4,4'-methylenedi-2,6-xylenol

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 was >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 study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to low vapour pressure of the test chemical, which is reported to be 0.00000256 Pa. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

Acute Dermal toxicity:

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

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The aim of this study was to assess the toxicity potential of test chemical after single oral administration in rats.

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:Bharat Serum and Vaccines Limited.
- Age at study initiation:8- 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: 183 g and Maximum: 216 g (Individual body weights were within ± 5% prior to treatment after overnight fasting).
- Fasting period:The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 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 (Nutrivet Life Sciences, Pune) 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 five days, 4-6 for seven days, 7-9 for nine days and 10-12 for six days prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 20.00 °C and Maximum: 23.60 °C.
- Humidity (%):Minimum: 37.40% and Maximum: 61.80%
- Air changes (per hr):More than 12 changes per hour.
- Photoperiod (hrs dark / hrs light):12:12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Amount of vehicle:10 ml
- Justification for choice of vehicle:Corn oil was selected because test item was not soluble in distilled water.
- Lot/batch no. (if required):MKBD4650
- Purity:N/A

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

Doses:

G1 = 300 mg/kg bw
G2 = 2000 mg/kg bw

No. of animals per sex per dose:

Twelve 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). Additionally, on day 0 animal nos. 7-9 were observed at 6 hours post dosing. Subsequently, all the 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. Animals were weighed immediately after found dead.

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

Statistics:

No data

Preliminary study:

No data

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Non toxic to rat.

Mortality:

No mortality was observed in the animals treated with 300 and 2000 mg/kg dose throught out the 14 days observation period post dosing.

Clinical signs:

other: At 300 mg/kg, all the six animals (animal nos. 1-6) were observed normal throughout experimental period. At 2000mg/kg, animal no. 7 was observed normal at 30 minutes and 1 hour post dosing, mild to moderate lethargy at 2, 3, 4 and 6 hours and mild ataxia

Gross pathology:

No external and internal gross pathological changes were seen in all the six animals treated with 300 and 2000 mg/kg body weight during terminal sacrifice

Other findings:

No data

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/ 300	210	223	234	6.19	11.43
2		204	229	234	12.25	14.71
3		200	230	237	15.00	18.50
4		216	240	253	11.11	17.13
5		204	240	246	17.65	20.59
6		198	237	218	19.70	10.10
7	G2/ 2000	210	219	229	4.29	9.05
8		197	228	224	15.74	13.71
9		195	220	235	12.82	20.51
10		187	217	229	16.04	22.46
11		183	219	228	19.67	24.59
12		185	215	229	16.22	23.78

 

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

 

Sex:Female

Group/ Dose (mg/kg)		Rats Body Weight (g)			Body Weight Changes (%)
		Day 0	Day 7	Day 14	0-7	0-14
G1/ 300	Mean	205.33	233.17	237.00	13.65	15.41
	SD	6.65	6.91	11.97	4.87	4.10
	n	6	6	6	6	6
G2/ 2000	Mean	192.83	219.67	229.00	14.13	19.02
	SD	10.09	4.46	3.52	5.29	6.25
	n	6	6	6	6	6

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

Table 3: Individual Animal Clinical Signs and Symptoms

 

Sex:Female

Animal No.	Group/ Dose (mg/kg)	Hours (Day 0)
		1/2	1	2	3	4	6
1	G1/ 300	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	-
7	G2/ 2000	1	1	99^+	99^++ 21^+	99^++ 21^+	99^++ 21^+
8		1	1	99^+	99^++ 21^+	99^+++ 21^+	99^+++ 21^++
9		1	1	99^++ 21^+	99^+++ 21^++	99^+++ 21^++	99^+++ 21^++
10		1	1	99^+	99^++ 21^+	99^++ 21^+	-
11		1	1	99^+	99^++ 21^+	99^++ 21^+	-
12		1	1	99^+	99^++ 21^+	99^++ 21^++	-

Interpretation of results:

other: Not classified

Conclusions:

Under the conditions of this; acute oral toxicity study of test chemical in female rats is as given below:
The acute oral LD50 value of test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that test chemical does not exhibit acute oral toxicity i.e it is acutely non toxic to animals.

