Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.
- Weight at study initiation: Body weight range was 199.1 to 219.9 grams.
Body weights at the start : Female Mean: 206.81 g (= 100 %); Minimum : 199.1 g (- 3.73 %); Maximum : 219.9 g (+ 6.33 %)
- Fasting period before study: Approximately 16 hours or more.
- Housing: The rats were housed in polycarbonate cages.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 23.2 degree centigrade.
- Humidity (%): 55.1% to 58.6%.
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
IN-LIFE DATES: 26-09-2016 to 15-10-2016

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(Distilled water)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 300 mg/kg, 300 mg/kg, 2000 mg/kg and 2000 mg/kg
MAXIMUM DOSE VOLUME APPLIED: 10 ml per kg of body weight.

Doses:

Dose Group I : 300 mg/kg
Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
Dose Group II : 2000 mg/kg

No. of animals per sex per dose:

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 therea fter for 14 day. Daily observation was done as far as possible at the same time.
Body weights: Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.
Gross Pathology: Necropsy was performed on all animals at the end of the study period on day 15. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique.
Histopathology: No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.

Statistics:

No data

Preliminary study:

No data

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was obseved

Mortality:

All animals treated at the dose level of 300 mg/kg body weight and 2000mg/kg body weight survived through the study period of 14 days.

Clinical signs:

Group I Step I : Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Group I Step II : Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Group II Step I : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Group II Step II : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Staining of the stool is attributed to the black colour of the test item.

Body weight:

Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.45% and 12.76% respectively.
Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.56% and 12.49% respectively.
Group II Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.06% and 10.98% respectively.
Group II Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 2.17% and 9.86% respectively.

Gross pathology:

Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.

Other findings:

No data

Table No. I

 

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

Group I :   

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From - to	Mortality
I	300	No clinical signs observed	3	1 - 3	0 to 14	0/3

 

 

Group I :

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From - to	Mortality
II	300	No clinical signs observed	3	4 - 6	0 to 14	0/3

 

 

Group II :

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From - to	Mortality
I	2000	Diarrhoea (Black colour stools)	3	7,9 8	4 hrs. - 6 hrs. 2 hrs. - 6 hrs.	0/3

 

Group II :

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From - to	Mortality
II	2000	Diarrhoea (Black colour stools)	3	10,11,12	4 hrs. - 6 hrs.	0/3

 

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

 

 

Table No.II

 

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	300	Mean	201.47	210.43	4.45	227.17	7.95	12.76
		± SD	2.63	2.15	0.39	2.19	0.25	0.68

 

 

Group I :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
II	300	Mean	203.23	210.47	3.56	228.60	8.62	12.49
		± SD	1.35	1.86	0.76	1.71	0.87	1.33

 

 

Group II :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	2000	Mean	207.00	213.33	3.06	229.73	7.69	10.98
		± SD	2.52	2.51	0.67	3.33	0.29	0.81

 

 

Group II :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
II	2000	Mean	215.53	220.20	2.17	236.77	7.52	9.86
		± SD	4.45	3.22	0.67	3.50	0.29	0.91

 

 

Table No.III

 

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	300	1 - 3	TS	No abnormality detected

 

Group I :

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
II	300	4 - 6	TS	No abnormality detected

 

Group II :

 

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	2000	7 - 9	TS	No abnormality detected

 

    Group II :

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
II	2000	10 - 12	TS	No abnormality detected

  TS = Terminal Sacrifice

Interpretation of results:

other: Not classified

Conclusions:

Under the condition of the study, the acute oral LD50 of the test chemical was >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category Not classified” criteria of CLP.

