Phenethyl phenylacetate

Endpoint summary

Description of key information

Acute Toxicity: Oral

Acute Oral Toxicity Study of the test chemical was conducted as per OECD 423 Guidelines in rats. Six female Wistar rats were selected for acute oral toxicity study. No external and internal gross pathological changes were seen in all the six animals treated with 2000 mg/kg body weight during terminal sacrifice. Under the conditions of this acute oral toxicity study, the acute oral LD50 value was greater than 2000 mg/kg. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.

Acute Toxicity: Inhalation

The acute inhalation study of test chemical was conducted in Wistar rats. The study was performed according to OECD-Guideline -403. The necropsy performed on all the animals at the termination of study did not show any gross pathological changes. The acute inhalation toxicity dose (LC50) was considered to be >5 mg/L (>5000 mg/m3), when male and female Wistar rats were exposed with the test chemical via inhalation route by nose only exposure for 4 hours. Hence the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Acute Toxicity: Dermal

Acute dermal toxicity study for the 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. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Under the conditions of the study, the acute dermal toxicity dose (LD50) was considered to be >2000 mg/kg bw, when rats were treated with the test chemical by dermal application.

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 reports

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The aim of this study was to assess the toxicity potential of the given test chemical after single oral administration in rats and an observation period of 14 days.

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house animals, bred at Animal House.
- Age at study initiation: 9-11 weeks at the time of dosing.
- Health Status: Healthy young adult animals were used for the study. Females were nulliparous and non pregnant.
- Weight at study initiation: Minimum: 148 g and Maximum: 165 g (Individual body weights were within ± 4% prior to treatment after overnight fasting)
- Fasting period:The animals were fasted for minimum 16-18 hours prior to dosing and for 4 hours post dosing.
- Housing: The animals were housed individually in polycarbonate cages.
- Bedding: All cages were provided with corn cobs.
- Room Sanitation: The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle: All the cages and water bottles were changed at least twice every week.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rodent diet, ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum via drinking bottles.
- Acclimation period: Animal nos. 1-3 were acclimatized for seven days and 4-6 for nine days prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.60°C and Maximum: 21.40°C.
- Humidity (%):Minimum: 47.40 % and Maximum: 58.60 %.
- Air changes (per hr): More than 12 changes per hour.
- Photoperiod (hrs dark / hrs light):12:12

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2000 mg/kg body weight
- Amount of vehicle (if gavage): No data
- Justification for choice of vehicle: based on solubility testing
- Lot/batch no.: MKBG9426V

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight

DOSAGE PREPARATION: Added slowly vehicle in to the test item and mixed well. Transferred the formulation to the measuring cylinder and made the volume up to desired quantity of 10 ml. The dosing solution was prepared freshly, shortly prior to dose administration

Doses:

G1 = 2000 mg/kg body weight

No. of animals per sex per dose:

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 survived rats were euthanised by overdose of CO2 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.
Body weight - All surviving rats were weighed on days 0 (prior to dosing), 7 and 14. Animals were weighed immediately after found dead.
Mortality - All surviving animals were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period.

Statistics:

not specified

Preliminary study:

not specified

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

No mortality was observed in the animals treated with 2000 mg/kg dose throught out the 14 days observation period

Clinical signs:

other: At 2000 mg/kg, all the animals were normal throughout the experimental period.

Gross pathology:

No external and internal gross pathological changes were seen in all the six animals treated with 2000 mg/kg body weight during terminal sacrifice

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	Day 0-7	Day 0-14
1	G1/ 2000	165	198	212	20.00	28.48
2		160	185	196	15.63	22.50
3		148	177	187	19.59	26.35
4		152	178	196	17.11	28.95
5		157	183	196	16.56	24.84
6		157	181	204	15.29	29.94

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

Group/ Dose (mg/kg)		Rats Body Weight (g)			Body Weight Changes (%)
		Day 0	Day 7	Day 14	0-7	0-14
G1/ 2000	Mean	156.50	183.67	198.50	17.36	26.84
	SD	5.96	7.63	8.53	2.00	2.82
	n	6	6	6	6	6

