Registration Dossier

Administrative data

Description of key information

Skin irritation

The dermal irritation potential of test chemical was assessedin various experimental studies which were conducted in rabbits.Based on the available key data and supporting studies,it can be concluded that the testchemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye irritation

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies for test chemical.Based on the available key data and supporting studies,it can be concluded thatchemical is able to cause eye irritation and considered as irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2 (irritating to eyes)”.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
The objective of the study was to assess the irritant and/or corrosive effects of test chemical after topical application on the intact skin in rabbits.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Sodium salicylate
- Name of test material (as cited in study report): Sodium salicylate
- Molecular Formula: C7H6O3.Na
- Molecular Weight: 160.105 g/mol
- Smiles Notation: c1(c(cccc1)O)C(=O)[O-].[Na+]
- InChI: 1S/C7H6O3.Na/c8-6-4-2-1-3-5(6)7(9)10;/h1-4,8H,(H,9,10);/q;+1/p-1
- Substance Type: Organic
- Physical State: Solid
- AI Content (assay): 99.8%
- Manufactured data: March, 2013
- Expiry Date : February, 2015
- Storage conditions: Room temperature (20 - 30 °C)
Handling and Disposal
Safety precautions : Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.
Disposal : The remaining unused test item was disposed as per internal SOPs
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: Male
- Source: LIVEON BIOLABS PVT LTD
- Age at study initiation: 3 to 4 Months (Approximately)
- Weight at study initiation: Minimum: Minimum: 1.764 kg & Maximum: 2.384 kg (Prior to Treatment)
- Housing: The animals were housed individually in stainless steel 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 minimum twice a week.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rabbit diet ad libitum
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum
- Acclimation period: Rabbits were acclimatised to the test conditions for a period of 6 days (Animal No.-1) and 8 days (Animal No.-2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 20.30 °C Maximum: 22.70 °C
- Humidity (%):Minimum: 48.30 % Maximum: 68.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12 hours
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g moistened with 0.5 ml distilled water


VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 ml
- Concentration (if solution):no data
- Lot/batch no. (if required): no data
- Purity: no data
Duration of treatment / exposure:
4-hour exposure period
Observation period:
72 hours
Number of animals:
Three male rabbits
Details on study design:
TEST SITE
- Area of exposure: dorsal lumbar region at contralateral sites
- % coverage: approximately 6 X 6 cm
- Type of wrap if used: porous gauze dressing and non-irritating tape (Micropore 3”)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was removed by using cotton soaked in distilled water
- Time after start of exposure: 4 hrs

SCORING SYSTEM: Grading of irritation lesions was carried out as per Draize Method
Irritation parameter:
erythema score
Basis:
mean
Remarks:
Animal 1,2,3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Remarks:
Animal 1,2 and 3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Animal No. 1, showed very slight erythema (barely perceptible) and no oedema at 1 hour of observation. At 24, 48 and 72 hour observation no erythema and oedema was observed in animal No 1.
In Animals No. 2 and 3 at 1 hour observation post patch removal, revealed very Slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed no erythema and no oedema..
The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively

Other effects:
Clinical Observation
No systemic toxicity was observed at treated rabbits during the experimental period.


Mortality
No mortality was observed during the observation period


Body Weights
Body weights were increased as compared to day 0 in all the three animals



Table 1: Skin Reaction

 

In Treated area 

Dose:500 mg of test item (moistened with 0.5 ml distilled water)            

Sex:Male 

Animal

No.

Test

Treated

 area*

Erythema score

Oedema score

1h

24h

48h

72h

1h

24h

48h

72h

1

Initial

Left

1

0

0

0

0

0

0

0

2

Confirmatory

Right

1

0

0

0

0

0

0

0

3

Right

1

0

0

0

0

0

0

0

 

In Control area

Dose:0.5 ml of distilled water                                                              

Sex:Male 

Animal

No.

