Registration Dossier

Administrative data

Description of key information

Acute Toxicity:Oral

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

Acute toxicity:Inhalation:

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account the low vapour  pressure of the test substance, which is reported as 0.00234 mm Hg. Thus, exposure to inhalable dust,  mist and vapour of the chemical is highly unlikely.  The particle size distribution of the test substance was found to vary in the size of 106-150 µm, so the potential for the generation of inhalable forms is low.Therefore this study is considered for waiver.

Acute toxicity:Dermal

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

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Experimental result performed using standard test methods
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Principles of method if other than guideline:
To assess the acute oral toxicity potency of test chemical when exported to rats via oral route
GLP compliance:
no
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Institute for Industrial Research & Toxicology
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 200±20g
- Fasting period before study: Fasted overnight prior to treatment. Food was offered three hours after dosing
- Housing: Groups of three 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): Aqua Guard filter water was kept in PVC bottles, ad libitum
- Acclimation period: One week in experimental room after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25° C
- Humidity (%): 40-60%
- Air changes (per hr): 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:
oral: drinking water
Vehicle:
water
Details on oral exposure:
Dose preparation of the test article was done freshly, few minutes prior to dosing. Test substance was dissolved in 0.1% Tween 80 in distilled water to obtain final concentration of 200 mg/ml.
Doses:
Group I: Dist. water, 10ml/kg body wt.
Group II: 2000 mg/kg body wt.
Group III: 2000 mg/kg body wt
The test compound was administered by oral route by using of oral cannula at the dose volume of 10 ml/kg b.wt.
No. of animals per sex per dose:
Three + one vehicle control
Control animals:
yes
Details on study design:
Body weight:
The body weight of all the animals was observed weekly on day 0 (pre treatment), 7th and 14th (post treatment).
Mortality:
The test compound was administered at different dose level in wistar albino rats observed for mortality at the time interval of 30 minutes, 1hr, 2hr, 4 hr, and 6hr time interval on the day of test compound administration and thereafter twice a day for 14 days.
Clinical Signs
The treated animals were closely observed for clinical signs of intoxication, first 4 hours and every 1 hrs interval for 24 hrs after dosing and thereafter twice a day for 14 days. All the rats were observed at least twice daily to observe any clinical signs or behavioral changes. These observations included changes in skin and fur, in the eyes and mucous membranes, respiratory, circulatory, central nervous and autonomic nervous systems, somatomotor activity and behavioral changes. The following clinical signs were observed in female mice to characterize the various systemic studies: Salivation, lacrimation, pale mucous membrane, diarrheal feaces, hunched posture, scratching, polyuria, hypoactivity etc. The clinical sign will be graded as 0 = Normal, + = Mild, ++= Moderate, +++ =High and ++++ = Severe.
Necropsy:
Necropsy was carried out on all the animals which died during the study or surviving animals were sacrificed at the end of the study to observe any gross pathological changes.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no other details available
Mortality:
The test compound administered at the dose level of 2000 mg/kg b.wt. did not show any mortality throughout the observation period
Clinical signs:
Test compound did not produce any clinical signs of intoxication in wistar albino rats throughout the observation period of 14 days at the dose level of 2000 mg/kg b.wt.
Body weight:
All the animals treated with the test compound CAS No. 25013-16-5 at the dose level of 2000 mg/kg b.wt on day 7th and 14th showed normal gain in body weight as compared to control group
Gross pathology:
NECROPSY FINDING
ABDOMINAL CAVITY
i. Opening and general examination- In the abdominal cavity all the organs were present in normal position.
ii. Spleen- Normal at 2000 mg/kg b.wt.
iii. Digestive system- No gross changes were observed.
iv. Liver and biliary ducts- No gross pathological changes were observed
v. Excretory system- No gross pathological changes were observed at the dose level of 2000 mg/kg b.wt.
vi. Adrenal- Observed normal.
vii. Male/female genital organs – Showed normal colour, consistency and no inflammatory changes.
2. THORACIC CAVITY
i. Opening and general examination- Thoracic cavity was found to be normal without any fluid, mucous or blood etc.
ii. Lungs- observed normal.
iii. Heart- No changes were observed in color and consistency. Heart found normal upto highest tested dose level 2000 mg/kg b.wt.
iv. Thyroid- Normal in shape, size and surface.
3. CRANIAL CAVITY
i. Brain- Normal in size.
Other findings:
NECROPSY FINDING
EXTERNAL
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 nodes- No change in mesenteric lymph node.
Interpretation of results:
not classified
Conclusions:
Under the condition of the study, the acute oral LD50 value of test chemical was considered to be >2000 mg/kg body weight. Thus, it was concluded that the acute toxicity study of test chemical when administered via oral route in Wistar rats falls into the “Category Not classified” criteria of CLP.
Executive summary:

