Registration Dossier

Administrative data

Description of key information

Skin Irritation

A study was designed and conducted to determine the dermal reaction profile of the test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

Eye Irritation

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be not irritating to eyes. Thus it can be further classified under the category “Not Classified” as per CLP regulation.

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:
comparable to guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
To assess the toxicological profile of the test chemical on application as a single semi-occlusive dermal application to rats
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Detailas on test animals and environmental conditions:

Sex: females, Females were nulliparous and non-pregnant
Source : Geniron Biolabs Pvt. Ltd., Bengaluru, India
Age at treatment: 11 to 12 weeks
Body weight range at treatment: Females: 211.56 to 231.27 g
Identification :By rat accession number. Identification of individual rats is by cage card and crystal violet colour body markings. The temporary body marking during acclimatization period was done with crystal violet. The rat accession numbers were allotted during the course of the study and was included in raw data and reported.
Housing: Animals were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as enrichment objects and were changed along with the cage once a week.
Steam sterilized corn cob was used and changed once a week along with the cage.
Diet: Rat & Mice pellet feed, manufactured by Krishna Valley Agro Tech LLP, manufactured at Sangli, Maharashtra, was provided to ad libitum to animals
Water: Purified water in polycarbonate bottles with stainless steel sipper tubes was provided ad libitum
Acclimatization: After physical examination for good health and suitability for experiment, the rats were acclimatized for six, eight, ten and twelve days before treatment for dose range finding studies and main study respectively under standard laboratory conditions. Animals were observed once daily during acclimatization period.

Environmental Conditions:
Temperature: 22 to 24°C
Relative humidity: 66 to 68%
Air changes: air conditioned with adequate fresh air supply (13.0 air changes/hour)
Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
Dose range finding study: 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000 (2 mL/kg body weight)
Main Study: 2000 mg/kg
Duration of treatment / exposure:
24 hours
Observation period:
14 days
Number of animals:
5 females (3 females for dose range finding study followed by 2 females for main study)
Details on study design:
TEST SITE
- Area of exposure: clipped skin of the torso
- % coverage: 10% of the body surface of the animal (semi-occlusive).
- Type of wrap if used: The area of application was covered with cotton gauze (size: Females: 8 x 5 cm
of 6 ply) and it was secured in position by adhesive tape wound around the torso
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: After the 24 hours contact period, the dressing was removed and the
applied area was washed with deionized water and wiped dry using clean towels
OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : All the rats were observed for clinical signs of toxicity and mortality
for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after
removal of test item using the Draize criteria
SCORING SYSTEM:
- Method of calculation: Draize method
Irritation parameter:
erythema score
Basis:
mean
Time point:
14 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
14 d
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method).
Other effects:
There were no clinical signs and pre-terminal deaths (mortality) observed during the study. All rats gained body weight throughout the observation period. No abnormality was detected at necropsy.

APPENDIX 1.      Individual test item application, clinical signs, skin reactionand necropsy findings

Dose range finding study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mL)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

 min

1 h

2 h

3 h 

4 h

 

5 h

6 h

 

*

Er

@

Ed

@

*

Er

@

Ed

@

*

Er

@

Ed

@

G1 and

200

DRF

 

24 April 2018

and

10.41 AM

Rm9127

F

231.27

0.05

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observation

Necropsy

Findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and

200

DRF

 

Rm9127

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes       DRF: Dose Range Finding           NAD: No abnormality detected          Er: Erythema             Ed: Edema           

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

Dose range finding study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mL)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

 min

1 h

2 h

3 h 

4 h

 

5 h

6 h

 

*

Er

@

Ed

@

*

Er

@

Ed

@

*

Er

@

Ed

@

G2 and

1000

DRF

 

26 April 2018

and

10.40 AM

Rm9128

F

218.19

0.22

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observation

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G2 and

1000

DRF

 

Rm9128

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes       DRF: Dose Range Finding               NAD: No abnormality detected      Er: Erythema             Ed: Edema           

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

Dose range finding study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mL)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

 min

1 h #

2 h #

3 h #

4 h

#

5 h #

6 h

#

*

Er

@

Ed

@

*

Er

@

Ed

@

*

Er

@

Ed

@

G3 and

2000

DRF

 

28 April 2018

and

10.43 AM

Rm9129

F

211.56

0.42

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observation

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and

2000

DRF

Rm9129

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes       DRF: Dose Range Finding               NAD: No abnormality detected      Er: Erythema             Ed: Edema           

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

Main study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mL)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G3 and

2000

Main

 

30 April 2018

and

11.01AM to 11.02 AM

Rm9130

F

214.99

0.43

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Rm9131

F

213.42

0.43

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observations

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and

2000

Main

 

Rm9130

F

N

N

N

N

N

N

N

N

N

N

NAD

Rm9131

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema          

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

 

Interpretation of results:
other: not irritating
Conclusions:
There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.
Executive summary:

A study was designed and conducted to determine the dermal reaction profile of the test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

The test chemical was tested in 5 females (3 females for dose range finding study followed by 2 females for main study) Wistar rats at the doses of 200, 1000 and 2000 mg/kg body weight. Based on the individual body weight, the undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000 (2 mL/kg body weight) - based on the density of the test item1.01 g/cm3(as per TIDS) was applied directly to the clipped skin of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The applied area was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towel. 