Test

Treated area*

Erythema score

Oedema score

1h

24h

48h

72h

1h

24h

48h

72h

1

Initial

Right

0

0

0

0

0

0

0

0

2

Confirmatory

Left

0

0

0

0

0

0

0

0

3

Left

0

0

0

0

0

0

0

0

Key: h = Hour.

Erythema                                                                                                       Oedema

0 =No erythema                                                                                           0 =No oedema

1 = Very slight erythema(barely perceptible)

Mean Individual Animal Score at 24, 48 and 72 hours

 

                     Animal Number                  

Observations                      

1

2

3

Erythema

0.00

0.00

0.00

Oedema

0.00

0.00

0.00

 

Table 2: Individual Animal BodyWeight

Sex:Male

Animal

No.

Body Weight (kg)

Prior to Dosing

At termination

1

2.314

2.360

2

2.384

2.420

3

1.764

1.836

Individual Animal Clinical Signs

Sex:Male

Animal

No.

Days (Post dosing Observation)

0

1

2

3

1

1

1

1

1

2

1

1

1

1

3

1

1

1

1

Key: ./. = Not Applicable. 1 = Normal.

Interpretation of results:
other: not irritating
Conclusions:
No erythema and oedema (skin irritation) were found at the end of 72 hour observation period after patch removal.The individual mean score at 24, 48 and 72 hoursfor Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively
Hence, it was concluded that Sodium salicylate was Non-Irritating to the skin of Male New Zealand White rabbits under the experimental conditions tested
Executive summary:

Acute Dermal Irritation/corrosion Study of Sodium Salicylate (CAS: 54 -21 -7) in Rabbits, was performed in an OECD GLP laboratory (Sustainability Support Services (Europe) AB has the letter of access,2014) as per OECD guideline No. 404. Three healthy young adult male rabbits were used for conducting acute dermal irritation/corrosion study.

The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 g of test item (moistened with 0.5 ml distilled water) was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis. Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure in animal no. 1, very slight erythema (barely perceptible) and no oedema observed at 1 hour of observation. At 24, 48 and 72 hours observation no erythema and oedema was observed in animal no 1. No severe skin lesions were observed hence a confirmatory test was conducted on additional two rabbits (No. 2 and 3) after 24 hours to confirm the non irritant nature of the test item. The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours post patch removal, evaluated and graded as per Draize method. In animal no. 2 and 3 at 1 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed no erythema and no oedema. The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively

Hence, it was concluded that Sodium salicylate was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions tested and classified as "Not Classified" as per CLP criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
The objective of the study was to assess the irritant and/or corrosive effects of “Sodium Salicylate (CAS: 54-21-7)” on eye, when exposed by the ocular route in rabbits.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Sodium salicylate
- Molecular Formula: C7H6O3.Na
- Molecular Weight: 160.105 g/mol
- Smiles Notation: c1(c(cccc1)O)C(=O)[O-].[Na+]
- InChI: 1S/C7H6O3.Na/c8-6-4-2-1-3-5(6)7(9)10;/h1-4,8H,(H,9,10);/q;+1/p-1
- Substance Type: Organic
- Physical State: Solid
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LIVEON BIOLABS PVT. LTD
- Age at study initiation: 3 to 4 Months (Approximately)
- Weight at study initiation: Minimum: 2.120 kg and Maximum: 2.448 kg (Prior to Treatment)
- Housing: The animals were housed individually in stainless steel 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 minimum twice a week
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rabbit diet
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum
- Acclimation period: Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 9 days (Animal No. 2 and 3) prior to the application of the test item

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 20.30 °C Maximum: 22.70 °C
- Humidity (%): Minimum: 48.30 % Maximum: 68.30 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg



Duration of treatment / exposure:
24 hours
Observation period (in vivo):
All the animals were observed at 1, 24, 48 and 72 hours and on day 7 after instillation of test item.
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
Three female rabbits
Details on study design:
Details on study design
REMOVAL OF TEST SUBSTANCE
- Washing (if done):The treated eye of rabbit was washed with normal saline.
- Time after start of exposure:24 hours
SCORING SYSTEM:Grading of irritation lesions was carried out as per Draize Method
TOOL USED TO ASSESS SCORE: Ophthalmoscope and fluorescent strips.REMOVAL OF TEST SUBSTANCE
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Animal 1,2 and 3
Time point:
24/48/72 h
Score:
> 0.67 - <= 1
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild irritation
Irritation parameter:
iris score
Basis:
mean
Remarks:
Animal 1,2 and3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
not specified
Remarks on result:
probability of mild irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 1,2 and 3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild irritation
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
Animal 1, 2 and 3
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days observation period
Remarks on result:
probability of mild irritation
Irritant / corrosive response data:
The following were observed in treated rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals.
Observation at 24 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in animal no. 1 whereas animal no. 2 and 3 showed no ulceration or opacity; Area of Opacity- One quarter (or less) but not zero in animal no. 1 and Zero in animal no. 2 and 3; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 20%, 20% damage in animal no. 1, 2 and 3 respectively.
Observation at 48 hour after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all animals; Area of Opacity- One quarter (or less) but not zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
Observation at 72 hour after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all animals; Area of Opacity- One quarter (or less) but not zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Diffuse, crimson color; individual vessels not easily discernible was observed in all animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all animals.
Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all 3 animals; Chemosis: No swelling (Normal) was observed in all 3 animals.
The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively
Other effects:
Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period

Mortality
No mortality was observed during the observation period


Body weight
All rabbits were weighed on test day 0 (prior to application) and at termination



Table 1 : Individual Animal Eye Irritation Scores

 

In Treated area Dose:100 mg (0.1gm) of test item                                              

 Sex:Female

Animal Numbers

1

2

3

Application Side

Left

Left

Left

Eye Reactions

At hour

At hour

At hour

*

1

24

48

72

Day 7

*

1

 24

48

72

Day 7

*

1

24

48

72

Day 7

Corneal Opacity

0

0

1

1

1

0

0

0

0

1

1

0

0

0

0

1

1

0

Area of Opacity

0

0

1

1

1

0

0

0

0

1

1

0

0

0

0

1

1

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctiva

0

1

2

2

2

0

0

1

2

2

2

0

0

1

2

2

2

0

Chemosis

0

1

1

1

1

0

0

1

1

1

1

0

0

1

1

1

1

0

Corneal Damage%

40

20

20

 

Dose:Untreated (Control Eye)                                                                       Sex:Female

 

Animal Numbers

1

2

3

Application Side

Left

Left

Left

Eye Reactions

At hour

At hour

At hour

*

1

24

48

72

Day 7

*

1

 24

48

72

Day 7

*

1

24

48

72

Day 7

Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Area of Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctiva

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Corneal Damage%

0

0

0

 

Key:*= Pre-exposure eye examination.

 

 

Eye Irritation Scores - Mean Values after 24, 48, 72 Hours (Treated eye)

            Animal No.

 Eye Reaction

1

2

3

Cornea

1.00

0.67

0.67

Iris

0.00

0.00

0.00

Conjunctiva

2.00

2.00

2.00

Chemosis

1.00

1.00

1.00

 

 Formula :

 Mean Irritation Score= Sum of the Individual Animal Score for eye reactions at 24, 48 and 72 hours

Number of the Observations (3)

Table 2 : Individual Animal Clinical Signs

 

Sex:Female

Animal No.

Days (Post application observation)

0

1

2

3

4

5

6

7

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

Key:1 = Norma

Individual Animal Body Weight

Sex :Female

Animal No.

Animal Body Weight (kg)

Prior to application

At termination

1

2.448

2.612

2

2.120

2.156

3

2.236

2.402

Key:kg = Kilogram

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7.
Hence, under the experimental test conditions, “Sodium salicylate (CAS No. – 54-21-7) is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes and is being classified "Category 2"as per the CLP
regulation.
Executive summary:

Acute Eye Irritation/Corrosion Study of Sodium Salicylate (CAS: 54 -21 -7) in Rabbits, was performed in a OECD GLP laboratory (Sustainability Support Services (Europe) has letter of access).The study was performed according to OECD 405 Guidelines. 3 female young adult rabbits were used for the study.

Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mgof test itemwas instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour and on day 7 after test item instillation.Ophthalmoscope was used for scoring of eye lesions.

In the initial test,100 mg of test item (pulverized form)was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3);100 mgof test itemwas instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 at 1, 24, 48 and 72 hour observation which recovered at day 7.

 

Untreated eye of all the three rabbits was normal throughout the experiment.

 

The following grading scores were observed in treated eye of treated rabbits.

Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was observed in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all the animals.

Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in animal no. 1 whereas animal no. 2 and 3 showed no ulceration or opacity; Area of Opacity-One quarter (or less) but not zero in animal no. 1 and Zero in animal no. 2 and 3;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 20%, 20% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hour after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

Observation at 72 hour after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all animals; Area of Opacity-One quarter (or less) but not zero in all 3 animals;Iris:Normal in all the animals.Conjunctivae –Diffuse, crimson color; individual vessels not easily discernible was observed in all animals;Chemosis:Some swelling above normal (includes nictitating membranes) was observed in all animals.

Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all 3 animals; Chemosis: No swelling (Normal) was observed in all 3 animals.

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7.  

Hence, under the experimental test conditions, “Sodium salicylate (CAS No. – 54-21-7) is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes and is being classified "Category 2"as per the CLP regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Various studies has been investigated for the test chemical to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for target chemical which have been summarized as follows;

 

Acute Dermal Irritation/corrosion Study of Sodium Salicylate (CAS: 54 -21 -7) was performed as per OECD guideline No. 404 using three healthy young adult male. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 g of test item (moistened with 0.5 ml distilled water) was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis. Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure in animal no. 1, very slight erythema (barely perceptible) and no oedema observed at 1 hour of observation. At 24, 48 and 72 hours observation no erythema and oedema was observed in animal no 1. No severe skin lesions were observed hence a confirmatory test was conducted on additional two rabbits (No. 2 and 3) after 24 hours to confirm the non-irritant nature of the test item. The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours post patch removal, evaluated and graded as per Draize method. In animal no. 2 and 3 at 1 hour observation post patch removal, revealed very slight erythema (barely perceptible) and no oedema. At 24, 48 and 72 hour post patch removal, animals showed no erythema and no oedema. The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively Hence, it was concluded that Sodium salicylate was Non-Irritating to the skin of male New Zealand White rabbits under the experimental conditions tested.

 

The above result was supported by another dermal irritation study conducted on New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of Sodium Salicylate. Three female New Zealand White rabbits were used for the study. The animals were prepared 24 hours prior to application of test product. The furs from the dorsal area of trunk of animals were removed with electric clippers exposing an area measuring approximately 6 cm2 of body surface area of animal. The care was taken such that abrasion penetrated the Stratum corneum only and not dermis. 0.5 gm of the test compound was applied on a small area 6 square cms of the intact test site. Each site of application was covered with an impervious dressing which was secured with an adhesive tape. The animals were housed individually and restrained by the use of plastic collars. After patch removal [4 hours later] the unabsorbed test chemical was removed and the test site was washed with lukewarm water. The intact skin site of application was observed for erythema and edema at 1, 24,48 and 72 hours till 14 days after application and scored according to Draize method. Sodium Salicylate produced slight redness to skin at 4 hours of observation. Furthermore, no clinical signs of skin irritation were observed at 24 hours of observation. Sodium Salicylate did not produce any clinical signs of toxicity throughout the examination period of 14 days. The dermal irritation index was calculated as 0.00 and test compound can be considered as not irritating to the skin of treated rabbit.