The reported study was designed and conducted to determine the acute oral toxicity profile test chemical in wistar rats. The study was conducted in accordance with OECD guideline 423 for testing of chemicals. Initially, three female animals were treated at the dose level of 2000 mg/kg body weight of the test item (Step - I). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality throughout the observation period.No clinical sings and mortality were observed in vechicle group.The necropsy was conducted on all the animals at the termination of the study did not reveal any gross pathological changes .After 72hours ,the result of step I was confirmed by administration of same dose level of the test chemical in additional three animals of same sex.

It is concluded that test chemical following the guideline OECD-423 is non-toxic to Wistar albino rats at the dose level of 2000 mg/kg bw.According to Globally Harmonized system of classification,the chemical is considered as “Not classified”

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The data is from study report of K2 level

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
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:
Experimental result performed usinf standard test methods
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
-Species:Rat (Rattus norvegicus)
-Strain:Wistar
-Sex:Male and Female
-Number of Animals:10 (Five per sex)
-Health Status :Healthy young adult animals were used for the study. Females were nulliparous and non pregnant
-Body weight of animals:Male:Minimum: 218 g and Maximum: 230 g
(Prior to Treatment)Female: Minimum: 200 g and Maximum: 223 g
-Diet:All animals were provided conventional laboratory rodent diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No 400004.
-Bedding:All cages were provided with corn cobs (Sparconn Life Sciences Bangalore) Batch No.: SPAR – 25/2014
-Water:Aqua guard filtered tap water was provided ad libitum via drinking bottles.
-Husbandry-:The animals were housed individually in polycarbonate cages.
-Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
-Cages and water bottle:All the cages and water bottles were changed at least twice every week
-Acclimatisation:All animals were acclimatized to the test conditions for 6 days prior to administration of the test item.
-Identification :During Acclimatization, animals were temporarily marked by permanent marker, on their tails. After acclimatization, the animals were marked by toe pad micro tattooing and cage cards. Individual cage cards were labeled with study no., study type, test system, group, dose, sex, animal number, experimental start and completion date.
-Randomization:Animals were selected manually. No computer generated randomization program was used.



ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.90 °C Maximum: 23.10 °C
- Humidity (%):Minimum: 53.30% Maximum: 66.50%
- 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: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
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied (volume or weight with unit):0.2 ml distilled water
- Concentration (if solution):N/A
- Lot/batch no. (if required):N/A
- Purity:N/A
Duration of exposure:
24 hrs
Doses:
2000 mg/kg body weight.
No. of animals per sex per dose:
5: male
5: female
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.
Key result
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:
All the animals were observed with normal clinical signs throughout the experimental period
Body weight:
Mean body weight of male and female animals was observed with increase on day 7 and 14, as compared to day 0
Gross pathology:
The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality

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

227

252

268

11.01

18.06

2

218

234

254

7.34

16.51

3

230

258

283

12.17

23.04

4

227

252

280

11.01

23.35

5

226

242

262

7.08

15.93

6

Female

208

211

223

1.44

7.21

7

200

203

196

1.50

-2.00

8

201

211

218

4.98

8.46

9

203

211

215

3.94

5.91

10

223

233

248

4.48

11.21

                                                                                                     

Keys:* = Based on day 0 body weight taken prior to dose application.


Table 2: Individual Animal Clinical Signs and Symptoms

 

Dose:2000 mg/kg body weight

 

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

146

146

146

146

146

146

146

7

146

146

146

146

146

146

146

8

65+

146

146

146

146

146

146

146

9

146

146

146

146

146

146

146

10

1

1

1

1

1

1

1

Keys: 1 = Normal, 65 = Erythema, 146 = Scab, 147 = Scale, 172 = Vocalization, + =Mild (slight),                  ++ = Moderate, +++ = Severe


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

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



Executive summary:

Acute Dermal Toxicity Study of test chemical inWistar Rats was 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. Ratsfree 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, anamount oftestitem moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the porous gauze dressing was put on to the intact skin of clipped area.This porous gauze dressing was covered with a non-irritating tape.After the 24-hour application period, the dressings were removed and theskin was gently wiped with distilled water.The skin reactions were assessed.