There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence based on various test chemicals
Justification for type of information:
Weight of evidence based on various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Weight of evidence based on various test chemicals
Principles of method if other than guideline:
Weight of evidence based on various test chemicals.The study 2,3 are referred as 1,2
GLP compliance:
not specified
Species:
rabbit
Strain:
not specified
Vehicle:
other: 1. distilled water; 2. undiluted
Controls:
not specified
Amount / concentration applied:
1. 10mg/ml [0.1-1% test chemical in distilled water]
2. 0.1ml
Duration of treatment / exposure:
1. no data available
2. single
Observation period (in vivo):
1. no data available
2. 1, 4, 24, 48, 72 and 96 h and daily thereafter for a total of 7 days
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
1.no data available
2. 6 New Zealand White rabbits
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
no signs of irritation observed
Interpretation of results:
other: not irritating
Conclusions:
Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be not irritating to eyes. Thus it can be further classified under the category “Not Classified” as per CLP regulation.
Executive summary:

The ocular irritation potential of the test chemical was assessed based on the available results from the various test chemicals.

The study was conducted to evaluate the acute eye irritation potential of the test chemical in rabbit. 0.1 -1% test chemical in distilled water was instilled into the eyes of rabbits and observed for effects [duration of exposure, observation period not specified]. The test chemical at dose levels up to 10mg/ml (1%) was non-irritating to rabbit eyes. Hence, it can be considered to be not irritating to eyes.

This is supported by the results of another ocular irritation study performed to determine the irritation potential of the test chemical in rabbits.0.1ml of the test chemical was instilled in one eye of 6 New Zealand White rabbits and observed for signs of irritation. The untreated eyes served as controls. Observations were made at 1, 4, 24, 48, 72 and 96 h and daily thereafter for a total of 7 days. Conjunctival irritation was observed in 1/6 rabbits for 24-h.Under the conditions of this study,the test chemical was considered to be not irritating.

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be not irritating to eyes. Thus it can be further classified under the category “Not Classified” as per CLP regulation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

Various studies have been performed to evaluate the dermal irritation potential of the test chemical in living organisms. These include in vivo experimental studies in guinea pigs, humans, rabbits, rats. The studies are summarized as follows:

Skin irritation and capacity for allergic sensitisation was determined by the Open Epicutaneous Test (OET) on guinea pigs.

The substance was applied undiluted as well as dissolved in acetone, ethanol or diethylphthalate etc. at concentrations of 30, 10, 3, 1, 0.3, 0.1 and 0.3% in order to establish a dose response curve which would permit the determination of the minimal irritating and the maximal tolerated concentrations on an ‘’ all or none basis’’. For each concentration, 6 to 8 animals were used.

To determine skin irritation after one and after 21 daily applications, 0.1 ml of the undiluted substance and its lower concentrated solutions were applied homogenously with a pipette to an area measuring 8 cm2, previously marked with a circular stamp on the clipped flank skin of groups of 6 to 8 animals. After the solvent had evaporated, the application site was left uncovered and reactions read after 24 hours.

To investigate the capacity to induce skin irritation after repeated applications, the undiluted substance and its lower concentrations were applied to the same animal groups during 21 consecutive days, always using the same skin site, thr reactions being read and evaluated similarly on day 21.

To determine whether allergic contact dermatitis was induced, all groups of experimental animals mentioned above as well as 6 to 8 untreated controls were tested on days 21 and 35 on the contralateral flank with the substance at the primary non toxic and at some lower concentrations. These tests were performed by applying 0.025 ml of each concentration to skin areas measuring 2 cm2 with a pipette, the reactions being read after 24, 48 and 72 hours. This procedure allowed us to determine the minimal concentration necessary to induce contact hypersensitivity and the minimal one to elicit allergic reactions. The total amount of the compound administered in the OET varies from 2100 mg to 6 mg, depending on the concentration at which the compound was applied.

The highest non irritant concentration of test chemical after a single application was 10%. Repeated application cause slight skin irritation. The 3% alcoholic solution was well tolerated in the first week, but only very slight skin reactions on the second and third week of the treatment are seen. The test chemical does not sensitize the guinea pigs in the OET.

Hence, it was concluded that test chemical was considered to be slightly irritating to guinea pig skin and being classified as ''Irritating to skin in Category 2” as per CLP Regulation.

This is supported by the study designed and conducted to determine the dermal reaction profile of the test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

The test chemical was tested in 5 females (3 females for dose range finding study followed by 2 females for main study) Wistar rats at the doses of 200, 1000 and 2000 mg/kg body weight.

Based on the individual body weight, the undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000 (2 mL/kg body weight) - based on the density of the test item1.01 g/cm3(as per TIDS) was applied directly to the clipped skin of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The applied area was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towel. 

