9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and hexyl and octyl derivs.

Endpoint summary

Description of key information

Acute oral toxicity: 

The acute oral toxicity dose (LD50) was considered based on different studies conducted on rats for the given test chemical. The studies concluded that the LD50 value is >2000 mg/kg bw, for acute oral toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity.

Acute Inhalation Toxicity:

The acute inhalation toxicity dose (LC50) was considered based on experimental study conducted on rats for the given test chemical. The study concluded that the LC50 value is >5 mg/L (>5000 mg/m3), for acute inhalation toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute inhalation toxicity.

Acute Dermal toxicity:

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

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data from various test chemicals

Justification for type of information:

Data is summarized based on the available information from various read across test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on 3 acute oral toxicity studies as - WoE 2, WoE 3 and WoE 4.
Acute Oral toxicity test was carried out to study the effects of the test chemicals on rodents.

GLP compliance:

not specified

Test type:

other: not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing.
- Weight at study initiation: Body weight range was 199.8 to 211.7 grams.
Body weights at the start :
Female
Mean : 204.54 g (= 100 %)
Minimum : 199.8 g (- 2.32 %)
Maximum : 211.7 g (+ 3.50 %)
Total No. of animals : 12
- Identification: Each female rat was individually identified by the picric acid marking.
- Fasting period before study: Approximately 16 hours or more.
- Housing: The rats were housed in polycarbonate cages.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 21.9 degree centigrade.
- Humidity (%): 56.3% to 59.8%
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: 14-07-2017 to 02-08-2017

3.. not specified

Route of administration:

oral: gavage

Vehicle:

other: 2. corn oil 3. not specified

Details on oral exposure:

2.VEHICLE
- Concentration in vehicle: 300 mg/kg, 300 mg/kg, 2000 mg/kg and 2000 mg/kg
- Amount of vehicle (if gavage): No data available
- Justification for choice of vehicle: No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight.

DOSAGE PREPARATION (if unusual): No data available

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: No data available

3. not specified

Doses:

2. Dose Group I : 300 mg/kg
Dose Group I : 300 mg/kg
Dose Group II : 2000 mg/kg
Dose Group II : 2000 mg/kg
3. 3660 mg/kg

No. of animals per sex per dose:

2. Three females were used at each step.
3. not specified

Control animals:

not specified

Details on study design:

2. - Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily
- Necropsy of survivors performed: Yes
- Other examinations performed:
Clinical Observations and General Appearance:
Animals were observed for clinical signs, mortality and morbidity, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.

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

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

Histopathology:
No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.
3. not specified

Statistics:

2. not specified
3. not specified

Preliminary study:

2. not specified
3. not specified
4. not specified

Sex:

female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Sex:

not specified

Dose descriptor:

LD50

Effect level:

3 660 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 50% mortality was observed

Mortality:

2. Group I
Step I :
Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days.

Group I
Step II :
Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days.

Group II
Step I :
Animals treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.

Group II
Step II :
Animals treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.


3. 50% mortality was observed at 3660 mg/kg bw.

Clinical signs:

2.Group I
Step I :
Animals treated at the dose level of 300 mg/kg body weight resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 2 after the dosing.

Group I
Step II :
Animals treated at the dose level of 300 mg/kg body weight resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 2 after the dosing.

Group II
Step I :
Animals treated at the dose level of 2000 mg/kg body weight resulted in test item coloured faeces with onset on day 1 after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 3 after the dosing.

Group II
Step II :
Animals treated at the dose level of 2000 mg/kg body weight resulted in test item coloured faeces with onset on day 1 after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 3 after the dosing.

3. not specified

Body weight:

2.Group I
Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 6.27% and 13.49% respectively.

Group I
Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.98% and 11.96% respectively.

Group II
Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.73% and 11.36% respectively.

Group II
Step II (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.90% and 13.15% respectively.

3. not specified

Gross pathology:

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

Other findings:

2. not specified
3. not specified

Interpretation of results:

other: Not classified

Conclusions:

According to CLP regulation, the test chemical cannot be classified for acute oral toxicity, as the LD50 value is >2000 mg/kg bw.