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

Table 3: Individual Animal Clinical Signs and Symptoms

Animal No.	Group/ Dose (mg/kg)	Hours (Day 0)
		1/2	1	2	3	4
1	G1/ 2000	1	1	1	1	1
2		1	1	1	1	1
3		1	1	1	1	1
4		1	1	1	1	1
5		1	1	1	1	1
6		1	1	1	1	1

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

Keys: 1 = Normal

Table 4: Individual Animal Mortality Record

Animal No.	Group/ Dose (mg/kg)	Day of Observation (Day 0 to 14)
		Morning Observations	Evening Observations
1	G1/ 2000	No mortality and morbidity	No mortality and morbidity
2		No mortality and morbidity	No mortality and morbidity
3		No mortality and morbidity	No mortality and morbidity
4		No mortality and morbidity	No mortality and morbidity
5		No mortality and morbidity	No mortality and morbidity
6		No mortality and morbidity	No mortality and morbidity

Table 5: Gross Necropsy Observation

Animal No.	Group/ Dose (mg/kg)	Mode of Death	Gross Observation
			External	Internal
1	G1/ 2000	TS	No abnormality detected	No abnormality detected
2		TS	No abnormality detected	No abnormality detected
3		TS	No abnormality detected	No abnormality detected
4		TS	No abnormality detected	No abnormality detected
5		TS	No abnormality detected	No abnormality detected
6		TS	No abnormality detected	No abnormality detected

Keys: TS =Terminal Sacrifice

Interpretation of results:

other: Not classified

Conclusions:

No external and internal gross pathological changes were seen in all the six animals treated with 2000 mg/kg body weight during terminal sacrifice. Under the conditions of this acute oral toxicity study, the acute oral LD50 value was greater than 2000 mg/kg. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.

Executive summary:

Acute Oral Toxicity Study of the test chemical was conducted as per OECD 423 Guidelines in rats. Six 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, food was withheld but drinking water provided ad libitum. Three rats of first group were dosed with starting dose of 2000 mg/kg body weight and the animals did not show any mortality so another three animals of the same group were dosed with 2000 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped.Body weights were recorded on day 0 (prior to dosing) 7 and 14. Mean body weight of all the animals treated with 2000 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0. At 2000 mg/kg, all the animals were normal throughout theexperimental period. No external and internal gross pathological changes were seen in all the six animals treated with 2000 mg/kg body weight during terminal sacrifice. Under the conditions of this acute oral toxicity study, the acute oral LD₅₀ value was greater than 2000 mg/kg. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

Kilmisch Rating 1

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 with acceptable restrictions

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Principles of method if other than guideline:

The study was conducted to find out the LC50, clinical effects and histopathological effect of test chemical at different aerosol concentration in Wistar albino rats.

GLP compliance:

no

Test type:

other: Acute Inhalation Toxicity

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: in bred in lab facility
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: Range of 200 ±20g
- Housing: Groups of 5 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): conventional Laboratory diets, Pelleted feed supplied
- Water (e.g. ad libitum):Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad-libitum
- Acclimation period:20 healthy albino rats were selected and acclimatized for standard laboratory condition for period of 1 week in experimental room under veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 degree 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: water (migrated information)

Remarks:

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 aerosol was generated by the used of Nanotek aerosol generator (particle size less than 1 micron)
- Method of conditioning air: 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.
- System of generating particulates/aerosols: Nanotek aerosol generator
- Treatment of exhaust air: The exhaust air was decontaminated by subsequent passage through 1% NaOH solution, silica gel and activated charcoal filters.

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.
Body weight: The body weight of all the animals was observed weekly on day 0 (Before treatment), 7th and 14th (post treatment).
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.
- Necropsy of survivors performed: yes, Necropsy was carried out on all the animals that died during the study or surviving animals were sacrificed at the end of the study to observe any gross pathological changes.
- Other examinations performed: All the animals were observed for mortality and signs of intoxication at various intervals for first 6 hours on the day of dosing and thereafter twice a day for 14 days.