Executive summary:

The acute oral toxicity study was designed and conducted as per OECD 423 for the test chemical using Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours and no mortality after the dosing. As no mortality were observed at 24 hours after the dosing, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool is attributed to the black colour of the test item. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. The acute oral LD50 of test chemical was >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category Not classified” criteria of CLP.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Principles of method if other than guideline:

According to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

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:

Species:Rattus norvegicus (Rat)
Strain:Wistar
Number and Sex:Six Females
Supplier / Source: In-house animals, bred.
Health Status :Healthy young adult animals were used for the study. Females were nulliparous and non pregnant.
Body weight of animals:Minimum: 141 g Maximum: 171 g (Individual body weights were within ± 20 % prior to treatment after overnight fasting)
Age: 08 - 11 weeks at the time of dosing.
Acclimatisation:Animal nos. 1-3 were acclimatized for 6 days and 4-6 for 10 days, prior to administration of the test item.
Identification :The animals were marked temporarily on tail, permanently on toe pad micro tattooing and cage cards. Individual cage cards were labelled with study no., study type, test system, group, dose, sex, animal number experimental start and completion date.
Husbandry Conditions
Diet:All animals were provided conventional laboratory rodent diet.
Bedding:All cages were provided with corn cobs (Sparconn Life Sciences Bangalore) SPAR – 27 /2014.
Water: Aqua guard filtered tap water was provided ad libitum via drinking bottles.
Husbandry:The animals were housed individually in polycarbonate cages.
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.
Experimental Room Condition
Temperature:Minimum: 19.40 °C Maximum: 22.10 °C
Relative humidity:Minimum: 46.60% Maximum: 65.40%
Light-dark-rhythm: 12 hour light and 12 hour dark
Air Changes: More than 12 changes per hour

Route of administration:

oral: unspecified

Vehicle:

water

Remarks:

Distilled Water

Details on oral exposure:

VEHICLE
Distilled water was selected as a vehicle based on solubility testing.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

2000 mg/kg bw- 6 female rats

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, mortality, body weight, pathology

Mortality: All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period

Clinical signs: 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 surviving animals were observed once a day during the 14 day observation period.

Body weight: All surviving rats were weighed on days 0 (prior to dosing), 7 and 14.

Gross pathology: At the end of 14 day observation period, all the survived rats were euthanised by overdose of CO2. All the animals were observed for external and internal gross pathology.

Statistics:

No Data Available

Preliminary study:

No Data Available

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

No mortality was observed in the animals treated with 2000 mg/Kg dose throughout the 14 days observation period

Clinical signs:

At 2000 mg/Kg bw, all the animals were normal thorughout the experimenatl period

Body weight:

Mean body weight of all surviving animals treated with 2000 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0

Gross pathology:

No external and internal gross pathology changes were seen in all the six animals treated with 2000 mg/Kg bw during terminal sacrifice

Other findings:

No Data Available

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

Sex:Female

Animal No.	Group/ Dose (mg/kg)	Body Weight (gram)				Body Weight Change (%)
		Dose Volume*	Day 0	Day 7	Day 14	Day 0-7	Day 0-14
1	G1/ 2000	1.6	162	189	208	16.67	28.40
2		1.4	141	184	199	30.50	41.13
3		1.6	164	207	210	26.22	28.05
4		1.7	171	205	212	19.88	23.98
5		1.7	165	190	198	15.15	20.00
6		1.7	171	191	207	11.70	21.05

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

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

 

Sex:Female

Group/ Dose (mg/kg)		Rats Body Weight (g)			Body Weight Changes (%)
		Day 0	Day 7	Day 14	0-7	0-14
G1/ 2000	Mean	162.33	194.33	205.67	20.02	27.10
	SD	11.09	9.37	5.82	7.11	7.70
	n	6	6	6	6	6

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

Interpretation of results:

not classified

Conclusions:

Based on all the observations and results, it was concluded that the acute oral medial lethal dose (LD50) of the test chemical was >2000 mg/kg bw and thus was not acutly toxic to the female Wistar rats.

Executive summary:

An acute toxicity study as per OECD 423 guideline was conducted to examine the effects of the test chemical through a single exposure. Female wistar rats were used for this experiment. The starting dose was selected as 2000 mg/kg bw. All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period. 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 surviving animals were observed once a day during the 14 day observation period. All surviving rats were weighed on days 0 (prior to dosing), 7 and 14. At the end of 14 day observation period, all the survived rats were euthanized by overdose of CO2. All the animals were observed for external and internal gross pathology. However, no mortality or any other changes were observed at any dose group. Based on all the observations and results, it was concluded that the acute oral medial lethal dose (LD50) of the test chemical was >2000 mg/kg bw and thus was not acutly toxic to the female Wistar rats.

