Ethyltriphenylfosfonium bromide

Endpoint summary

Description of key information

Acute oral toxicity: 

An acute oral toxicity dose (LD50) of the test chemical was considered based on experimental study conducted on rats, the LD50 value was considered in between 50-300 mg/kg bw in rats. 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: 

An acute inhalation toxicity dose (LC50) of the test chemical was considered based on experimental study conducted on rats, the LC50 value was considered to be >5.0 mg/L (>5000 mg/m3) in rats. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute inhalation toxicity.

Acute Dermal Toxicity:

An acute Dermal toxicity dose (LD50) of the test chemical was considered based on experimental study conducted on rats, the value was considered to be >2000 mg/kg bw in rats. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute dermal toxicity.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

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

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

- Name of test material: ETHYL TRI-PHENYL PHOSPHONIUM BROMIDE
- Appearance: White to off-white crystalline powder
- Substance type: Organic
- Physical state: solid
- AI Content (purity): 99.7%
- Storage conditions : Room temperature (20 - 30 °C)
- Batch number: ETPB1/B/14016
- Manufactured date: February, 2014
- Expiry Date : January, 2015 (Retest date)
- Other: Handling and Disposal
- Safety precautions:Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the Personnel.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-House Bred
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8- 11 weeks at the time of dosing. Healthy young adult animals were used for the study.
- Weight at study initiation: Minimum: 136 g Maximum: 155 g (Individual body weights were within ± 4% prior to treatment after overnight fasting)
- Fasting period before study: The animals were fasted for minimum 16-18 hours prior to dosing and for 4 hours post dosing, with food withheld but drinking water provided ad libitum.
- 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 7 days and 4-6 for 9 days, 7-9 for 10 days and 10-12 for 6 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 19.60°C Maximum: 23.30°C
- Humidity (%): Minimum: 51.60 % Maximum: 64.60 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: April 26, 2014 To: May 26, 2014

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2000 mg/kg, 300 mg/kg and 50 mg/kg
- Amount of vehicle (if gavage):10 ml/kg body weight.
- Justification for choice of vehicle: Corn oil was selected as a vehicle because test item was not soluble in the distilled water during solubility testing.

Doses:

2000 mg/kg bw, 300 mg/kg bw and 50 mg/kg bw

No. of animals per sex per dose:

2000 mg/kg bw-3 female rats
300 mg/kg bw-3 female rats
50 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, at the end of 14 day observation period, all the surviving rats were euthanised by overdose of CO2. All the animals were subjected for external and internal observations.
- Other examinations performed: 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 surviving animals were observed once a day during the 14 day observation period.
Mortality - All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period.
Body weight - All rats were weighed on days 0 (prior to dosing), 7 and 14. Animals were weighed immediately after found dead.

Statistics:

not specified

Preliminary study:

not specified

Sex:

female

Dose descriptor:

LD50

Effect level:

> 50 - < 300 mg/kg bw

Based on:

test mat.

Mortality:

At 2000 and 300 mg/ kg, all the animals were found dead on day 0 post dosing. No mortality was observed in the animals treated with 50 mg/kg dose throughtout the 14 days observation period.

Clinical signs:

other: At 2000 mg/kg, animal no. 1 was observed with salivation, mild tremors, mild abdominal breathing, sternal recumbency and found dead at 30 minutes post dosing. Animal no. 2 was observed with salivation, mild tremors, mild abdominal breathing, lateral recum

Gross pathology:

At 2000 mg/kg, all three animals were observed with wet area around mouth during external gross pathological examination.
At 300 mg/kg, animal nos. 4 and 5 were observed with no abnormality and animal no. 6 with red area around nose.
At 50 mg/kg, all the six animals were observed with no abnormality.
At 2000 mg/kg, all the animals were observed with moderate red discolouration of all lobes of the lungs, test item in stomach and mild dark colored liver during internal gross pathological examination.
At 300 mg/kg, Animal no. 4 was observed with mild red discolouration of all lobes of the lungs, test item in stomach and mild congestion of Intestine. Animal no. 5 was observed with mild red discolouration of all lobes of the lungs and test item in stomach. Animal no. 6 was observed severe hemorrhages and congestion of lungs, test item in stomach and mild congestion of Intestine.
At 50 mg/kg, all the six animals were observed with no abnormality.