Statistics:

not specified

Preliminary study:

not specified

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality at the tested dose level of 5.0 mg/L was observed throughout the period of observation after exposure.

Clinical signs:

other: other:

Body weight:

- other body weight observations: The body weight of all the animals recorded individually on day 7th and 14th (post treatment) showed normal gain as compared to day 0th.

Gross pathology:

No gross pathological changes were observed

Other findings:

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 inmesenteric 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 normalcolour, 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
i. Brain- Normal in shape and size.

TABLE -1

EXPOSURE ATMOSPHERE DATA

 

Parameters	Group-I (5 mg/L) (limit test)	Group-II (5 mg/L) (confirmatory test)
Chamber temperature °C (Mean ± S.E.)	23.19±1.46	24.21±1.63
Relative humidity (%)  (Mean ± S.E.)	46.27±2.35	45.27±2.69
Oxygen content (%) (Mean ± S.E.)	19.46±1.64	19.14±1.2

 

TABLE – 2

Mean Body Weight (gm)

 

S.No.	Groups	BODY WEIGHT (gm)
		DAY 0	DAY 7^th	% gain or loss	DAY14^th	% gain or loss
1.	Group-I (5.0 mg/L)	199.36	205.51	3.0 %	213.89	7.28 %
2.	Group-II (5.0 mg/L)	207.57	214.66	3.41 %	223.49	7.66 %

TABLE – 3

CLINICAL SIGNS AND MORTALITY

Group: I                                                                                           Dose: 5.00 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

 

0         =   No clinical sign (Normal)

+         =   Mild

++        =   Moderate

+++      =   High

++++    =    Severe

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
20167-1	TS	Day 14	NAD
20167-2	TS	Day 14	NAD
20167-3	TS	Day 14	NAD
20167-4	TS	Day 14	NAD
20167-5	TS	Day 14	NAD
20167-6	TS	Day 14	NAD
20167-7	TS	Day 14	NAD
20167-8	TS	Day 14	NAD
20167-9	TS	Day 14	NAD
20167-10	TS	Day 14	NAD

 

Day 0 is the day of exposure

TS=Terminal Sacrifice

NAD=No Abnormality Detected

FD=Fg/L

Group II- 5 mg/l 

WISTAR ALBINO RAT

Animal ID	Fate	Time	Gross Findings
20167-11	TS	Day 14	NAD
20167-12	TS	Day 14	NAD
20167-13	TS	Day 14	NAD
20167-14	TS	Day 14	NAD
20167-15	TS	Day 14	NAD
20167-16	TS	Day 14	NAD
20167-17	TS	Day 14	NAD
20167-18	TS	Day 14	NAD
20167-19	TS	Day 14	NAD
20167-20	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:

The acute inhalation toxicity dose (LC50) was considered to be >5 mg/L (>5000 mg/m3), when male and female Wistar rats were exposed with the test chemical via inhalation route by nose only exposure for 4 hours. Hence the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Executive summary:

The acute inhalation study of test chemical was conducted in Wistar rats. The study was performed according to OECD-Guideline -403. In the 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 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 chemical aerosol exposure and later on twice a day throughout the experimentation period of 14 days.The necropsy was performed on all the animals which were died during the exposure or were sacrificed at termination of experiment. All the albino rats exposed to aerosol at the concentration of 5 mg/Ldid 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 sex body weight 200±20 gm were selected for study after acclimatization. The test groups of animals were exposed to aerosol 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 chemical 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 5mg/L did not show any clinical signs of intoxication. Furthermore, no mortality was observed throughout the period of observation.The body weight of animals exposed to test compound, observed on day 0th (pre treatment) and day 7th (post treatment) did not differ significantly as compared to day 0th. Whereas, body weight of animals observed on day 14th showed normal increase as compared to day 0th. The necropsy was performed on all the animals at the termination of study did not show any gross pathological changes.