Based on the available data for key and supporting study, it can be concluded that test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye irritation:

In different studies, the test chemical has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo and in-vitro experiments in rabbits for target chemical that have been summarized as below;

 

Acute Eye Irritation/Corrosion Study of Sodium Salicylate (CAS: 54 -21 -7) was performed according to OECD 405 Guidelines using three female young adult rabbits. The Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mg of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour and on day 7 after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test item (pulverized form)was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3);100 mg of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 and 3 at 1, 24, 48 and 72 hour observation which recovered at day 7. Untreated eye of all the three rabbits was normal throughout the experiment. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.00; 0.67, 0.00, 2.00, 1.00 and 0.67, 0.00, 2.00, 1.00 respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbits was observed within day 7.  Hence, under the experimental test conditions, “Sodium salicylate (CAS No. – 54-21-7) is “Mildly Irritating to eyes” of New Zealand White female rabbit eyes. 

The Vitrigel-EIT method used to assess the skin sensitization study of test chemical. It is composed of two parts, i.e., the construction of a human corneal epithelium (HCE) model in a collagen vitrigel membrane chamber and the prediction of eye irritancy by analyzing the time-dependent profile of transepithelial electrical resistance values for 3 min after exposing a chemical to the HCE model. A SV40-immortalized HCE cell strain (HCE-T cells, RCB no. 2280) was obtained from the RIKEN BioResource Center (Tsukuba, Japan). The cells were maintained in the following culture medium: 1: 1 mixture of Dulbecco’s modified eagle medium and nutrient mixture F-12 supplemented with 5% heat-inactivated fetal bovine serum, 5 μgml–1 recombinant human insulin, 10 ngml–1 recombinant human epidermal growth factor, 0.5% dimethyl sulfoxide 100 units/ ml– penicillin and 100 μg/ml streptomycin. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air. The test chemical was selected according to the globally harmonized system of classification and labeling (GHS) classification for eye irritation (United Nations, 2013). Every test chemical solution was prepared in a culture medium at a concentration of 2.5 (weight/volume) % appropriate for measuring TEER values without being influenced by the test chemical-dependent electrical resistance. Here, the chemicals were dissolved in the medium by using an appropriate technique(s) as follows: vortex mixing within 1 min, sonication within 20 min and/or heating in a water bath <70 °C. In case test chemical was insoluble or immiscible by the above technique(s), the test chemical solution was prepared as a homogeneous suspension that the chemical was mixed well in the medium by vortex within 1 min immediately before use. The pH level of each 2.5 w/v % test chemical solution was measured using Universal pH test paper from ADVANTEC (Tokyo, Japan). The HCE models on day 6 were subjected to the exposure experiment of test chemicals. At first, 500 μl of culture medium was poured in the chamber and the value of the R sample, before chemical exposures, was measured to obtain the initial TEER value of each model. Next, the medium inside the chamber was changed to 500 μl of test chemical solution and the periodical values of R sample were measured by the TEER recorder at intervals of 10 s for 3 min after exposure of each test solution. Three independent models were subjected to the exposure experiment for each test solution to plot the average time-dependent profile of TEER values on a chart. The chemical exposure experiment was conducted in the ambient temperature of 28 ± 2 °C. The sensitivity, specificity and accuracy of the Vitrigel-eye irritancy test method were 82.3%, 89.5% and 83.7%, respectively. The score plateau level for Sodium Salicylate in the Vitrigel-eye irritancy test method was 80. According to classification criteria of Vitrigel-eye irritancy test method, Sodium Salicylate can be regarded as an Eye irritant.

 

The Draize test was conducted according to the OECD guidelines for Sodium salicylate. In the first validation, 100 ml aqueous solution or a suspension of Sodium salicylate(10%) was applied to the right eyes of male New Zealand white rabbits (2.30±2.98 kg, 13 wk of age). Left eyes remained untreated as a control. Eyes were observed at 1 hr, 4 hr, and every 24 hr thereafter for 7 days. Three rabbits were used for test. MAS [Maximum average scores] and scores at 24 hr after application calculated for cornea, iris, conjunctivae and the sum of these scores (total average score) were used for comparison with in vitro data. Isotonic sodium chloride solution was also used as a negative control. According to the classification by Kay and Calandra (1962), chemicals can be classified as follows based on their MAS scores. The Maximum Average Score[MAS] obtained for 100% Sodium salicylate instilled in rabbits eyes was 83.7; whereas for 10% solution the MAS score was 0.0 According to the classification based on the MAS scores Sodium Salicylate can be considered to be an eye irritant at higher concentrations.