Other findings:

not specified

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

 

Sex:Female

Animal No.	Group/ Dose (mg/kg)	Body Weight (gram)				Body Weight Change (%)
		Day 0	Day 7	Day 14	Found dead	Day 0-7	Day 0-14
1	G1/ 2000	146	-	-	147	-	-
2		143	-	-	147	-	-
3		142	-	-	143	-	-
4	G2/ 300	155	-	-	153	-	-
5		153	-	-	154	-	-
6		150	-	-	146	-	-
7	G3/ 50	152	189	202	-	24.34	26.46
8		146	185	197	-	26.71	27.57
9		136	153	157	-	12.50	13.73
10		148	182	201	-	22.97	29.12
11		143	181	196	-	26.57	29.28
12		146	179	195	-	22.60	27.37

Key:- = Not applicable

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	143.67	-	-	-	-
	SD	2.08	-	-	-	-
	N	3	-	-	-	-
G2/ 300	Mean	152.67	-	-	-	-
	SD	2.52	-	-	-	-
	N	3	-	-	-	-
G3/ 50	Mean	145.17	178.17	191.33	22.62	25.59
	SD	5.38	12.81	17.05	5.25	5.91
	N	6	6	6	6	6

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

Table 3: Individual Animal Clinical Signs and Symptoms

 

Sex:Female

Animal No.	Group/ Dose (mg/kg)	Hours (Day 0)
		1/2	1	2	3	4
1	G1/ 2000	145 166^+ 4^+ 155 2	-	-	-	^-
2		145 166^+ 98 4^+ 2	-	-	-	-
3		145 166^++ 4^+ 98 2	-	-	-	-
4	G2/ 300	99^++	98 4^+ 166^+ 2	-	-	-
5		99^++	166^+ 4^+ 98 2	-	-	-
6		1	99^+ 64	99^+ 64	98 4^+ 64	2
7	G3/ 50	1	1	1	49^+	49^+ 99^+
8		1	1	1	64^+ 49^+	64^+ 49^+
9		1	1	1	49^+	49^+
10		1	1	1	49^+	49^+
11		1	1	1	49^+	49^+
12		1	1	1	1	1

Keys:- = Not applicable, 1 = Normal, 2 = Dead, 4 = Abdominal breathing, 49 = Diarrhoea, 64 = Epistaxis, 98 = Lateral recumbency, 99 = Lethargy, 145 = Salivation, 155 = Sternal recumbency, 166 = Tremors,⁺= Mild, ++ = Moderate.

Table 3: Individual Animal Clinical Signs and Symptoms(Contd...)

 

Animal No.		Group/ Dose (mg/kg)		Days post dosing
			1		2		3		4		5		6		7		8		9		10		11		12		13		14
1		G1/ 2000		-		-		-		-		-		-		-		-		-		-		-		-		-		-
2			-		-		-		-		-		-		-		-		-		-		-		-		-		-
3			-		-		-		-		-		-		-		-		-		-		-		-		-		-
4		G2/ 300		-		-		-		-		-		-		-		-		-		-		-		-		-		-
5			-		-		-		-		-		-		-		-		-		-		-		-		-		-
6			-		-		-		-		-		-		-		-		-		-		-		-		-		-
7		G3/ 50		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			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

Keys:- = Not applicable, 1 = Normal

Table 4: Individual Animal Mortality Record

 