The acute inhalation toxicity dose (LC50) was considered to be >5 mg/L (>5000 mg/m3), when male and female Wistar rats were exposed with the given test chemical via inhalation route by nose only exposure for 4 hours. From the results obtained from present investigation, it can be concluded that test chemical was non-toxic at the aerosol concentration of 5.0 mg/L under the test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 5 000 mg/m³ air

Quality of whole database:

Klimisch Rating 1

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 reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

The objective of the study was to assess the dermal toxicity of the test chemical after single dose application by dermal route in rats and 14 day observation period.

GLP compliance:

yes

Test type:

other: Acute Dermal Toxicity

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-House Bred at Animal Facility.
- Age at study initiation:Healthy young adult animals were used for the study.
- Females were nulliparous and non pregnant.
- Weight (Prior to Treatment):Male:Minimum: 250 g and Maximum: 277 g ,Female:Minimum: 240 g and Maximum: 256 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.
- Randomization : Animals were selected manually. No computer generated randomization program was used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.70 °C and Maximum: 21.40 °C
- Humidity (%):Minimum: 47.40% and Maximum: 58.60%
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: The test item was applied uniformly over clipped dorsal area of rat skin.
- % coverage: Approximately 10% body surface area of rat.
- Type of wrap if used: The porous gauze dressing and non-irritating tape.

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

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A limit dose of 2000 mg/ kg body weight of test item was applied.
- Constant volume or concentration used: yes

Duration of exposure:

24 hrs

Doses:

2000 mg/kg body weight.

No. of animals per sex per dose:

10 (Five per sex)

Control animals:

no

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 and subjected to gross pathology examination, for external and internal observations.
- Other examinations performed:
- Clinical signs : After test item administration, individual animals were frequently observed at 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all animals were observed once a day during the 14 day observation period.
- Body weight: All rats were weighed on days 0 (prior to dosing), 7 and 14.
other:
- Local Signs/Skin Reactions
All animals were observed once daily during days 1-14 (in common with clinical signs).
- Mortality
Animals were observed twice daily for any mortality during the experimental period.

Statistics:

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

Preliminary study:

No data available

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

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

Clinical signs:

other: At 2000 mg/kg, all the animals were observed normal 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:

No data available

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

Dose: 2000 mg/ kg bodyweight                                                                            Density: 1.07513

Animal No.	Sex	Dose Volume* (ml)	Body Weight (gram)			Body Weight Change (%)
			Day 0	Day 7	Day 14	Day 0-7	Day 0-14
1	Male	0.49	263	270	278	2.66	5.70
2		0.47	250	257	269	2.80	7.60
3		0.52	277	281	294	1.44	6.14
4		0.48	260	271	285	4.23	9.62
5		0.49	265	279	293	5.28	10.57
6	Female	0.48	256	259	262	1.17	2.34
7		0.45	240	242	247	0.83	2.92
8		0.47	252	256	261	1.59	3.57
9		0.47	253	255	257	0.79	1.58
10		0.45	244	247	251	1.23	2.87

Keys: * = based on the test item density and day 0 body weight

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

Key: 1 = Normal

Table 3: Individual Animal Mortality Record

Dose: 2000 mg/kg body weight

Animal No.	Sex	Days of Observation (0 to 14)
		Morning Observations	Evening Observations
1	Male	No mortality and morbidity	No mortality and morbidity
2		No mortality and morbidity	No mortality and morbidity
3		No mortality and morbidity	No mortality and morbidity
4		No mortality and morbidity	No mortality and morbidity
5		No mortality and morbidity	No mortality and morbidity
6	Female	No mortality and morbidity	No mortality and morbidity
7		No mortality and morbidity	No mortality and morbidity
8		No mortality and morbidity	No mortality and morbidity
9		No mortality and morbidity	No mortality and morbidity
10		No mortality and morbidity	No mortality and morbidity

Table 4: Summary of 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	263.00	271.60	283.80	3.28	7.92
	SD	9.72	9.48	10.52	1.49	2.13
	n	5	5	5	5	5
Female	Mean	249.00	251.80	255.60	1.12	2.66
	SD	6.71	7.05	6.47	0.33	0.74
	n	5	5	5	5	5

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

Table 5: Gross Necropsy Observation

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

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

Interpretation of results:

other: not classified

Conclusions:

Under the conditions of the study, the acute dermal toxicity dose (LD50) was considered to be >2000 mg/kg bw, when rats were treated with the test chemical by dermal application.