Executive summary:

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

 

The study reported was designed and conducted to determine the acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing and no mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in test item coloured faeces with onset on day 1 after the dosing and no mortality after the dosing. As no mortality were observed at 24 hours after the dosing, additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in test item coloured faeces with onset on day 1 after the dosing and no mortality after the dosing. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. The acute oral LD50 (Cut-off value) of test chemical was 5000 mg/kg body weight.  Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in Sprague Dawley rats falls into the Category "Not classified" criteria of CLP.

 

The above study is further supported with the study mentioned in database and handbook for the test chemical. The acute oral toxicity was conducted in rats at the concentration of 3660 mg/kg bw. 50% mortality was observed in treated rats at 3660 mg/kg bw. Therefore, LD50 was considered to be 3660 mg/kg bw, when rats were treated with the test chemical via oral route.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that LD50 value is >2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 2 and from various read across test chemicals.

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 experimental 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 mortality(LC50), clinical effect and histopathological effect of test compound at different aerosol concentration in Wistar albino rats.

GLP compliance:

no

Test type:

other: not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Institute for Industrial Research and Toxicology.
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 200±20 g
- Fasting period before study: No data available.
- 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 supplied by Pranav agro Industries Ltd., B7/6 Ramesh Nagar, Delhi, India.
- 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: Twenty healthy albino rats were selected and acclimatized for standard laboratory condition for period of one week in experimental room under vaterinary 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 degC
- 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: 0.1% Tween 80 in Distilled water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nanotek aerosol generator
- 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 chamb er, so that only the snouts and nostrils of the animals were exposed to the aerosol.
- Source and rate of air: No data available
- 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
- Method of particle size determination: No data available
- 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:
Temperature: Group I : 22.15±1.14 and Group II: 22.37±1.41,
Relative humidity (%): Group I : 47.32±3.15 and Group II: 48.15±3.27

TEST ATMOSPHERE
- Brief description of analytical method used: No data available
- Samples taken from breathing zone: Yes

VEHICLE
- Composition of vehicle (if applicable): 0.1% Tween 80 in Distilled water
- Concentration of test material in vehicle (if applicable): 5 mg/L
- Justification of choice of vehicle: No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Less than 1 micron
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): No data available

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: No data available

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

4 h

Concentrations:

Two groups
Group I - Limit test (5 mg/L)
Group II - Confirmatory test (5 mg/L)

No. of animals per sex per dose:

Total: 20
Group I – Limit test (5 mg/L) : 5 males and 5 females
Group II – Confirmatory test (5 mg/L): 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: daily
- Necropsy of survivors performed: yes
- Other examinations performed:
Body weight:
The body weight of all the animals was observed weekly on day 0 (Before treatment), 7th and 14th (post treatment).

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.

Necropsy:
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:

No data available

Preliminary study:

No data

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:
A.EXTRENAL:
i:skin: skin and hair coat was observed wet
ii:all external orifices: normal
B.INTERNAL:
i:subcutaneous: no change was observed
ii:superficial and deep lymph node: 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 observed
iii. digestive sysytem: no gross changes were observed in stomach and intestine
iv. liver and biliary ducts: no gross pathological changes were observed
v. excretory sysytem: no gross pathological changes were observed
vi. adrenal: observed normal
vii. male/female genital organs: showed normal color,consistancy and no inflammartory 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 observed
iii. heart: no changes were observed in color and consistancy. heart found normal
iv. thyroid: normal in shape,size and surface
3. CRANIAL CAVITY:
brain: normal in shape and size

Other findings:

No data

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.)	22.15±1.14	22.37±1.41
Relative humidity (%) (Mean ± S.E.)	47.32±3.15	48.15±3.27
Oxygen content (%) (Mean ± S.E.)	19.54±1.14	20.27±1.41

TABLE - 2

MEAN BODY WEIGHT (gm)

S. No.	Groups	Body Weight (gm)
		Day 0	Day 7th	% gain or loss	Day 14th	% gain or loss
1.	Group – I (5.0 mg/L)	202.43	208.21	2.85	213.51	5.47
2.	Group – II (5.0 mg/L)	200.46	206.61	3.06	213.12	6.31

TABLE – 3

CLINICAL SIGNS AND MORTALITY

GROUP – I                                                                                                                                 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	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe 

 

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

 

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE – 4

SUMMARY OF NECROPSY FINDINGS

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 and necropsy findings

Group:I                                                                               5.0 mg/l

WISTAR ALBINO RATS

Animal ID	Fate	Time	Gross findings
20171-1	TS	Day 15	NAD
20171-2	TS	Day 15	NAD
20171-3	TS	Day 15	NAD
20171-4	TS	Day 15	NAD
20171-5	TS	Day 15	NAD
20171-6	TS	Day 15	NAD
20171-7	TS	Day 15	NAD
20171-8	TS	Day 15	NAD
20171-9	TS	Day 15	NAD
20171-10	TS	Day 15	NAD

DAY 0 is the day of exposure

TS= terminal sacrifice

NAD=no abnormality

FD=found dead

 

Table-5 contd……………….