Sex:Female

Animal No.	Group/ Dose (mg/kg)	Day of Observation (Day 0 to 14)
		Morning Observations	Evening Observations
1	G1/ 2000	Found dead on day 0 post dosing	Not applicable
2		Found dead on day 0 post dosing	Not applicable
3		Found dead on day 0 post dosing	Not applicable
4	G2/ 300	Found dead on day 0 post dosing	Not applicable
5		Found dead on day 0 post dosing	Not applicable
6		Found dead on day 0 post dosing	Not applicable
7	G3/ 50	No mortality and morbidity	No mortality and morbidity
8		No mortality and morbidity	No mortality and morbidity
9		No mortality and morbidity	No mortality and morbidity
10		No mortality and morbidity	No mortality and morbidity
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                                                                                                                                        

Animal No.	Group/ Dose (mg/kg)	Mode of Death	Gross Observation
			External	Internal
1	G1/ 2000	Found dead	Wet area around mouth	Lungs: Red discolouration, all lobes (moderate) Stomach: Test item observed Liver: Dark colored (mild)
2		Found dead	Wet area around mouth	Lungs: Red discolouration, all lobes (moderate) Stomach: Test item observed Liver: Dark colored (mild)
3		Found dead	Wet area around mouth	Lungs: Red discolouration, all lobes (moderate) Stomach: Test item observed Liver: Dark colored (mild)
4	G2/ 300	Found dead	No abnormality detected	Lungs: Red discolouration, all lobes (mild) Stomach: Test item observed Intestine: Congestion (mild)
5		Found dead	No abnormality detected	Lungs: Red discolouration, all lobes (mild) Stomach: Test item observed
6		Found dead	Red area around nose	Lungs: Hemorrhages and congestion (severe) Stomach: Test item observed Intestine: Congestion (mild)
7	G3/ 50	Terminal Sacrifice	No abnormality detected	No abnormality detected
8		Terminal Sacrifice	No abnormality detected	No abnormality detected
9		Terminal Sacrifice	No abnormality detected	No abnormality detected
10		Terminal Sacrifice	No abnormality detected	No abnormality detected
11		Terminal Sacrifice	No abnormality detected	No abnormality detected
12		Terminal Sacrifice	No abnormality detected	No abnormality detected

Interpretation of results:

other: Category '3' as per CLP criteria of classification and labeling.

Conclusions:

The acute oral LD50 value of test chemical was considered in between 50-300 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical exhibits acute oral toxicity in “Category 3” LD50 >50 to ≤300 mg/kg body weight.

Executive summary:

Acute oral toxicity study of the given test chemical was conducted as per OECD No. 423 in 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 food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of group G1 were dosed with starting dose of 2000 mg/kg body weight and all the animals died post dosing on day 0. At 2000 mg/kg, animal no. 1 was observed with salivation, mild tremors, mild abdominal breathing, sternal recumbency and found dead at 30 minutes post dosing. Animal no. 2 was observed with salivation, mild tremors, mild abdominal breathing, lateral recumbency and found dead at 30 minutes post dosing. Animal no. 3 was observed with salivation, moderate tremors, mild abdominal breathing, lateral recumbency and found dead at 30 minutes post dosing. So, three rats of group G2 were dosed with 300 mg/kg weight and all animals died on day 0 post dosing. At 300 mg/kg, animal nos. 4 and 5 were observed with moderate lethargy at 30 minutes, mild tremors, mild abdominal breathing, lateral recumbency and found dead at 1 hour post dosing. Animal no. 6 was observed with normal sign at 30 minutes, mild lethargy at 1 and 2 hours, epistaxis at 1, 2 and 3 hours, lateral recumbency and mild abdominal breathing at 3 hours and found dead at 4 hours post dosing. So, three rats of G3 were dosed with 50 mg/kg body weight and no mortality was observed. So, another three animals of the same group G3 were dosed with 50 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped. Body weights of surviving animals were re­corded on day 0 (prior to dosing) 7 and 14. Mean body weight of animals treated with 50 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0. At 50 mg/kg, animal no. 7 was observed with mild diarrhoea at 3 and 4 hours and mild lethargy at 4 hours, animal no. 8 with mild epistaxis and diarrhoea at 3 and 4 hours, animal nos. 9, 10 and 11 with mild diarrhoea at 3 and 4 hours and normal rest of the observation period. Animal no. 12 was observed normal throughout the experiment period. At 2000 mg/kg, all three animals were observed with wet area around mouth during external gross pathological examination. At 300 mg/kg, animal nos. 4 and 5 were observed with no abnormality and animal no. 6 with red area around nose. At 50 mg/kg, all the six animals were observed with no abnormality. At 2000 mg/kg, all the animals were observed with moderate red discolouration of all lobes of the lungs, test item in stomach and mild dark colored liver during internal gross pathological examination. At 300 mg/kg, Animal no. 4 was observed with mild red discolouration of all lobes of the lungs, test item in stomach and mild congestion of Intestine. Animal no. 5 was observed with mild red discolouration of all lobes of the lungs and test item in stomach. Animal no. 6 was observed severe haemorrhages and congestion of lungs, test item in stomach and mild congestion of Intestine. At 50 mg/kg, all the six animals were observed with no abnormality. Under the conditions of this; the acute oral LD50 value of test chemical was considered in between 50-300 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical exhibits acute oral toxicity in “Category 3” LD50 >50 to ≤300 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