Executive summary:

Acute dermal toxicity study for the 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. Approximately, twenty four hours prior to dermal application of test item, greater than 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight based on the test item density (1.07513) and latest body weight was applied by single dermal application and observed for 14 days after treatment. On test day 0, calculated volume of test item 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 and post dosing till the termination. 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 re¬corded 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. At 2000 mg/kg, all the animals were observed normal throughout the experimental period. Mean body weight was observed with gain on day 7 and 14 of male and female animals, 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 the study, the acute dermal toxicity dose (LD50) was considered to be >2000 mg/kg bw, when rats were treated with the test chemical by dermal application.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

Klimisch Rating 1

Additional information

Acute Toxicity: Oral

Various studies have been reviewed to determine the acute oral toxicity of the test chemical. These include in vivo experimental studies performed on rats, mice, guinea pigs for the test chemical. The results are mentioned below:

Acute Oral Toxicity Study of the test chemical was conducted as per OECD 423 Guidelines in rats. Six 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, food was withheld but drinking water provided ad libitum. Three rats of first group were dosed with starting dose of 2000 mg/kg body weight and the animals did not show any mortality so another three animals of the same group were dosed with 2000 mg/kg body weight and no mortality was observed. Hence, further dosing was stopped. Body weights were recorded on day 0 (prior to dosing) 7 and 14. Mean body weight of all the animals treated with 2000 mg/kg body weight was observed with gain on day 7 and 14, as compared to day 0. At 2000 mg/kg, all the animals were normal throughout the experimental period. No external and internal gross pathological changes were seen in all the six animals treated with 2000 mg/kg body weight during terminal sacrifice. Under the conditions of this acute oral toxicity study, the acute oral LD50 value was greater than 2000 mg/kg. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.

The result from the Guideline study is supported by an acute oral toxicity study conducted for the test chemical in rats. A group of 10 male and female Osborne-Mendel rats were dosed the test chemical at the dose concentration of 12830 -18470 mg/kg bw via stomach tube. All animals were maintained under close observation for recording toxic signs and time of death. Such observation was continued until animals appeared normal and showed weight gain. The usual observation period was 2 weeks. LD50's were computed by the method of Litchfield & Wilcoxon (1949). Mortality was observed between 4hrs to 5 days. Clinical signs were observed such as, depression soon after treatment and scrawny appearance for several days. Under the condition of the study, the acute oral median lethal dose (LD50) was considered to be 15390 mg/kg bw, with 95% confidence limits of 12830 -18470 mg/kg bw, when group of 10 male and female Osborne-Mendel rats were treated with the test chemical via oral gavage route.

These results are further supported by a similar acute oral toxicity study performed on mice. The test chemical was administered at the dose concentration of 3190 mg/kg bw in mice. Animals were observed for mortality. 50% mortality was observed at dose 3190 mg/kg bw in mice. Hence, the acute oral median lethal dose (LD50) was considered to be 3190 mg/kg of body weight, when mice were treated with the test chemical via oral route.

The above results are also supported by another similar study performed on guinea pigs. Guinea pigs were orally dosed with 3190 mg/kg bw of the test chemical and observed for mortality and other other clinical changes on a regular basis (observation duration not mentioned). 50% mortality was observed at dose 3190 mg/kg bw in guinea pigs. Hence, the acute oral median lethal dose (LD50) was considered to be 3190 mg/kg of body weight, when guinea pigs were treated with the test chemical via oral route.