 

Another eye irritation study in rabbits was conducted to determine the degree of eye damage and histopathological changes when exposed to sodium salicylate for shorter durations. 3 New Zealand White rabbits of both sexes were used for the study. Sodium Salicylate was dissolved in Physiological saline. 100mg/ml of Sodium Salicylate was instilled in the right conjunctival sac of the rabbits. The lower lid was pulled away from the eye ball and the chemical solution was added drop wise into the conjunctival sac. The eyelids were held together for a few seconds so as to ensure contact with the ocular tissue. The left eye was the control (for scoring and histopathology).After 24 hours, the eyes were thoroughly washed with physiological saline. The animals were then kept for further observation for 72 hours and scored periodically (0, 24, 48, and 72 h). At the end of 72 hours blood was collected from the animal for hematological and biochemical parameters and then sacrificed. The mean scores at the end of 72 hours of exposure to sodium salicylate for corneal opacity, iris, conjunctival redness, chemosis and discharge were 1,2,2,3,2,2 respectively. Sodium salicylate showed highest degree of irritation when compared to values of control animals. Histopathologically, Animals treated with Sodium Salicylate showed corneal morphology similar to control. However, chronic inflammation was visible in the ciliary body and mild inflammation was evident in the choroid and retina of Sodium Salicylate treated animals. Hence, based on the scores and histopathological observations, Sodium Salicylate was regarded as a severe to highly irritating chemical to rabbit eyes.

 

The next ocular irritation study to determine the degree of eye damage and histopathological changes when exposed to sodium salicylate for longer durations. 3 New Zealand White rabbits of both sexes were used for the study. Sodium Salicylate was dissolved in Physiological saline. 15mg/ml/day of Sodium Salicylate was instilled in the right conjunctival sac of the rabbits. The lower lid was pulled away from the eye ball and the chemical solution was added drop wise into the conjunctival sac. The eyelids were held together for a few seconds so as to ensure contact with the ocular tissue.The left eye was the control (for scoring and histopathology).The test chemical was dosed daily for 7 days.At the end of the 7th day, the animals were kept for observation for 72 hr. A score from 0-3 (3-being the most severe) was given to eyes of each animal as per Draize test. Then they were sacrificed by administering high dose of thiopentone through the marginal ear vein and eyes, spleen, liver and brain were collected for different experiments. The mean scores at the end of 7 days of repeated exposure to sodium salicylate for corneal opacity, iris, conjunctival redness, chemosis and discharge were 1, 3, 2, 3, 2, 2 respectively. It can be seen that prolonged treatment caused swelling in the cornea. Redness, chemosis and discharge from the conjunctiva was evident. Sodium salicylate showed highest degree of irritation when compared to values of control animals. Histopathologically, Animals treated with Sodium Salicylate the cornea had increased in size in comparison to control, due to oedema. Mild acute inflammation of the ciliary body was also noticed. Hence, based on the scores and histopathological observations, Sodium Salicylate was regarded as a severe to highly irritating chemical to rabbit eyes.

 

Another Driaze study was conducted for test chemical, in which undiluted test chemical was instilled in the eyes of 3rabbit and observed for signs of irritation till 21 days. The reactions observed were scored according to Draize method. Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical. The study was terminated on Day 14 as corneal opacity was 4 in 2 of 3 rabbits tested which was not reversible by day 21. Iris and conjunctival chemosis, conjunctival redness scores on day 14 were 0 and 2, 2 respectively. Based on these observations and scores, Sodium salicylate can be considered as highly irritating to eyes.

