test.

Rats treated with doses less than 225 mg/kg recovered within 24 hour but all rats treated with 370 and 600 mg/kg died within 2 days. Depending on the dose administered, loss of spontaneous activity, ataxic gait, eyelid closure, lacrimation, paralytic gait, prone position and coma were observed. Slight congestion in lung, congestion in glandular stomach mucosa, and retention of administered oil in the stomach through to the colon were observed in animals which died within 3 days of dosing. Rats that survived for 14 days exhibited no abnormalities.

Therefore, the LD50 value was considered to be 300 mg/kg when Wistar male and female rats were treated with the given test chemical orally by gavage.

 

These studies are further supported with the data available in authoritative database and conducted to determine acute oral toxicity dose for the given test chemical. In an acute oral toxicity study, Sprague-Dawley male and female rats were treated with the given test chemical at the concentration of 0, 79, 119, 178, 267, 400 and 600 mg/kg orally by gavage in 0.5% aqueous CMC solution. Deaths were observed until 6 hours after administration, and more deaths were seen 2 days after administration than 1 day after administration.

One male in the 178 mg/kg group died on the afternoon of 1 day after administration and one female died on 2 days after administration. One male in 267 mg/kg group died on the afternoon of 1 day after administration. Females in the 267 mg/kg group died on the second day after administration. One female in the 600 mg/kg group died 1st day after administration and one each sex of the 600 mg/kg group died on the same afternoon.

On the 2nd day after administration, all remaining males and females in the group of 400 mg/kg or more died. At 400 mg/kg or more, in both males and females, all cases died by 2 days after administration. Deaths were more common on 2 days after administration than 1 day after administration.

In the control group, abnormality was not observed in both males and females in observation of general condition. No abnormal symptoms were observed in males and females within 30 minutes after administration in the group of 267 mg/kg or less. In males and females of 79 mg/kg group, salivation was observed in each case after 2 hours after administration, but disappeared in 6 hours after administration in males and disappeared on 1 day after administration in females.

In the 119 mg/kg group, in 2 to 3 cases of salivation and spontaneous locomotion in 2 hours after administration in females, the decline in locomotor activity was observed in 1 case on 1 day after administration. No abnormal symptoms were observed after 2 days. In males and females of 178 mg/kg group and 267 mg/kg group, symptoms such as a decrease in locomotor activity, prone, salivation, epidermal decline and respiratory depression were observed in 1 to 5 cases after 2 hours from the administration. In both sexes of male and female, 1 to 5 cases of lacrimation, 178 mg/kg and 267 mg/kg in addition to symptoms such as reduction of locomotor activity, prone, epidermal decline and respiratory depression on the first day after administration were observed.

In males and females at 400 mg/kg or more, there is a decrease in spontaneous locomotion after 20 minutes from the administration, staggering walking, prone, salivation, epidermal decline, breathing slowing, coat pollution around the nose and mouth, lacrimation Symptoms were seen in 1 to 5 cases. One day after administration, symptoms such as abdomen, menstrual contamination around the nose and mouth, epidermal decline, respiratory depression, and lacrimation were observed in 1 to 5 cases in males and females.

In the group of 267 mg/kg or more, both the sexes decreased in body weight on the 1st day after administration compared with that before administration, and in male, a significant difference was observed in each group as compared with the control group. In the group of 178 mg/kg or less in both sexes, there was a tendency to suppress increase until 1 to 7 days after administration, but no significant difference was observed, and at the time of necropsy, it was almost the same value as the control group at autopsy. In the surviving cases, there was no marked change in any of the sexes of the control group and each test group administered group.

In death cases, almost all of the males and females on the 2nd day after administration contained green liquid retention in the bladder, dark reddening of the glandular gastric mucosa and diffuse thymic darkness in the thymus in half or more of sexes on 1 and 2 days after administration Red spots were observed. In addition, dark reddening of the bladder mucosa was observed in a few cases of males and females dead on 1 and 2 days after administration, and one male with death on the first day after administration showed reddish brown ascetic fluid retention Moderate to severe bleeding was observed in cortex and medulla of Thymus in males and females who had diffuse dark red spots at necropsy. Moderate bleeding was observed in mucous membranes and sub mucosa of Bladder in female deaths where dark reddening of the mucosa was observed at necropsy. In the stomach, there was no remarkable change in any of the cases.

Therefore, LD50 was considered to be 218 mg/kg bw, with 95% confidence limits of 166 to 287 for male and 208 mg/kg bw for female when Sprague-Dawley male and female rats were treated with the given test chemical orally by gavage. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical is classified into the “Category 3”.

 

All these studies are contradicted with the study mentioned in handbooks and secondary source for the test chemical. The acute oral toxicity study was conducted in rats at the dose concentration of 476 mg/kg bw. Animals were observed for mortality. 50% mortality was observed at 476 mg/kg bw in treated rats. Hence, the acute oral LD50 value was considered to be 476 mg/kg bw, when rats were treated with the given test chemical via oral route.