Available results indicate that the test chemical is not likely to cause any oral toxicity to the test animals when dosed above 2000 mg/kg. Hence, the acute oral median lethal dose (LD50) can be considered to be greater than 2000 mg/kg, and the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Acute Toxicity: Inhalation

The acute inhalation study of test chemical was conducted in Wistar rats. The study was performed according to OECD-Guideline -403. In the 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 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 chemical aerosol exposure and later on twice a day throughout the experimentation period of 14 days.The necropsy was performed on all the animals which were died during the exposure or were sacrificed at termination of experiment. All the albino rats exposed to aerosol at the concentration of 5 mg/Ldid 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 sex body weight 200±20 gm were selected for study after acclimatization. The test groups of animals were exposed to aerosol 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 chemical 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 5mg/L did not show any clinical signs of intoxication. Furthermore, no mortality was observed throughout the period of observation. The body weight of animals exposed to test chemical, 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 performed on all the animals at the termination of study did not show any gross pathological changes. The acute inhalation toxicity dose (LC50) was considered to be >5 mg/L (>5000 mg/m3), when male and female Wistar rats were exposed with the test chemical via inhalation route by nose only

exposure for 4 hours. Hence the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Acute Toxicity: Dermal

Various studies have been reviewed to determine the acute oral toxicity of the test chemical. These include in vivo experimental studies performed on rats, mice, guinea pigs for the test chemical. The results are mentioned below:

Acute dermal toxicity study for the 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. Approximately, twenty four hours prior to dermal application of test item, greater than 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight based on the test item density (1.07513) and latest body weight was applied by single dermal application and observed for 14 days after treatment. On test day 0, calculated volume of test item 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 and post dosing till the termination. 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. At 2000 mg/kg, all the animals were observed normal throughout the experimental period. Mean body weight was observed with gain on day 7 and 14 of male and female animals, 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 the study, the acute dermal toxicity dose (LD50) was considered to be >2000 mg/kg bw, when rats were treated with the test chemical by dermal application.

The above result from the Guideline study is supported by another similar study performed according to OECD 402 Guidelines on Wistar albino rats. A limit test was conducted using ten healthy Wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was shaved 24 hrs prior to application of test chemical. The test chemical was applied dermally at the dose level of 2000 mg/kg for each animal. The treated animals were closely observed for clinical signs of intoxication and mortality for 4 hrs and 1 hr interval for period of 24 hrs. Thereafter, all the animals were observed twice daily for mortality and clinical signs of toxicity throughout the experimentation 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 study. The test chemical when 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. No mortality was observed throughout the period of observation (14 days). After 72 hours a confirmatory test was conducted using ten healthy Wistar albino rats of both sex (ranging b.wt 200±20 gm). Approximate 10 percent back skin of total body surface area was shaved 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 closely observed for clinical signs of intoxication and mortality for 4 hrs and 1 hr interval for period of 24 hrs. Thereafter, all the animals were observed twice daily for mortality and clinical signs of toxicity throughout the experimentation 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 study. The test chemical did not produce any mortality at the tested dose level of 2000 mg/kg in Wistar albino rats throughout the period of observation. The test chemical did not elicit any clinical signs of toxicity during the entire observation period. No skin reaction was observed after 24 hours of patch removal. The body weight of each animal recorded on day 7th and 14th (post treatment) showed normal gain in body weight as compared to day 0 (Pre-treatment). From the results obtained from present investigation, it can be concluded that the test chemical was non toxic to Wistar albino rats at the dose level of 2000 mg/kg b.wt. Hence, the acute dermal median lethal dose (LD50) for the test chemical can be considered to be greater than 2000 mg/kg. Based on these results the test chemical is likely to classify under the category "Not Classified" as per CLP Regulation.

Results from both the Guideline studies indicate that the test chemical is not likely to cause any dermal toxicity to the test animals when dosed above 2000 mg/kg. Hence, the acute dermal median lethal dose (LD50) can be considered to be greater than 2000 mg/kg, and the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Justification for classification or non-classification

Based on the available results, the test chemical was comparatively non-toxic cia oral, inhalation, dermal route to living organisms. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.