Executive summary:

Acute Oral Toxicity Study was conducted by using the given test chemical in Rats, This study was performed as per OECD No. 423.

Twelve female Wistar rats were selected for acute oral toxicity study. The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 hours post dosing, with food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of group G1 were dosed with starting dose of 300 mg/kg body weight and the animals did not show any mortality so another three animals of the group G1 were dosed with 300 mg/kg body weight and no mortality was observed. Three animals of the group G2 were dosed with 2000 mg/kg body weight and no mortality was observed, therefore another three animals of same group G2 were dose with 2000 mg/kg and no mortality was observed. Hence, further dosing was stopped. Body weights were re¬corded on day 0 (prior to dosing) 7 and 14. Mean body weight was observed with gain was observed in the animals treated with 300 and 2000 mg/kg body weight, during the 14 day observation period, as compared to day 0. At 300 mg/kg, all the six animals (animal nos. 1-6) were observed normal throughout experimental period. At 2000 mg/kg, animal no. 7 was observed normal at 30 minutes and 1 hour post dosing, mild to moderate lethargy at 2, 3, 4 and 6 hours and mild ataxia at 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal no. 8 was observed normal at 30 minutes and 1 hour post dosing, mild to severe lethargy at 2, 3, 4 and 6 hours and mild to moderate ataxia at 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal no. 9 was observed normal at 30 minutes and 1 hour post dosing, moderate to severe lethargy and mild to moderate ataxia at 2, 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal nos. 10-12 were observed normal at 30 minutes and 1 hour post dosing, mild to moderate lethargy at 2, 3 and 4 hours and mild ataxia at 3 and 4 hours followed by normal clinical sign till day 14. 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 of test chemical in female rats is as given below:

The acute oral LD50 value of test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that test chemical does not exhibit acute oral toxicity i.e it is acutely non toxic to animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 1 and from study report.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size

Clinical signs:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

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 test chemical after single dose application by dermal route in rats.

GLP compliance:

yes

Test type:

standard acute method

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 sa-Ford, Animal Facility
- Age at study initiation:Healthy young adult animals were used for the study
- Females (if applicable) nulliparous and non-pregnant: yes, Females were nulliparous and non pregnant.
- Weight (Prior to Treatment):Male:Minimum: 238 g and Maximum: 274 g ,Female:Minimum: 234 g and Maximum: 250 g
- Fasting period before study:N/A
- 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 (Nutrivet Life Sciences, Pune) 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 5 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: 20.40 °C and Maximum: 23.10 °C
- Humidity (%):Minimum: Minimum: 38.40% and Maximum: 58.70%
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12

Type of coverage:

semiocclusive

Vehicle:

other: distilled water

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: Test item was held in contact with the skin with a porous gauze dressing. The test site was further covered in a suitable manner to retain the gauze dressing and test item and ensured that the animals cannot ingest the test item.
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
- For solids, paste formed: yes
VEHICLE
- Amount(s) applied (volume or weight with unit):0.2 ml distilled water.

Duration of exposure:

24-hour

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.
- 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:

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

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Non toxic to animals.

Mortality:

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

Clinical signs:

other: No systemic or local signs of toxicity were observed at limit dose of 2000 mg/kg body weight of test item during 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

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

 

Dose:2000 mg/ kg bodyweight                                                                                                         

Animal No.	Sex	Body Weight (gram)			Body Weight Change (%)
		Day 0	Day 7	Day 14	Day 0-7	Day 0-14
1	Male	245	247	229	0.82	-6.53
2		245	264	280	7.76	14.29
3		238	248	257	4.20	7.98
4		274	297	269	8.39	-1.82
5		251	244	272	-2.79	8.37
6	Female	249	249	265	0.00	6.43
7		238	254	262	6.72	10.08
8		246	250	267	1.63	8.54
9		234	227	244	-2.99	4.27
10		250	254	263	1.60	5.20