Data source

Reference

Reference Type:

other: other company data

Title:

WoE of acute oral toxicity study for CAS no 73507-17-2

Author:

Sustainability Support Services (Europe) AB

Year:

2019

Bibliographic source:

WoE report, Sustainability Support Services (Europe) AB, 2019

Materials and methods

Test guideline

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:

not specified

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

EC Number:

277-492-0

EC Name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Cas Number:

73507-17-2

Molecular formula:

C52H28Cl2CrN14O18S44Na

IUPAC Name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Details on test material:

- Name of the test chemical: Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
- Molecular formula: C52H28Cl2CrN14O18S4.H.4Na
- Molecular weight: 2693.61 g/mol
- Substance type: Organic

Test animals

Species:

rat

Strain:

other: 2. Sprague-Dawley ; 3. Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

2. TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used.
- Weight at study initiation: Body weight range was 199.1 to 219.9 grams.
Body weights at the start : Female Mean: 206.81 g (= 100 %); Minimum : 199.1 g (- 3.73 %); Maximum : 219.9 g (+ 6.33 %)
- Fasting period before study: Approximately 16 hours or more.
- Housing: The rats were housed in polycarbonate cages.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 23.2 degree centigrade.
- Humidity (%): 55.1% to 58.6%.
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
IN-LIFE DATES: 26-09-2016 to 15-10-2016

3. Species:Rattus norvegicus (Rat)
Strain:Wistar
Number and Sex: Six Females
Supplier / Source: In-house animals, bred.
Health Status :Healthy young adult animals were used for the study. Females were nulliparous and non pregnant.
Body weight of animals:Minimum: 141 g Maximum: 171 g (Individual body weights were within ± 20 % prior to treatment after overnight fasting)
Age: 08 - 11 weeks at the time of dosing.
Acclimatisation:Animal nos. 1-3 were acclimatized for 6 days and 4-6 for 10 days, prior to administr ation of the test item.
Identification :The animals were marked temporarily on tail, permanently on toe pad micro tattooing and cage cards. Individual cage cards were labelled with study no., study type, test system, group, dose, sex, animal number experimental start and completion date. Husbandry Conditions
Diet:All animals were provided conventional laboratory rodent diet.
Bedding:All cages were provided with corn cobs (Sparconn Life Sciences Bangalore) SPAR – 27 /2014.
Water: Aqua guard filtered tap water was provided ad libitum via drinking bottles.
Husbandry:The animals were housed individually in polycarbonate cages.
Room Sanitation:The experimental room floor and work tops were swept and mopped with dis infectant solution every day.
Cages and water bottle:All the cages and water bottles were changed at least twice every week.
Experimental Room Condition
Temperature:Minimum: 19.40 °C Maximum: 22.10 °C
Relative humidity:Minimum: 46.60% Maximum: 65.40%
Light-dark-rhythm: 12 hour light and 12 hour dark
Air Changes: More than 12 changes per hour

Administration / exposure

Route of administration:

other: oral: 1. Gavage; 2. Unspecified

Vehicle:

water

Remarks:

(Distilled water)

Details on oral exposure:

2. VEHICLE
- Concentration in vehicle: 300 mg/kg, 300 mg/kg, 2000 mg/kg and 2000 mg/kg
MAXIMUM DOSE VOLUME APPLIED: 10 ml per kg of body weight.