 

The Short time exposure (STE) test was performed for predicting eye irritation potential of Sodium Salicylate. SIRC (ATCC CCL-60) cells were obtained from American Type Culture Collection (Manassas, VA, USA). SIRC cells were cultured in Eagle’s MEM containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. When the cells proliferated in the culture flask to confluence, the cells were dispersed with trypsin-EDTA solution. The dispersed cells were spread into 96-well flat-bottomed plates at 3.0 × 103cells/well. After incubation (37°C, 5% CO2) for 5 days or (6.0 × 103cells/well for 4 days), the cells reached confluence. Briefly, physiological saline was used as first vehicle for test chemicals. The cells cultured in 96-well plates were exposed to 200 microliters of 5% and 0.05% test chemical solution. After exposure, the cells were washed with phosphate buffered saline (-) (PBS (-))twice and 200 μl of methylthiazolydiphenyl-tetrazolium bromide (MTT, Sigma Aldrich) solution (0.5 mg MTT/ml of medium) was added. After a 2-hr reaction time, MTT formazan was extracted with 0.04 N HCl-isopropanol (Kanto Chemical Co., Inc.) for 30 min, and the absorbance of the extract was measured at 570 nm with a plate reader. The ratio of absorbance (%) on each test sample to that of control was represented as relative viability (triplicate determinations). The control group cells were exposed to physiological saline, saline with 5% DMSO, or mineral oil. The mean of three wells for each test concentration was calculated. This was the mean relative viability for one independent test. A total of three independent tests were conducted for each concentration of a test material, and the calculated overall mean of three independent tests was used for estimation of eye irritation. Irritation results from the STE test were converted to a point system in order to establish a potency ranking. A lower rank point score was given to the experimental condition that had a lower cytotoxicity. For the lower test concentration results, a higher rank point score was awarded due to the potential cytotoxicity observed with the lower concentration. Once a score was given for each test condition (i.e., with 5% or 0.05% test concentrations), the total points for each test material were added to get a rank category of a 1, 2, or 3. A Rank of 1 meant that the test material was a ‘‘minimal irritant” while a Rank of 2 meant that the material was a ‘‘moderate irritant”, and a Rank of 3 meant that the test material was a ‘‘severe irritant”. The GHS eye irritation ranking were classified as NI(Not classified; not an eye irritant),Category 2(Cat.2, irritating to eyes) or category 1 (Cat. 1, irreversible effects on the eye) based on the Draize eye irritation test . To evaluate the predictive capacity of the STE test, the correspondence between STE Rank 1 and GHS NI; STE Rank 2 and GHS Cat. 2;and STE Rank 3 and Cat. 1 were calculated. The mean viability for 5% Sodium Salicylate was 89.7, 88.7, 84.5 and that of 0.05% Sodium Salicylate was 100.7, 95.4, and 101.3 for the three labs. The CVs of the cells treated with Sodium Salicylate were 2.7, 3.2 for 5% and 0.05% respectively. Based on the STE scores and ranking, Sodium Salicylate was considered to be an irritant and the GHS Classification was correlated to be Category 1.

 