There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

These results are supported by a skin irritation potential of the test chemical were investigated in rabbits. The test chemical applied full strength to intact or abraded rabbit skin for 24 hr under occlusion was moderately irritating. Hence, it was concluded that test chemical was considered to be moderately irritating to rabbit skin and being classified as ''Irritating to skin in Category 2” as per CLP Regulation.

Irritation caused by the test chemical was also evaluated during the induction phase in a human repeated insult patch test (HRIPT). 0.3 ml aliquot of a 4% solution of the test chemical in 3:1 DEP:EtOH was applied to 25 mm Hill Top Chambers. Following a 10–30 min volatilization period, the patches were placed on the subjects skin between the left scapula and spinal mid-line for 24 hour under occlusion. After a 24– 48-hour rest period, subjects were again patched at the same site. A total of nine applications were made over a three-week period. No irritation was observed in 54 male and female volunteers. Hence, the test chemical can be considered to be not irritating to skin.

This is supported by the results of a similar study performed on humans. 0.3 ml aliquot of a 4% solution of the test chemical in 3:1 DEP:EtOH was applied to 25 mm Hill Top Chambers. Following a 10–30 min volatilization period, the patches were placed on the subjects skin between the left scapula and spinal mid-line for 24 hour under occlusion. After a 24– 48-hour rest period, subjects were again patched at the same site. A total of nine applications were made over a three-week period. No irritation was observed in 55 male and female volunteers. Hence, the test chemical can be considered to be not irritating to skin.

These results are supported by A Pre-test for a human maximization study performed to assess the dermal irritation potential of the test chemical. 10% test chemical in petrolatum was applied to the backs of 33 male and 24 male volunteers for 48 hours under occlusion. In a pre-test for a human maximization study, no irritation was observed after a 48-hour closed patch test to 33 and 24 male human volunteers.

Hence, the test chemical can be considered to be not irritation skin.

These results are further supported by a similar study performed on humans for determining the irritation potential of the test chemical.10% test chemical in hydrophilic ointment was applied to the backs of 25 male and female volunteers for 48 hours under occlusion.

No signs of irritation observed in 25 male and female volunteers after 48 hours of exposure to the test chemical in hydrophilic ointment. Hence, the test chemical can be considered to be not irritating to skin.

 

All the above results are supported by a patch test performed to determine the highest non-irritant concentrations of the test chemical. The test chemical were tested in a small series of rising concentrations[10% in petrolatum] by means of an occlusive cellophane material. The amount of test chemical applied to the filter paper disk of the AL-test patch-testing strips was standardized to some extent by limiting the strip of ointment extruded from the syringe in which the test substances are stored to half the diameter of the filter paper disk. AL-test (Imeco, Stockholm 42, Sweden) in strips of 2-5 patches were placed in vertical rows from the spina scapulae downward. The strips were fixed with waterproof Leukoplast (Beiersdorf), of 5 cm width. The patches were applied on the healthy-looking skin of the back of 50 volunteers till 2 days. The readings were of the tests were made 2 and 3 days after removal of patches.

The highest concentration of the test chemical not giving rise to any reaction in any of the 50 volunteers was 10% in petrolatum.

Hence, the test chemical can be considered to be not irritating to skin when tested till 10% in petrolatum.

Even though the test chemical caused signs of irritation to the skin of guinea pigs and rabbits, but it failed to cause any irritation to skin of humans, rats.Also, when tested at the maximum dose of 2000mg/kg in rats; no signs of irritation were observed till end of observation than the concentrations tested on humans.

Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Eye Irritation

The ocular irritation potential of the test chemical was assessed based on the available results from the various test chemicals.

The study was conducted to evaluate the acute eye irritation potential of the test chemical in rabbit. 0.1 -1% test chemical in distilled water was instilled into the eyes of rabbits and observed for effects [duration of exposure, observation period not specified]. The test chemical at dose levels up to 10mg/ml (1%) was non-irritating to rabbit eyes. Hence, it can be considered to be not irritating to eyes.

This is supported by the results of another ocular irritation study performed to determine the irritation potential of the test chemical in rabbits.0.1ml of the test chemical was instilled in one eye of 6 New Zealand White rabbits and observed for signs of irritation. The untreated eyes served as controls. Observations were made at 1, 4, 24, 48, 72 and 96 h and daily thereafter for a total of 7 days. Conjunctival irritation was observed in 1/6 rabbits for 24-h.Under the conditions of this study,the test chemical was considered to be not irritating.

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be not irritating to eyes. Thus it can be further classified under the category “Not Classified” as per CLP regulation.

Justification for classification or non-classification

Even though the test chemical caused signs of irritation to the skin of guinea pigs and rabbits, but it failed to cause any irritation to skin of humans, rats.Also, when tested at the maximum dose of 2000mg/kg in rats; no signs of irritation were observed till end of observation than the concentrations tested on humans.

Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Based on the available data for the various test chemicals and applying the weight of evidence approach, it can be concluded that the test chemical will also behave in similar manner and was estimated to be not irritating to eyes. Thus it can be further classified under the category “Not Classified” as per CLP regulation.