3. Distilled water was selected as a vehicle based on solubility testing.

Doses:

2. Dose Group I : 300 mg/kg
Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
Dose Group II : 2000 mg/kg

3. 2000 mg/kg bw

No. of animals per sex per dose:

2. Three females were used at each step.
3. 2000 mg/kg bw- 6 female rats

Control animals:

not specified

Details on study design:

2. - Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: yes
- Other examinations performed: Clinical Observations and General Appearance: Animals were observed for clinical signs, mortality and morbidity, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily therea fter for 14 day. Daily observation was done as far as possible at the same time.
Body weights: Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.
Gross Pathology: Necropsy was performed on all animals at the end of the study period on day 15. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique.
Histopathology: No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.

3. - Duration of observation period following administration:
14 days - Frequency of observations and weighing: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, mortality, body weight, pathology
Mortality: All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period
Clinical signs: After test item administration, individual animals were frequently observed at 30 m inutes, 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all surviving animals were observed once a day during the 14 day observation period.
Body weight: All surviving rats were weighed on days 0 (prior to dosing), 7 and 14.
Gross pathology: At the end of 14 day observation period, all the survived rats were euthanised by overdose of CO2. All the animals were observed for external and internal gross pathology.

Statistics:

No data

Results and discussion

Preliminary study:

No data

Effect levels

Effect levels 1

Sex:

female

Dose descriptor:

LD50

Remarks:

2

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was obseved

Effect levels 2

Sex:

female

Dose descriptor:

LD50

Remarks:

3

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

2. All animals treated at the dose level of 300 mg/kg body weight and 2000mg/kg body weight survived through the study period of 14 days.
3. No mortality was observed in the animals treated with 2000 mg/Kg dose throughout the 14 days observation period

Clinical signs:

2. Group I Step I : Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Group I Step II : Animals treated at the dose level of 300 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Group II Step I : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Group II Step II : Animals treated at the dose level of 2000 mg/kg body weight resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.
Staining of the stool is attributed to the black colour of the test item.

3. At 2000 mg/Kg bw, all the animals were normal thorughout the experimenatl period

Body weight:

2. Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.45% and 12.76% respectively.
Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.56% and 12.49% respectively.
Group II Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 3.06% and 10.98% respectively.
Group II Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 2.17% and 9.86% respectively.

3. Mean body weight of all surviving animals treated with 2000 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0

Gross pathology:

2. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups.
3. No external and internal gross pathology changes were seen in all the six animals treated with 2000 mg/Kg bw during terminal sacrifice

Other findings:

No data

Applicant's summary and conclusion

Interpretation of results:

other: Not classified

Conclusions:

Under the condition of the study, the acute oral LD50 of the test chemical was >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in rats falls into the “Category Not classified” criteria of CLP.

Executive summary:

Data available for the various test chemicals was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

The acute oral toxicity study was designed and conducted as per OECD 423 for the test chemical using Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg did not result in any signs of toxicity and mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in diarrhoea (black colour stools) in all animals with onset at 2 hours and no mortality after the dosing. As no mortality were observed at 24 hours after the dosing, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in diarrhoea (black colour stools) in all animals with onset at 4 hours and no mortality after the dosing. All animals from 300 mg/kg and 2000 mg/kg dose groups survived through the study period of 14 days. Staining of the stool is attributed to the black colour of the test item. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. The acute oral LD50 of test chemical was >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in Sprague Dawley rats falls into the “Category Not classified” criteria of CLP.

An acute toxicity study as per OECD 423 guideline was conducted to examine the effects of the test chemical through a single exposure. Female wistar rats were used for this experiment. The starting dose was selected as 2000 mg/kg bw. All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period. 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 surviving animals were observed once a day during the 14 day observation period. All surviving rats were weighed on days 0 (prior to dosing), 7 and 14. At the end of 14 day observation period, all the survived rats were euthanized by overdose of CO2. All the animals were observed for external and internal gross pathology. However, no mortality or any other changes were observed at any dose group. Based on all the observations and results, it was concluded that the acute oral medial lethal dose (LD50) of the test chemical was >2000 mg/kg bw and thus was not acutly toxic to the female Wistar rats.

Based on the data available and applying the weight of evidence approach, the acute oral median lethal dose (LD50) of the test chemical was >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in rats falls into the “Category -Not classified” criteria of CLP.