 

 

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	8	9	10	11	12	13	14
1	Male	1	1	1	1	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	1	1	1	1
3		1	1	1	1	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	1	1	1	1
5		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
6	Female	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
7		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
8		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
9		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
10		1	1	1	1	1	1	1	1	1	1	1	1	1	1	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

Interpretation of results:

other: Not classified

Conclusions:

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

Executive summary:

Acute Dermal Toxicity Study was conducted by using the given test chemical in Wistar Rats performed as per OECD No.402. Five male and five female healthy young adult rats were randomly selected and used for conducting acute dermal toxicity study. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item moistened with 0.2 ml distilled water was applied by single dermal application and observed for 14 days after treatment. On test day 0, an amount of test item moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the surgical gauze patch was put on to the intact skin of clipped area. This gauze patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non-irritating adhesive tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1 14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were 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. No clinical signs and any skin reaction were observed throughout the experimental period in all treated animals. The male and female animals were observed with body weight gain compared to day 0 throughout the experiment. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 1 and from 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 the given test chemical. The studies are summarized as below –

 

Acute Oral Toxicity Study was conducted by using the given test chemical in Rats. This study was performed as per OECD No. 423. Twelve female Wistar rats were selected for acute oral toxicity study. The animals were fasted for minimum 16-18 hours prior to dosing and for 3-4 hours post dosing, with food withheld but drinking water provided ad libitum. The time intervals between dosing were determined by the onset, duration and severity of toxic signs. Three rats of group G1 were dosed with starting dose of 300 mg/kg body weight and the animals did not show any mortality so another three animals of the group G1 were dosed with 300 mg/kg body weight and no mortality was observed. Three animals of the group G2 were dosed with 2000 mg/kg body weight and no mortality was observed, therefore another three animals of same group G2 were dose with 2000 mg/kg 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 was observed with gain was observed in the animals treated with 300 and 2000 mg/kg body weight, during the 14 day observation period, as compared to day 0. At 300 mg/kg, all the six animals (animal nos. 1-6) were observed normal throughout experimental period. At 2000 mg/kg, animal no. 7 was observed normal at 30 minutes and 1 hour post dosing, mild to moderate lethargy at 2, 3, 4 and 6 hours and mild ataxia at 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal no. 8 was observed normal at 30 minutes and 1 hour post dosing, mild to severe lethargy at 2, 3, 4 and 6 hours and mild to moderate ataxia at 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal no. 9 was observed normal at 30 minutes and 1 hour post dosing, moderate to severe lethargy and mild to moderate ataxia at 2, 3, 4 and 6 hours followed by normal clinical sign till day 14. Animal nos. 10-12 were observed normal at 30 minutes and 1 hour post dosing, mild to moderate lethargy at 2, 3 and 4 hours and mild ataxia at 3 and 4 hours followed by normal clinical sign till day 14. 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 of test chemical in female rats is as given below:

The acute oral LD50 value of test chemical was considered to be >2000 mg/kg body weight. Thus by considering the CLP criteria for acute toxicity rating for the chemicals, it infers that test chemical does not exhibit acute oral toxicity i.e. it is acutely non toxic to animals.

 

The above study was supported with another study mentioned in study report for the given test chemical. The acute oral toxicity study was conducted under the OECD Guideline-423 for testing of chemicals in wistar albino rats. The healthy wistar albino rats of body weight 200+20 gm were selected for study after acclimatization to standard laboratory condition and divided into test compound and vehicle control group each having three animals. The study was conducted stepwise as follow: starting dose 2000 mg/kg body weight:

Step-l - The test compound was mixed with distilled water and administered orally at the dose level of 2000 mg/kg body weight (dose volume 10mi/kg) to three female rats. However; vehicle control group treated with the distilled water at the dose level of 10 ml/kg b.wt. The treated animals were closely observed for clinical signs of intoxication during first four hours of test compound administration. Thereafter, all the animals were observed periodically at one hour interval for 24 hrs and twice daily for a period of 14 days. The necropsy was performed on all animals at the termination of the study. The test compound did not elicit any clinical signs of intoxication throughout the period of observation at the tested dose level 2000 mg/kg body weight. Furthermore, no mortality was recorded at test dose level. Vehicle control group of animals were also free from any mortality and Clinical signs. The necropsy finding did not reveal any gross pathological changes at the tested dose level. Furthermore, no gross pathological change was observed in vehicle control group.