 

The above contradicted study is supported with another study mentioned in databases and handbook for the test chemical. In acute oral toxicity study, mice were treated with the given test chemical at the dose concentration of 750 mg/kg bw orally. Animals were observed for mortality. 50% mortality observed at 750 mg/kg bw. Therefore, the LD50 value was considered to be 750 mg/kg bw when mice were treated with the given test chemical orally.

 

These studies are supported with the data available in handbook and secondary source for the given test chemical. The acute toxicity study was conducted in groups of 5 male and 5 female Wistar rats at the dose concentrations of 1160, 1390, 1670, 2000 and 2400 mg/kg bw.

The given test chemical was dissolved as 20% (w/v) suspension in polyethylene glycol and administered via oral gavage route. Animals were observed for mortality, clinical signs and gross pathology for 14 days. Necropsy of survivors performed.

Deaths occurred from 20 hours to 11 days after treatment. Signs of intoxication observed in all dose groups within a few hours after dosing included sluggishness and decreased activity. The survivors recovered rapidly and looked healthy again at the end of 14 day observation period. At autopsy of the survivors, there were no indications for pathological alterations were observed.

Hence, the acute oral LD50 value was considered to be 1230 mg/kg bw when Wistar male and female rats were treated with the given test chemical orally by gavage.

 

All the above contradicted studies are further supported with the study mentioned in different handbooks and secondary source for the test chemical. The acute oral toxicity study was conducted in white mice at the dose concentration of 1250 mg/kg bw. Animals were observed for mortality and signs of toxicity. 50% mortality observed at 1250 mg/kg bw in treated animals. No signs of intoxication were observed in treated mice. Hence, the acute oral LD50 value was considered to be 1250 mg/kg bw when white mice were treated with the given test chemical orally.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that the LD50 value is between 50-300 mg/kg bw. Thus, comparing this range with the criteria of CLP regulation, the given test chemical can be classified in “Category 3” 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 4.66E-6 mm Hg. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

In one case, the acute inhalation toxicity study was conducted by using the given test chemical in rats at the dose concentration of 1270 mg/m3 via inhalation route for 4 hours of exposure. Animals were observed for mortality. No mortality was observed at 1270 mg/m3 in treated animals. Hence, the LC50 value was considered to be >1270 mg/m3 when rats were treated with the given test chemical by inhalation route for 4 hours.

 

From the above study, the details on possible symptoms or findings were not provided. Hence, this study is difficult to classify for acute inhalation toxicity.

Acute Dermal Toxicity:

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

 

The reported study was designed and conducted to determine the acute dermal toxicity profile of the given test chemical as per OECD Guideline 402 (Acute Dermal Toxicity) in Sprague Dawley rats.

The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. 

It was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to male and female Sprague Dawley rats was considered to be >2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not classify as an acute dermal toxicant. CLP Classification: “Not classified”.

 

The above study is supported with another study mentioned in study report and 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% 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.

The test compound 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). The necropsy was performed on all animals at the termination of the study did not show any 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% 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.

The test compound did not produce any mortality at the tested dose level of 2000 mg/kg b.wt in wistar albino rats throughout the period of observation. Furthermore, the test compound did not elicit any clinical signs of toxicity during the entire the observation period. The body weight of each animal treated with test compound observed on day 0th (pre treatment) and then 7thand 14th (post treatment) showed normal gain as compared to day 0. Necropsy was conducted on day 15th (end of study) did not reveal any significant gross pathological changes related to compound toxicity.

Based on the results obtained from present investigation, it can be concluded that the given test compound is non toxic to Wistar albino rats at the dose level of 2000 mg/kg body weight. The acute dermal LD50 of test compound considered to be >2000 mg/kg b.wt. Thus, according to CLP criteria the given test compound can be classify under non toxic category at the tested dose level of 2000 mg/kg body weight.

 

These studies are supported with the study mentioned in secondary literature for the given test chemical. The acute dermal toxicity study was conducted by using the given test chemical in rabbits at the dose concentration of 5010 mg/kg bw. Animals were observed for mortality. No mortality was observed in treated animals at 5010 mg/kg bw. Hence, the acute dermal LD50 value was considered to be >5010 mg/kg bw, when rabbits were treated with the given test chemical by dermal application.

 

All the above studies are further supported with the data available in secondary literature for the given test chemical. The acute dermal toxicity study was conducted by using the given test chemical in rabbits at the dose concentration of 7940 mg/kg bw. Animals were observed for mortality. No mortality was observed in treated animals at 7940 mg/kg bw. Hence, the acute dermal LD50 value was considered to be >7940 mg/kg bw, when rabbits were treated with the given test chemical by dermal application.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that the 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 dermal toxicity.

 

Justification for classification or non-classification

Based on the above studies on test chemical, it can be concluded that LD50 value is between >50 - ≤300 mg/kg bw, for acute oral toxicity and LD50 value is >2000 mg/kg bw, for acute dermal toxicity. Thus, comparing this range and value with the criteria of CLP regulation, the given test chemical can be classified in “Category 3” for acute oral toxicity and cannot be classified for acute dermal toxicity. For acute inhalation toxicity wavier was added so, not possible to classify.

Categories Display