150 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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

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 assess the toxicity potential of given test chemical after inhalation exposure in rats and an observation period of 14 days.

GLP compliance:

no

Test type:

other: Acute Inhalation Toxicity

Limit test:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report):ethyltriphenylfosfonium bromide
- Molecular formula :C20H20BrP
- Molecular weight :371.25656
- Substance type:Organic
- Physical state:powder

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation:7 to 9 weeks
- Weight at study initiation:200 ±20g
- Identification-By cage tag and corresponding colour body marking
- Housing:Groups of five animals of similar 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, ad libitum
- Water (e.g. ad libitum): Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad-libitum
- Nutritional conditions:For feeding conventional Laboratory diets may be used with an unlimited supply of drinking water.
- Acclimation period:Twenty healthy albino rats were selected and acclimatized for standard laboratory condition for period of one week in experimental room under veterinary examination.
- Randomization:After acclimatization and veterinary examination all the selected rats randomly divided into two groups of five females and five males.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-25 deg. C
- Humidity (%):30-60%
- 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.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Distilled water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:cylindrical chamber built from stainless steel and glass.
- Exposure chamber volume: 8 liters
- Method of holding animals in test chamber:For the inhalation purpose the rats were placed in polycarbonate holder tubes positioned radically around exposure chamber, so that only the snouts and nostrils of the animals were exposed to the aerosol.
- Source and rate of air: The chamber had a inner and outer chamber to minimize the fluctuation in concentration and temperature.
- Treatment of exhaust air: The exhaust air was decontaminated by subsequent passage through 1% NaOH solution, silica gel and activated charcoal filters.
- Temperature, humidity, pressure in air chamber: The chamber was maintained at a slightly negative pressure to prevent leakage of the test atmosphere from the system, as well as its dilution with outside air.

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

4 h

Concentrations:

Group I – Limit test (5 mg/L)
Group II – Confirmatory test (5 mg/L)

No. of animals per sex per dose:

10 (5 males and 5 females)

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical Signs: The treated animals were observed for signs of intoxication, at various intervals for first six hours after dosing and thereafter twice a day for 14 days. Any clinical signs in the treated group, if observed, recorded and mentioned in the report.
Mortality: All the animals were observed for mortality at various intervals for first six hours on the day of dosing and thereafter twice a day for 14 days.
Body weight: The body weight of all the animals was observed weekly on day 0 (Before 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.