An in-vitro EpiOcularTMEye Irritation Test (EpiOcularTMEIT) was performed to determine the degree of ocular damage caused by Sodium Salicylate. The study was conducted according to procedures mentioned in OECD 492 Guidelines.The EpiOcular™ tissue model OCL-200 (herein referred to as EpiOcular™) was produced using good manufacturing practice (GMP) in the MatTek production facilities. EpiOcular™ tissues were packaged on a proprietary agarose formulation and stored overnight at 4 ± 2°C to mimic shipping conditions to customers. The next day, the tissues were transferred from the agarose into 6-well plates containing 1mL of medium (provided with the OCL-200 kit) and pre-incubated for one hour under standard culture conditions (SCC), which are defined as an atmosphere with 90 ± 10% relative humidity, 5± 0.5% v/v) CO2, and a temperature of 37 ± 1°C. After 1 hour, the medium was changed and the EpiOcular™ cultures were further pre-incubated overnight (16–18 hours) under SCC. On day 1 of the test, the tissues were pre-treated for 30 minutes with 20 μL of calcium and magnesium-free DPBS. If the DPBS did not spread across the tissue surface.Next, 50 μL of the control substances (H2O and methyl acetate), or approximately 50mg of solid test material, were applied topically to the EpiOcular™ tissues, the latter by using a calibrated tool. Sodium Salicylate and control was tested in duplicate tissues.After a 30-minute exposure to the test chemical or controls, each pair of tissues was successively rinsed by dipping, swirling, and decanting through a set of three 150 ml beakers prefilled with DPBS; separate beakers were used for each test chemical or control. After the final rinse and decanting, the tissues were immersed in 5mL of EpiOcular™ assay medium in a 12-well plate for 12 minutes (post-soak) at room temperature. After the post-soak period, the medium was decanted from the CCIs and the CCIs were transferred to a 6-well plate containing 1mL of warm medium (37°C) and post-incubated for 2 hours under SCC.tissues exposed to solid test samples were post-incubated for 18 hours. After the 18-hour post-incubation period, tissue viability was determined by using the MTT assay. Tissue viability was determined with the MatTek MTT viability assay kit (MTT-100) which contains MTT Concentrate (5mg/mL), MTT Diluent (Dulbecco’s Modified Eagle Medium - DMEM) and MTT Extractant (Isopropanol p.a.). Approximately 1 hour prior to use, the MTT concentrate was thawed and diluted with the MTT Diluent resulting in a final concentration of 1mg/mL. A 300 μL aliquot of the diluted MTT solution was pipetted into each well of a 24-well plate. EpiOcular™ tissue inserts were rinsed in DPBS and placed in the MTT plate, making sure that no air bubbles were trapped underneath the CCI. The tissues were then incubated under SCC for 3 hours. After incubation, each EpiOcular™ insert was removed from the plate, blotted on an absorbent paper, and transferred into a 24-well plate containing 2mL of the MTT Extractant. Alternatively, tissues were extracted for 2 hours at room temperature on a plate shaker covered with foil. After the formazan extraction period was completed, the inserts were discarded and the extract was homogenised by pipetting up and down 2–3 times. Duplicate samples of the extract (200 μL) were pipetted into separate wells of a 96-well microtitre plate, and the optical density of the samples measured at 570 nm on a plate reader. The mean viability of the positive control (Methyl acetate) was 37.0 % and the mean OD for the Negative control in the established range of 1.0 – 2.5. The mean tissue viability for Sodium Salicylate was 5.0% and 5.1% respectively. The results of the EpiOcular EIT protocol were in concordance with the Draize test results, indicating a classification of Category 1 for Sodium Salicylate. Hence, Sodium salicylate was considered to be highly irritating to eyes.

 

The above results were further supported by the Draize ocular irritation test performed for Sodium Salicylate. 0.1 ml of each test solution was placed directly into the conjunctival cul-de-sac of one eye in each animal. The other eye remained untreated and served as control. Eyes were observed and scored, according to the scale of Draize et al. (1944), at 1, 3, 6, 24, 96, and 168 hour after dose. Three to six animals per dosage group were used, and at least three dosage groups were tested to obtain DS 0.Draize Score 0 (DS0) was defined as the maximum concentration of test compound at not showing any ocular irritation. The DS 0 values were calculated from the dose-response curves obtained. The Draize score 0(DS0) concentrations for Sodium Salicylate was obtained at 5% w/v. Sodium salicylate did not show any ocular irritation, even at the high concentrations which reached the limits of their solubilities and/or physiological osmotic pressure, and their DS0 values could not be obtained. Hence, it was considered that non-ocular irritation dose for Sodium salicylate which can be used for ophthalmic preparations was 5% w/v.

 

Thus based on the available data for the target as well as supporting studies, it can be concluded that test chemical is able to cause severe eye irritation and considered as irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”.

 

 

Justification for classification or non-classification

The skin and eye irritation potential of test chemical were observed in various studies. The results obtained from these studies indicates that the chemical is unlikely to cause skin irritation but can cause severe eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and “Category 2” for eye as per CLP.