The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically.

No mortality was observed in any animal till the end of the experimental period.

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

Mean body weight of male and female animals was observed with increase on day 7 and 14, as compared to day 0.

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

The acute dermal median lethal dose of test chemical was >2000 mg/kg body weight.Thus by considering the CLP criteria set by EU 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 from k1 study report

Additional information

Acute toxicity (Oral) :

In different experimental studies, 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 and rabbits for test chemical. The studies are summarized as below:

The reported study was designed and conducted to determine the acute oral toxicity profile test chemical in wistar rats. The study was conducted in accordance with OECD guideline 423 for testing of chemicals. Initially, three female animals were treated at the dose level of 2000 mg/kg body weight of the test item (Step - I). Administration of the test item at 2000 mg/kg did not result in any signs of toxicity and mortality throughout the observation period.No clinical sings and mortality were observed in vechicle group.The necropsy was conducted on all the animals at the termination of the study did not reveal any gross pathological changes .After 72hours ,the result of step I was confirmed by administration of same dose level of the test chemical in additional three animals of same sex.

It is concluded that test chemical following the guideline OECD-423 is non-toxic to Wistar albino rats at the dose level of 2000 mg/kg bw.According to Globally Harmonized system of classification,the chemical is considered as “Not classified”

The study is further supported by the result ,the acute oral median lethal dose (LD50) of test chemical in rat and rabbits was determined to be 2200 mg/kg of body weight. Acute oral toxicity of test chemical to rat by oral route indicates that the chemical is relatively non toxic.

From the above experimental studies, it is concluded that the LD50 value is >2000 mg/kg bw, with detailed and reliable data. Also, the test animal “rats” are most preferred in animal toxicity studies. Thus, comparing this value with the criteria of CLP regulation, test chemical cannot be classified for acute oral toxicity.

Acute toxicity: inhalation

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account the low vapour  pressure of the test substance, which is reported as 0.00234 mm Hg. Thus, exposure to inhalable dust,  mist and vapour of the chemical is highly unlikely.  The particle size distribution of the test substance was found to vary in the size of 106-150 µm, so the potential for the generation of inhalable forms is low.Therefore this study is considered for waiver.

Acute toxicity: dermal

In different experimental studies, 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 rabbits for test chemical. The studies are summarized as below:

Acute Dermal Toxicity Study of test chemical inWistar Rats was 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. Ratsfree 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, anamount oftestitem moistened with 0.2 ml distilled water was applied directly on the intact skin of clipped area of rats; the porous gauze dressing was put on to the intact skin of clipped area.This porous gauze dressing was covered with a non-irritating tape.After the 24-hour application period, the dressings were removed and theskin was gently wiped with distilled water.The skin reactions were assessed.

The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically.No mortality was observed in any animal till the end of the experimental period.All the animals were observed with normal clinical signs throughout the experimental period.Mean body weight of male and female animals was observed with increase on day 7 and 14, as compared to day 0.The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality.The acute dermal median lethal dose of test chemical was >2000 mg/kg body weight.Thus by considering the CLP criteria set by EU 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.

In a supporting study ,Acute dermal testing on rabbits of an eye make-up containing 0.1% of test chemical yielded a LD50 value of >2000 mg/kg.Based on the LD50 value of >2000 mg/kg for rabbit avaialble for test chemical,it can be concluded that test chemical is not toxic via dermal route.

From the above experimental studies, it is concluded that the LD50 value is >2000 mg/kg bw, with detailed and reliable data. Also, the test animal “rabbits” are most preferred in animal toxicity studies. Thus, comparing this value with the criteria of CLP regulation, test chemical cannot be classified for acute dermal toxicity.

Justification for classification or non-classification

According to new CLP regulation classification creiteria and based on the values of acute toxicity via oral, inhalative and dermal route it is concluded that the substance tert-butyl-4-methoxyphenol is not classified as toxic via oral, inhalation or dermal route.