Statistics:

not specified

Preliminary study:

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 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 compound 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.Necropsy findings: The necropsy was performed on all the animals at the termination of study did not show any gross pathological changes.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: no mortality was observed

Mortality:

The test compound did not elicit any mortality at the tested dose level of 5.0 mg/L throughout the period of observation after exposure.

Clinical signs:

other: The test compound did not elicit any clinical signs of intoxication at the tested aerosol concentration of 5 mg/L observed for the period of 14 days.

Body weight:

The body weight of all the animals recorded individually on day 0th and thereafter 7th and 14th (post treatment) showed normal increase as compared to day 0th.

Gross pathology:

NECROPSY FINDING
EXTERNAL
i.Skin- Skin and hair coat was observed wet.
ii.All external orifices- Normal
B. INTERNAL
i. Subcutaneous- No changes were 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 – 3

CLINICAL SIGNS AND MORTALITY

Group: I                                                                                           Dose: 5.0 mg/L

WISTAR ALBNINO RATS

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	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 TABLE – 3 Contd…………….

CLINICAL SIGNS AND MORTALITY

Group: II                                                                                          Dose: 5.0 mg/L

                                                                                               

WISTAR ALBINO RATS

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	0	0	0	0	6	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

0         =   No clinical sign (Normal)

+         =   Mild

++        =   Moderate

+++      =   High

++++    =   Severe

 

TABLE – 4

SUMMARY OF NECROPSY FINDING

S. No.	Fate	Wistar albino rats
		Dose (mg/l)
		5.0 (limit test)	5.0 (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                                                                                                       5.0 mg/L        

WISTAR ALBINO RATS

Animal ID	Fate	Time	Gross Findings
20173-1	TS	Day 14	NAD
20173-2	TS	Day 14	NAD
20173-3	TS	Day 14	NAD
20173-4	TS	Day 14	NAD
20173-5	TS	Day 14	NAD
20173-6	TS	Day 14	NAD
20173-7	TS	Day 14	NAD
20173-8	TS	Day 14	NAD
20173-9	TS	Day 14	NAD
20173-10	TS	Day 14	NAD

  

Day 0 is the day of exposure

TS=Terminal Sacrifice

NAD=No Abnormality Detected

FD=Found Dead

 

TABLE- 5 Contd………………

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

Group: II                                                                                                      5.0 mg/L       

 

WISTAR ALBINO RATS

Animal ID	Fate	Time	Gross Findings
20173-11	TS	Day 14	NAD
20173-12	TS	Day 14	NAD
20173-13	TS	Day 14	NAD
20173-14	TS	Day 14	NAD
20173-15	TS	Day 14	NAD
20173-16	TS	Day 14	NAD
20173-17	TS	Day 14	NAD
20173-18	TS	Day 14	NAD
20173-19	TS	Day 14	NAD
20173-20	TS	Day 14	NAD

 

Day 0 is the day of exposure

TS=Terminal Sacrifice

NAD=No Abnormality Detected

FD=Found Dead

Interpretation of results:

other: Not classified

Conclusions:

Based on the results obtained from above investigation, the acute inhalation toxicity dose (LC50) was considered to be >5.0 mg/l. Hence, it can be concluded that the test compound is non toxic to male and female Wistar rats by inhalation route via aerosol.

Executive summary:

The acute inhalation study of the given test chemical was conducted in albino rat according to OECD-Guideline -403 for testing of chemicals. In 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 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 compound 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. 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 animals of test group 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. Based on the results obtained from above investigation, the acute inhalation toxicity dose (LC50) was considered to be >5.0 mg/l. Hence, it can be concluded that the test compound is non toxic to male and female Wistar rats by inhalation route via aerosol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 000 mg/m³ air

Quality of whole database:

Data is Klimisch 2 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:

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:

According to OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

Identification : ETHYL TRI-PHENYL PHOSPHONIUM BROMIDE
Appearance : White to off-white crystalline powder
Batch number : ETPB1/B/14016
AI Content (purity) : 99.79%
Storage conditions : Room temperature (20 - 30 °C)
Manufactured date : February, 2014
Expiry Date : January, 2016 (Retest date)
Handling and Disposal
Safety precautions :Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the Personnel.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-House Bred
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Healthy young adult animals were used for the study
- Weight at study initiation: Male:Minimum: 264 g and Maximum: 298 g
(Prior to Treatment)Female:Minimum: 242 g and Maximum: 281 g
- 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:All animals were acclimatized to the test conditions for 7 days prior to administration of the test item.
- Identification:During Acclimatization, animals were temporarily marked by permanent marker, on their tails. After acclimatization, the animals were marked by toe pad micro tattooing and cage cards. Individual cage cards were labelled with study no., study type, test system, sex, animal number, experimental start and completion date.
- Randomization:Animals were selected manually. No computer generated randomization program was used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 19.70 °C Maximum: 23.30 °C
- Humidity (%): Minimum: 51.60% Maximum: 64.60%
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From:April 26, 2014 To: May 17, 2014

Type of coverage:

occlusive

Vehicle:

other: distilled water

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of rat skin
- % coverage: approximately 10%
- Type of wrap if used: Test item was held in contact with the skin with a porous gauze dressing and non-irritating tape

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

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.2 ml

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

10 (Five per sex)

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 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all animals were observed once a day during the 14 day observation period.
Mortality - Animals were observed twice daily for any mortality during the experimental period.
Body weight - All rats were weighed on days 0 (prior to dosing), 7 and 14.
Local Signs/Skin Reactions - All animals were observed once daily during days 1-14 (in common with clinical signs).
- Necropsy of survivors performed: yes, at the end of 14 day observation period, all the surviving rats were euthanised by overdose of CO2 and subjected to gross pathology examination, for external and internal observations.

Statistics:

No statistical analysis was performed since the study was terminated with limit test.

Preliminary study:

Limit Test - Five male and five female wistar rats were treated with test item by a single dermal application at the dose level of 2000 mg/kg body weight. Since no test item related mortality was observed, the study was terminated with limit test only.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

No mortality was observed at limit dose of 2000 mg/kg body weight of test item during the 14 day observation period.

Clinical signs:

other: All the animals were observed with normal clinical signs throughout the experimental period.

Gross pathology:

The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality.

Other findings:

not specified

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

Dose:2000 mg/ kg bodyweight                                                                                                         

Animal No.	Sex	Body Weight (gram)			Body Weight Change (%)
		Day 0	Day 7	Day 14	Day 0-7	Day 0-14
1	Male	264	283	316	7.20	19.70
2		298	318	340	6.71	14.09
3		287	299	319	4.18	11.15
4		289	305	330	5.54	14.19
5		275	274	290	-0.36	5.45
6	Female	249	244	247	-2.01	-0.80
7		274	272	282	-0.73	2.92
8		281	280	283	-0.36	0.71
9		242	242	253	0.00	4.55
10		256	255	255	-0.39	-0.39

Table 2: Individual Animal Clinical Signs and Symptoms

 

Dose:2000 mg/kg body weight

Animal No.	Sex	Hour(s) - Day 0				Day
		1	2	3	4	1	2	3	4	5	6	7
1	Male	1	1	1	1	1	1	1	1	1	1	1
2		1	1	1	1	1	1	1	1	1	1	1
3		1	1	1	1	1	1	1	1	1	1	1
4		1	1	1	1	1	1	1	1	1	1	1
5		1	1	1	1	1	1	1	1	1	1	1
6	Female	1	1	1	1	1	1	1	1	1	1	1
7		1	1	1	1	1	1	1	1	1	1	1
8		1	1	1	1	1	1	1	1	1	1	1
9		1	1	1	1	1	1	1	1	1	1	1
10		1	1	1	1	1	1	1	1	1	1	1