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

GROUP:II                                                       5.0 mg/l

Animal ID	Fate	Time	Gross findings
20171-11	TS	Day 15	NAD
20171-12	TS	Day 15	NAD
20171-13	TS	Day 15	NAD
20171-14	TS	Day 15	NAD
20171-15	TS	Day 15	NAD
20171-16	TS	Day 15	NAD
20171-17	TS	Day 15	NAD
20171-18	TS	Day 15	NAD
20171-19	TS	Day 15	NAD
20171-20	TS	Day 15	NAD

DAY 0 is the day of exposure

TS= terminal sacrifice

NAD=no abnormality

FD=found dead

Interpretation of results:

other: Not classified

Conclusions:

The LC50 value for acute inhalation toxicity test was considered to be >5 mg/l, when wistar albino rats were exposed with the given test chemical by inhalation route for 4 hour according to OECD guideline 403(Acute inhalation toxicity).

Executive summary:

The acute inhalation study was conducted by using the given test chemical performed according to OECD-Guideline – 403 for testing of chemicals in albino rat.

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 1hr, 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 necroscopy was performed on all the animals at the termination of study did not show any gross pathological changes.

CONFIRMATORY TEST: After 72 hrs, the result obtained from limit test was confirmed in another 10 animal of both sex at similar concentration following same guideline. Ten healthy Wistar albino rats of both sex (body weight 200±20 gm) were selected for study after acclimatization. The test group of animals were exposed to aerosol of the test compound at the concentration of 5 mg/L for period of 4 hrs. After exposure all the animals were closely observed for any clinical signs of toxicity at various intervals such as 1 hr, 2 hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day throughout a 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(pretreatment) and day 7th(post treatment) showed normal gain as compared to control group. The necroscopy was performed on all the animals at the termination of study did not show any gross pathological changes.

The result obtained from present investigation can be concluded that, the LC50 value for acute inhalation toxicity test was considered to be >5 mg/l, when wistar albino rats were exposed with the given test chemical by inhalation route for 4 hour according to OECD guideline 403(Acute inhalation toxicity).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 000 mg/m³

Quality of whole database:

Data is Klimisch 2 and from study report.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

The study was conducted to find out the mortality, clinical sign of toxicity and histopathological effect of the given test chemical at different dose level in Wistar albino rats.

GLP compliance:

no

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: Institute for Industrial Research and Toxicology.
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 200±20 g
- Fasting period before study: Animals were fasted overnight prior to test and food was offered 3 hours after dosing.
- Identification : By cage tag and corresponding colour body marking.
- Housing: Groups of two animals of same sex in polypropylene cages with stainless steel grill top, facilities for food and water bottle, and bedding of clean paddy husk.
- Diet (e.g. ad libitum): Pelleted feed supplied by Pranav agro Industries Ltd., B7/6 Ramesh Nagar, Delhi, India.
- Water (e.g. ad libitum): Fresh and clean water filtered through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad-libitum.
- Acclimation period: One week
- Randomization : After acclimatization and veterinary examination all the animals randomly divided into two groups and each group having five male and five female rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25 degC
- Humidity (%): 40-60 %
- Air changes (per hr): Air conditioned rooms with 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: Back skin of total body surface area.
- % coverage: Approximate 10% area.
- Type of wrap if used: The test compound was held in contact with the skiin with impervious dressing secured in place with an adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the site of application was cleaned with lukewarm water wiping the test compound.
- Time after start of exposure: 24 hrs

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg b.wt

Duration of exposure:

24 hours

Doses:

Group - I : 2000 mg/kg b.wt
Group - II : 2000 mg/kg b.wt

No. of animals per sex per dose:

Group - I : 2000 mg/kg b.wt : 5 female and 5 male
Group - II : 2000 mg/kg b.wt : 5 female and 5 male