Step -ll: After 72 hrs, the result of step-I was confirmed by administration of same dose level (2000 mg/kg. b.wt) of test compound in additional three animals of same sex (OECD- 423 guidelines) under same test condition. The test compound administered at the dose level of 2000 mg/kg b.wt did not produce any mortality and clinical sign of intoxication throughout the observation period of 14 days. Body weight of each rat was also recorded on day 7" and 14" showed normal gain as compared to control group. Necropsy was conducted at the end of the study (15 day) on all the animals which did not reveal any gross pathological changes.

Based on the results obtained from present investigation, it can be concluded that the test compound is non toxic to Wistar albino rats at the testing dose level of 2000 mg/kg body weight.

 

Thus, based on the above summarised studies on test chemical, it can be concluded that 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 study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to low vapour pressure of the test chemical, which is reported to be 0.00000256 Pa. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

Acute Dermal Toxicity:

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

Acute Dermal Toxicity Study was conducted by using the given test chemical in Wistar Rats performed as per OECD No.402. Five male and five female healthy young adult rats were randomly selected and used for conducting acute dermal toxicity study. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item moistened with 0.2 ml distilled water was applied by single dermal application and observed for 14 days after treatment. On test day 0, an amount of test item moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the surgical gauze patch was put on to the intact skin of clipped area. This gauze patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non-irritating adhesive tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1 14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were 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. No clinical signs and any skin reaction were observed throughout the experimental period in all treated animals. The male and female animals were observed with body weight gain compared to day 0 throughout the experiment. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality.

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

 

The above study was supported with another study mentioned in study report and conducted to determine the acute dermal toxicity dose of the given test chemical on Wistar albino rats. This study was conducted according to OECD guideline 402 for testing of chemicals. The summary of the study was as follows -

LIMIT TEST (2000 mg/kg body weight): Ten healthy wistar albino rats of both sexes (ranging b.wt 200±30 gm) were 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 compound 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 during first four hours of test compound administration. Thereafter, all the animals were observed periodically at one hour interval for 24 hrs and twice daily for a period of 14 days. The body weight of each rat was observed on day 0 (pre treatment), 7 and 14 (post treatment). The necropsy was performed on all animals at 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 mortality as well as clinical signs of toxicity throughout the observation period of 14 days. Necropsy finding did not reveal any gross pathological changes under test condition.

CONFIRMATORY TEST: After 72 hrs, a confirmatory test was conducted in same species of animals to confirm the results obtained from limit test (OQECD-402 guidelines). Ten healthy Wistar albino rats of both sexes (ranging b.wt 200+30 gm) were 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 compound 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 during first four hours of test compound administration. Thereafter, all the animals were observed periodically at one hour interval for 24 hrs and twice daily for a period of 14 days. The body weight of each rat was observed on day 0 (pre treatment), 7 and 14 (post treatment). The necropsy was performed on all animals at the termination of the study. No mortality was recorded in Wistar albino rats after administration of test compound at the dose level of 2000 mg/kg b.wt throughout the period of observation (14 days). The test compound did not elicit any clinical signs of toxicity during the observation period. No skin reaction was observed after 24 hrs of patch removal. The body weight of each animal recorded on day 7 and 14 showed normal increase as compared to day 0 (pre treatment). Necropsy was conducted at the end of the observation on all the animals (day 15) did not reveal any significant gross pathological changes related to compound toxicity.

Result obtained from present investigation can be concluded that the test compound is acutely non toxic at the tested dose level of 2000 mg/kg b.wt in Wistar albino rats when applied by dermal 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 dermal toxicity.

 

Justification for classification or non-classification

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