 

Animal No.	Sex	Day
		8	9	10	11	12	13	14
1	Male	1	1	1	1	1	1	1
2		1	1	1	1	1	1	1
3		1	1	1	1	1	1	1
4		1	1	1	1	1	1	1
5		1	1	1	1	1	1	1
6	Female	1	1	1	1	1	1	1
7		1	1	1	1	1	1	1
8		1	1	1	1	1	1	1
9		1	1	1	1	1	1	1
10		1	1	1	1	1	1	1

Keys: 1 = Normal

Table 4:Summaryof Animal Body Weight (g) and Body Weight Changes (%)

 

Dose:2000 mg/kg body weight

Sex		Body Weight (gram)			Body Weight Changes (%)
		Day 0	Day 7	Day 14	Day 0-7	Day 0-14
Male	Mean	282.60	295.80	319.00	4.65	12.92
	SD	13.24	17.51	18.79	3.04	5.19
	n	5	5	5	5	5
Female	Mean	260.40	258.60	264.00	-0.70	1.40
	SD	16.56	16.88	17.15	0.78	2.28
	n	5	5	5	5	5

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

Table 5: GrossNecropsyObservation

 

 Dose:2000 mg/kg body weight                                               Mode of Death:Terminal Sacrifice

Animal No.	Sex	Gross Observation
		External	Internal
1	Male	No abnormalities detected	No abnormalities detected
2		No abnormalities detected	No abnormalities detected
3		No abnormalities detected	No abnormalities detected
4		No abnormalities detected	No abnormalities detected
5		No abnormalities detected	No abnormalities detected
6	Female	No abnormalities detected	No abnormalities detected
7		No abnormalities detected	No abnormalities detected
8		No abnormalities detected	No abnormalities detected
9		No abnormalities detected	No abnormalities detected
10		No abnormalities detected	No abnormalities detected

Interpretation of results:

other: Not classified

Conclusions:

Under the conditions of this; acute dermal toxicity dose (LD50) value for given test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute dermal toxicity i.e it is acutely non toxic to animals.

Executive summary:

Acute dermal toxicity study of the given test chemical was conducted as per OECD No.402 in Wistar rats. Five male and five female healthy young adult rats were randomly selected and used for conducting acute dermal toxicity study. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item moistened with 0.2 ml distilled water was applied by single dermal application and observed for 14 days after treatment. On test day 0, an amount of test item (pulverized) moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the porous gauze dressing was put on to the intact skin of clipped area. This porous gauze dressing was covered with a non-irritating tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically. No mortality was observed in any animal till the end of the experimental period. All the animals were observed with normal clinical signs throughout the experimental period. In males, mean body weight was observed with increase on day 7 and 14, as compared to day 0. In females, mean body weight was observed with decrease on day 7 and increase on day 14, as compared to day 0. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Under the conditions of this; acute dermal toxicity dose (LD50) value for given test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute dermal toxicity i.e it is acutely non toxic to animals.

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:

An acute oral toxicity study of the given test chemical was conducted as per OECD TG No. 423 in 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 food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of group G1 were dosed with starting dose of 2000 mg/kg body weight and all the animals died post dosing on day 0. At 2000 mg/kg, animal no. 1 was observed with salivation, mild tremors, mild abdominal breathing, sternal recumbency and found dead at 30 minutes post dosing. Animal no. 2 was observed with salivation, mild tremors, mild abdominal breathing, lateral recumbency and found dead at 30 minutes post dosing. Animal no. 3 was observed with salivation, moderate tremors, mild abdominal breathing, lateral recumbency and found dead at 30 minutes post dosing. So, three rats of group G2 were dosed with 300 mg/kg weight and all animals died on day 0 post dosing. At 300 mg/kg, animal nos. 4 and 5 were observed with moderate lethargy at 30 minutes, mild tremors, mild abdominal breathing, lateral recumbency and found dead at 1 hour post dosing. Animal no. 6 was observed with normal sign at 30 minutes, mild lethargy at 1 and 2 hours, epistaxis at 1, 2 and 3 hours, lateral recumbency and mild abdominal breathing at 3 hours and found dead at 4 hours post dosing. So, three rats of G3 were dosed with 50 mg/kg body weight and no mortality was observed. So, another three animals of the same group G3 were dosed with 50 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped. Body weights of surviving animals were re­corded on day 0 (prior to dosing) 7 and 14. Mean body weight of animals treated with 50 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0. At 50 mg/kg, animal no. 7 was observed with mild diarrhoea at 3 and 4 hours and mild lethargy at 4 hours, animal no. 8 with mild epistaxis and diarrhoea at 3 and 4 hours, animal nos. 9, 10 and 11 with mild diarrhoea at 3 and 4 hours and normal rest of the observation period. Animal no. 12 was observed normal throughout the experiment period. At 2000 mg/kg, all three animals were observed with wet area around mouth during external gross pathological examination. At 300 mg/kg, animal nos. 4 and 5 were observed with no abnormality and animal no. 6 with red area around nose. At 50 mg/kg, all the six animals were observed with no abnormality. At 2000 mg/kg, all the animals were observed with moderate red discolouration of all lobes of the lungs, test item in stomach and mild dark colored liver during internal gross pathological examination. At 300 mg/kg, Animal no. 4 was observed with mild red discolouration of all lobes of the lungs, test item in stomach and mild congestion of Intestine. Animal no. 5 was observed with mild red discolouration of all lobes of the lungs and test item in stomach. Animal no. 6 was observed severe haemorrhages and congestion of lungs, test item in stomach and mild congestion of Intestine. At 50 mg/kg, all the six animals were observed with no abnormality. Under the conditions of this, all rats were died at 300 mg/kg bw, hence the LD100 was considered to be 300 mg/kg bw. Considering this value, the LD50 value can be assumed to be 150 mg/kg bw. Therefore, comparing this value with the criteria of CLP regulation, the given test chemical can be classified in “Category 3 (>50 to ≤300)” for acute oral toxicity.

Acute Inhalation toxicity:

The acute inhalation study of the given test chemical was conducted in albino rat according to OECD-Guideline -403 for testing of chemicals. In 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 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 compound 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. 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 animals of test group 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. Based on the results obtained from above investigation, the acute inhalation toxicity dose (LC50) was considered to be >5.0 mg/l. Hence, it can be concluded that the test compound is non toxic to male and female Wistar rats by inhalation route via aerosol.

Acute Dermal toxicity:

An acute dermal toxicity study of the given test chemical was conducted as per OECD No.402 in Wistar rats. Five male and five female healthy young adult rats were randomly selected and used for conducting acute dermal toxicity study. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item moistened with 0.2 ml distilled water was applied by single dermal application and observed for 14 days after treatment. On test day 0, an amount of test item (pulverized) moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the porous gauze dressing was put on to the intact skin of clipped area. This porous gauze dressing was covered with a non-irritating tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically. No mortality was observed in any animal till the end of the experimental period. All the animals were observed with normal clinical signs throughout the experimental period. In males, mean body weight was observed with increase on day 7 and 14, as compared to day 0. In females, mean body weight was observed with decrease on day 7 and increase on day 14, as compared to day 0. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Under the conditions of this; acute dermal toxicity dose (LD50) value for given test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not exhibit acute dermal toxicity i.e. it is acutely non toxic to animals.

Justification for classification or non-classification

Based on the above experimental studies on the test chemical, it can be concluded that LD50 value is between 50 to 300 mg/kg bw, for acute oral toxicity; LD50 value is >2000 mg/kg bw, for acute dermal toxicity; and LC50 value is >5 mg/L, for acute inhalation toxicity. Thus, comparing these values 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 and acute inhalation toxicity.