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, necropsy was carried out on all 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:
- Body weight: The body weight of all the animals was observed weekly on day 0 (pre treatment), 7th and 14th (post treatment).
- Clinical signs : The treated animals were closely observed for clinical signs of intoxication, first 4 hours and thereafter for every 1 hrs interval for 24 hours after dosing and twice a day for 14 days. All the rats were observed at least twice daily with the purpose of recording any symptoms of ill-health or behavioral changes. These observations included changes in skin and fur in the eyes and mucous membranes, respiratory, circulatory, central nervous and autonomous systems, somatomotor activity and behavior changes. The following clinical signs were observed in rats to characterize with erythema, hypersensitivity, edema etc.
- Mortality: All the animals were observed for mortality at 30 minutes time interval for first six hours on the day of test compound administration and thereafter twice a day for 14 days.

Statistics:

No data

Preliminary study:

no data

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: no mortality was observed

Mortality:

The test compound did not produce any mortality throughout the observation period of 14 days.

Clinical signs:

The test compound did not elicit any clinical signs at the dose level of 2000 mg/kg b.wt. in entire observation period.

Body weight:

All the animals treated with the test compound at the dose level of 2000 mg/kg b.wt. showed normal gain in body weight as compared to control group on day 0th (pre treatment).

Gross pathology:

Necropsy finding:
A.EXTRENAL:
i:skin: skin and hair coat was observed wet
ii:all external orifices: normal
B.INTERNAL:
i:subcutaneous: no chnage was observed
ii:superficial and deep lymph node: no chnage 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 observed
iii. digestive sysytem: no gross changes were observed in stomach and intestine
iv. liver and biliary ducts: no gross pathological changes were observed
v. excretory sysytem: no gross pathological changes were observed
vi. adrenal: observed normal
vii. male/female genital organs: showed normal color,consistancy and no inflammartory 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 observed
iii. heart: no changes were observed in color and consistancy. heart found normal
iv. thyroid: normal in shape,size and surface
3. CRANIAL CAVITY:
brain: normal in shape and size

Other findings:

no data

TABLE - 1

SUMMARY OF BODY WEIGHT (GM)

Group	Animal ID	Day 0	Day 7	% Gain/loss	Day 14	% Gain/loss
Group-I 2000 mg/kg b. wt	20171-1	198.26	204.73	3.11	211.85	6.85
	20171 -2	200.43	206.05	2.80	213.26	6.40
	20171 -3	202.82	208.96	3.02	215.58	6.29
	20171 -4	201.20	207.61	3.18	212.07	5.40
	20171 -5	203.56	209.84	3.08	215.35	5.79
	20171 -6	200.78	206.39	2.94	213.22	6.19
	20171 -7	198.53	204.75	3.13	211.60	6.58
	20171 -8	199.38	205.62	3.12	212.54	6.60
	20171 -9	202.64	208.08	2.68	214.37	5.78
	20171 -10	201.95	207.33	2.66	212.28	5.11
Group-II 2000 mg/kg b. wt	20171 -11	199.03	205.85	3.42	210.56	5.79
	20171 -12	198.77	204.74	3.00	211.93	6.62
	20171 -13	199.35	205.82	3.24	209.52	5.10
	20171 -14	202.64	207.51	2.40	213.26	5.24
	20171 -15	200.25	206.83	3.28	212.40	6.06
	20171 -16	203.75	208.59	2.37	214.28	5.16
	20171 -17	200.37	206.64	3.12	211.19	5.40
	20171 -18	201.82	207.93	3.02	213.61	5.84
	20171 -19	200.45	206.31	2.92	210.58	5.05
	20171 -20	204.52	209.63	2.49	217.11	6.15

TABLE - 2

CLINICAL SIGNS AND MORTALITY

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

Parameters	Incidence of Clinical Signs Observed after Dosing on																			Mortality
	Day 0					DAY
	Min	Hour
	30	1	2	4	6	1	2	3	4	5	6	7	8	9	10	11	12	13	14	Total	%
Mortality (total)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0/10	0
Clinical Signs- Local
Redness	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Pain	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Swelling	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Systemic signs
Clinical signs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE - 2 Contd...

CLINICAL SIGNS AND MORTALITY

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

                                                                                   

                                                           

Parameters	Incidence of Clinical Signs Observed after Dosing on																			Mortality
	Day 0					DAY
	Min	Hour
	30	1	2	4	6	1	2	3	4	5	6	7	8	9	10	11	12	13	14	Total	%
Mortality (total)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0/10	0
Clinical Signs- Local
Redness	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Pain	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Swelling	-	-	-	-	-	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Systemic signs
Clinical signs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

-          =Observed after 24 hrs

0        = No clinical signs

+        = Mild

++      = Moderate

+++    = High

++++  = Severe

TABLE – 3

SUMMARY OF NECROPSY FINDINGS

S.No.	Fate	Wistar albino rats
		Dose (mg/kg b.wt)
		2000 (limit test)	2000 (confirmatory test)
1	Terminal sacrifice	10/10	10/10
2	Found dead	0/10	0/10
3	Abnormalities detected	0/10	0/10

Table 4: individual animal fate and necropsy findings

Group:I  (limit test)                                                                            2000 mg/kg bw

Animal ID	Fate	Time	Gross findings
20171-1	TS	Day 15	NAD
20171-2	TS	Day 15	NAD
20171-3	TS	Day 15	NAD
20171-4	TS	Day 15	NAD
20171-5	TS	Day 15	NAD
20171-6	TS	Day 15	NAD
20171-7	TS	Day 15	NAD
20171-8	TS	Day 15	NAD
20171-9	TS	Day 15	NAD
20171-10	TS	Day 15	NAD

DAY 0 is the day of exposure

TS= terminal sacrifice

NAD=no abnormality

FD=found dead

 

Table-4 contd……………….

INDIVIDUAL ANIMAL FATE & NECROPSY FINDINGS

GROUP:II (confirmatory test)                                                   2000 mg/kg bw

Animal ID	Fate	Time	Gross findings
20171-11	TS	Day 15	NAD
20171-12	TS	Day 15	NAD
20171-13	TS	Day 15	NAD
20171-14	TS	Day 15	NAD
20171-15	TS	Day 15	NAD
20171-16	TS	Day 15	NAD
20171-17	TS	Day 15	NAD
20171-18	TS	Day 15	NAD
20171-19	TS	Day 15	NAD
20171-20	TS	Day 15	NAD

DAY 0 is the day of exposure

TS= terminal sacrifice

NAD=no abnormality

FD=found dead

 

Interpretation of results:

other: not classified

Conclusions:

The LD50 value was considered to be >2000 mg/kg bw, when Wistar albino rats were occlusively treated with the given test chemical by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

Executive summary:

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

LIMIT TEST (2000 mg/kg b.wt): Ten healthy wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test drug was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. The treated animals were observed for clinical signs of intoxication and mortality at different time interval for a period of 14 days. The body weight of each rat was observed on day 0 (pretreatment), 7thand 14th(post treatment). The necropsy was performed on all animals at the termination of the study. The test compound applied at the dose level of 2000 mg/kg b.wt in Wistar albino rats did not show any clinical signs of toxicity throughout the observation period of 14 days.  Furthermore, no mortality was observed throughout the period of observation (14 days). The necropsy was performed on all animals at the termination of the study did not show any gross pathological changes.

CONFIRMATORY TEST: After 72 hrs, a confirmatory test was conducted in same species of animals to confirm the limit test of test compound (OECD-402 guidelines). Ten healthy Wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test drug was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. The treated animals were obser ved for clinical signs of intoxication and mortality at different time interval for a period of 14 days. The body weight of each rat was observed on day 0 (pretre atment), 7thand 14th(post treatment). The necropsy was performed on all animals at the termination of the study. The test compound did not produce any mortality throughout the observation period of 14 days. Furthermore, the test compound did not elicit any clinical signs at the dose level of 2000 mg/kg b.wt. in entire observation period. All the animals treated with the test compound at the dose level of 2000 mg/kg b.wt. showed normal gain in body weight as compared to control group on day 0th (pre treatment). Necropsy was conducted on day 15th (end of study) did not reveal any significant gross pathological changes related to compound toxicity.

The results obtained from present investigation can be concluded that, the LD50 value was considered to be >2000 mg/kg bw, when Wistar albino rats were occlusively treated with the given test chemical by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 2 and from study report.

Additional information

Acute oral toxicity:

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

 

The study reported was designed and conducted to determine the acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in test item coloured faeces and diarrhoea with onset from 4 hours to day 1 after the dosing and no mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in test item coloured faeces with onset on day 1 after the dosing and no mortality after the dosing. As no mortality were observed at 24 hours after the dosing, additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in test item coloured faeces with onset on day 1 after the dosing and no mortality after the dosing. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg and 2000 mg/kg dose groups. The acute oral LD50 (Cut-off value) of test chemical was 5000 mg/kg body weight.  Thus, it was concluded that the acute toxicity study of test chemical, when administered via oral route in Sprague Dawley rats falls into the Category "Not classified" criteria of CLP.

 

The above study is further supported with the study mentioned in database and handbook for the test chemical. The acute oral toxicity was conducted in rats at the concentration of 3660 mg/kg bw. 50% mortality was observed in treated rats at 3660 mg/kg bw. Therefore, LD50 was considered to be 3660 mg/kg bw, when rats were treated with the test chemical via oral route.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that LD50 value is >2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity.

 

Acute inhalation toxicity:

The acute inhalation study was conducted by using the given test chemical performed according to OECD-Guideline – 403 for testing of chemicals in albino rat.

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 1hr, 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.

CONFIRMATORY TEST: After 72 hrs, the result obtained from limit test was confirmed in another 10 animal of both sex at similar concentration following same guideline. Ten healthy Wistar albino rats of both sexes (body weight 200±20 gm) were selected for study after acclimatization. The test group of animals were exposed to aerosol of the test compound at the concentration of 5 mg/L for period of 4 hrs. After exposure all the animals were closely observed for any clinical signs of toxicity at various intervals such as 1 hr, 2 hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day throughout a 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) showed normal gain as compared to control group. The necroscopy was performed on all the animals at the termination of study did not show any gross pathological changes.

The result obtained from present investigation can be concluded that, the LC50 value for acute inhalation toxicity test was considered to be >5 mg/l, when wistar albino rats were exposed with the given test chemical by inhalation route for 4 hour according to OECD guideline 403(Acute inhalation toxicity).

 

Acute Dermal Toxicity:

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

LIMIT TEST (2000 mg/kg b.wt): Ten healthy wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test drug was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. The treated animals were observed for clinical signs of intoxication and mortality at different time interval for a period of 14 days. The body weight of each rat was observed on day 0 (pretreatment), 7thand 14th (post treatment). The necropsy was performed on all animals at the termination of the study. The test compound applied at the dose level of 2000 mg/kg b.wt in Wistar albino rats did not show any clinical signs of toxicity throughout the observation period of 14 days.  Furthermore, no mortality was observed throughout the period of observation (14 days). The necropsy was performed on all animals at the termination of the study did not show any gross pathological changes.

CONFIRMATORY TEST: After 72 hrs, a confirmatory test was conducted in same species of animals to confirm the limit test of test compound (OECD-402 guidelines). Ten healthy Wistar albino rats of both sex (ranging b.wt 200±20 gm) selected for study after acclimatization. Approximate 10 percent back skin of total body surface area was prepared 24 hrs prior to application of test compound. The test drug was applied dermally at the dose level of 2000 mg/kg b.wt for each animal. The treated animals were observed for clinical signs of intoxication and mortality at different time interval for a period of 14 days. The body weight of each rat was observed on day 0 (pre-treatment), 7thand 14th (post treatment). The necropsy was performed on all animals at the termination of the study. The test compound did not produce any mortality throughout the observation period of 14 days. Furthermore, the test compound did not elicit any clinical signs at the dose level of 2000 mg/kg b.wt. in entire observation period. All the animals treated with the test compound at the dose level of 2000 mg/kg b.wt. showed normal gain in body weight as compared to control group on day 0th (pre treatment). Necropsy was conducted on day 15th (end of study) did not reveal any significant gross pathological changes related to compound toxicity.

The results obtained from present investigation can be concluded that, the LD50 value was considered to be >2000 mg/kg bw, when Wistar albino rats were occlusively treated with the given test chemical by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

 

 

Justification for classification or non-classification

Based on the above studies for the test chemical, it can be concluded that LD50 value is >2000 mg/kg bw, for acute oral toxicity and acute dermal toxicity; LC50 value is >5 mg/L (>5000 mg/m3), for acute inhalation toxicity. Thus, comparing these values with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral, acute inhalation and acute dermal toxicity.