Registration Dossier

Administrative data

Description of key information

Skin irritation:

The skin irritation potential of test chemical was assessed in various experimental studies conductedin rabbits, guinea pigsand humans. Based on the available data for the target and supporting studies, it can be concluded that the test chemical is unable to cause skin irritation and thus can be 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 experimental studies which were conducted in rabbits for test chemical.Based on the available studies,it can be concluded that the testchemical is unable to cause eye 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''.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data is from experimental study
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
The test is designed to investigate the irritation effects of substances when applied once to the intact skin under a semi-occlusive dressing.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
- Age at study initiation: 9-12 weeks
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.5 ml of each test and std. solution (0.2 gm for solid samples)
Duration of treatment / exposure:
7 days
Observation period:
24,48 and 72 hours after treatment, and occasionally up to 7 days
Number of animals:
8 animals
Details on study design:
TEST SITE
- Area of exposure: -clipped dorsum of each rabbit
- Type of wrap if used:Occlusive patches are prepared by attaching a piece of thin flexible polythene (3cm x 3cm) to a piece of zinc oxide plaster 9cm x 2.5cm. A 2.5 cm square of cotton gauze (8 ply folded in two) is laid on the polythene such that the edges of the pad are attached to the zinc oxide plaster

REMOVAL OF TEST SUBSTANCE
- Washing (if done): treated areas were wiped clean of excess material.
- Time after start of exposure: sites were evaluated at 24, 48 and 72 hours after treatment, and occasionally up to 7 days after treatment

SCORING SYSTEM: The wilcoxon matched - pairs signed ranks test, which consideres the size and direction of differences between reactions is used to test the significance of differences between pairs of treatment groups.

other: The whole dorsal surface is clipped 3-4 days before the test is due to start and animals in the telogen phase of hair growth are selected. Test solutions (0.5 ml) are applied to a dry patch. Test solids are applied to a moistened patch. Aerosol preparations are applied directly to the skin with two 1 second burst and the patches are saturated with spray which acts as a reservoir.

The patches are firmly attached to ensure good contact between the skin and test substance. The animals are immobilised in a canvas body sleeve for 4 hours after application of the patch. When the patches are removed, the treatment sites are wiped clean of excess material. The position of treatment sites are randomised.

Irritation parameter:
overall irritation score
Remarks:
Test chemical
Basis:
mean
Time point:
24/48/72 h
Score:
11
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: By 48 and 72 hours there were no visible macroscopic changes.
Irritation parameter:
other: Mean Score per site
Remarks:
test chemical
Basis:
mean
Time point:
other: 4/24/48/72 h
Reversibility:
fully reversible within: 72 hours
Remarks on result:
no indication of irritation
Irritation parameter:
other: Mean score per site per day
Basis:
mean
Time point:
other: 4/24/48/72 h
Score:
0.34
Reversibility:
fully reversible within: 72 hours
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
A single application of the test chemical produced marginal erythema in a few animals and marginal edema in one animal at 24 hours. By 48 and 72 hours there were no visible macroscopic changes.

Table 2: Daily microscopic assessment of irritation reaction of Test Chemical

Rabbit

Treatment site no.

4 Hours

24 Hours

48 Hours

72 Hours

Total

Total score

No.

Wt (Kg)

 

er

oe

cr

sc

 

 

er

oe

cr

sc

 

 

er

oe

cr

sc

 

 

er

oe

cr

sc

 

 

er

oe

cr

sc

 

 

 

1144

 

2

a

a

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

1

1

0

0

 

 

2

1145

 

3

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

0

0

0

0

 

 

0

1146

 

4

a

a

-

-

 

 

a

a

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

2

2

0

0

 

 

4

1147

 

5

a

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

1

0

0

0

 

 

1

1092

 

6

-

-

-

-

 

 

a

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

1

0

0

0

 

 

1

1101

 

1

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

0

0

0

0

 

 

0

1163

 

1

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

0

0

0

0

 

 

0

1164

 

4

a

b

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

-

-

-

-

 

 

1

2

0

0

 

 

3

Overall irritation score

11

Mean score per site

1.38

Mean score per site per day

0.34

 

Note:

er

Erythema

oe

Oedema

cr

Cracking

sc

Scaling

sb

Scabbing

ex

Exudate

hae

Haemorrahage

 

Reaction grades and scores:

Marginal/very slight

1

Slight

2

Fairly distinct

3

Quite distinct

4

Becoming well developed

6

Well developed

8

Becoming severe

10

Severe

12

Table 3: Total Irritation Scores

Rabbit number

Treatment groups

C

B

D

A

F

E

1144

0

2

1

26

21

27

1145

3

0

1

38

45

40

1146

1

4

2

34

26

33

1147

1

1

2

25

31

31

1092

0

1

2

18

30

31

1101

1

0

9

21

24

22

1163

0

0

3

26

44

39

1164

4

3

3

25

15

19

Overall irritation score

10

11

23

213

236

242

Mean score per site

1.25

1.38

2.88

26.63

29.5

30.25

Mean score per site per day

0.31

0.34

0.72

6.66

7.38

7.56

Mean Irritation Ranking

1.75

1.81

2.44

4.63

5.06

5.31

Reaction Grades and Score

a- Marginal/very slight = 1

b- Slight = 2

c- Fairly distinct = 3

d- Quite distinct = 4         

e- Becoming well developed = 6

f- Well developed = 8

g- becoming severe = 10

h- severe = 12

Interpretation of results:
other: Not Irritating
Conclusions:
A single application of the test chemical produced marginal erythema in a few animals and marginal edema in one animal at 24 hours. By 48 and 72 hours there were no visible macroscopic changes. The mean score per site per day for test chemical was calculated to be 0.34. Based on the scores and observations, the test material can be considered as not irritating to New Zealand White rabbit skin.
Executive summary:

The test is designed to investigate the irritation effects of when applied once to the intact skin under a semi-occlusive dressing. Eight New Zealand White rabbits were used for the study.

in this test, the dorsal surface was clipped 3 -4 days before the test was due to start. Occlusive patches were prepared by attaching a piece of thin flexible polythene (3*3 cm) to a piece of zinc oxide plaster 9cm*2.5cm. A 2.5 cm square of cotton gauze was laid on the polythene such that the edges of the pad are attached to the zinc oxide plaster. 0.2 gm of undiluted test chemical on a patch dampened with Diethyl Phthalate and 0.5ml of the standards for comparison were applied to the clipped dorsum of rabbits for 4 hours under individual semi-occlusive patches. Bromostyrene, Coumarin, Diethyl phthalate, Cyclamen aldehyde and Geraniol were used as standards for comparison.

Four hours after exposure, the patches were removed and the test sites were wiped cleaned of the excess test material. The sites were scored for erythema, edema, cracking and scaling and any other feature using an 8 point grading scale ranging from "a" (very slight) to "h"(very severe). At the end of the test the reaction grades which were used to calculate the total irritation score per site, a mean irritation score per site per day for each treatment group.

A single application of the test chemical produced marginal erythema in a few animals and marginal edema in one animal at 24 hours. By 48 and 72 hours there were no visible macroscopic changes. The response was similar to that produced by Coumarin and Diethyl phthalate, but considerably less than that produced by Geraniol and Cyclamen aldehyde.

The mean score per site per day for test chemical was calculated to be 0.34.

Based on the scores and observations, the test material can be considered as not irritating to New Zealand White rabbit skin.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from experimental report
Qualifier:
according to
Guideline:
other: Modified Federal Hazardous Labelling Act
Principles of method if other than guideline:
The test was designed to investigate the irritation effects of test chemical when applied to the one eye of single rabbits.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Age at study initiation: aged 7-9 weeks
- Weight at study initiation:weighing between 1-2 Kg
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
10 mg
Duration of treatment / exposure:
24 hrs.
Observation period (in vivo):
15 min. after application
24 hrs. after application
daily intervals for 5 days per week for 3 weeks
Number of animals or in vitro replicates:
6 animals
Details on study design:
The lower eyelid is gently pulled away from the globe and 10 mg of test substance applied into the cup so formed ;the eyelid then release. The rabbit was inspected with hand torch before any treatment and those with spontaneous eye lesions are rejected.Flurescein is applied to eye to assist in the detection of corneal lesions.Corneal thickness is also measured before the application ot test material
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Reversibility:
other: Not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The test chemical caused no corneal, conjunctival, or iridial damage 24 hours after treatment.

Table 1: Summary of test result

Experimental group

No of rabbits

Discomfort

Number of rabbits with stated ocular lesion

Positive conjunctival reactions

No. with iritis

No. with pannus

Maximum corneal opacity

Duration of opacity

Maximum corneal swelling

0.5

1

2

3

4

>1 on day 8

>0.5 on day 8

Nil (0-10%)

Slight (11-45%)

Moderate (46-80%)

Severe (81%+)

A

1

-

-

-

-

-

-

-

-

-

-

-

1

-

-

-

Interpretation of results:
not irritating
Conclusions:
The test chemical caused no corneal, conjunctival, or iridial damage 24 hours after treatment at concentration of 10mg. Hence, the test material can be considered to be not irritating to New Zealand White rabbit eyes.
Executive summary:

An eye irritation study was conducted in accordance with Modified Federal Hazardous Labelling Act Method by using a single rabbit.

The rabbit was inspected with a hand torch before any treatment and those without any serious corneal lesions were taken. Corneal thickness was also measured before the start of the test. The lower eyelid was gently pulled away from the globe and 10 mg of test substance applied on one eye into the cup so formed; the eyelid was then released.

Animal was watched during and immediately after the application of the test chemical. 15 minutes after the application of the test chemical the eyes were observed for conjunctival reactions. One day after the application of test material and then at daily intervals for five days per week for three weeks or until the eye is completely clear. Fluorescein was applied to eyes to assist in the detection of corneal lesions.

Each day after application of the test chemical, the corneal thickness was measured using a slit lamp. Corneal swelling was expressed as:

(Corneal thickness on day of measurement)/(Corneal thickness on before treatment)-1)*100%.

The test chemical caused no corneal, conjunctival, or iridial damage 24 hours after treatment at tested concentration of 10 mg.

Hence, the test chemical can be considered to be not irritating to New Zealand White rabbit eyes.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Various studieshas 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, guinea pigs and humans for target chemical that have been summarized as below;

 

The dermal irritation test was conducted on eight New Zealand White rabbits. The test is designed to investigate the irritation effects of when applied once to the intact skin under a semi-occlusive dressing. In this test, the dorsal surface was clipped 3 -4 days before the test was due to start. Occlusive patches were prepared by attaching a piece of thin flexible polythene (3*3 cm) to a piece of zinc oxide plaster 9cm*2.5cm. A 2.5 cm square of cotton gauze was laid on the polythene such that the edges of the pad are attached to the zinc oxide plaster. 0.2 gm of undiluted test chemical on a patch dampened with Diethyl Phthalate and 0.5ml of the standards for comparison were applied to the clipped dorsum of rabbits for 4 hours under individual semi-occlusive patches. Bromostyrene, Coumarin, Diethyl phthalate, Cyclamen aldehyde and Geraniol were used as standards for comparison. Four hours after exposure, the patches were removed and the test sites were wiped cleaned of the excess test material. The sites were scored for erythema, edema, cracking and scaling and any other feature using an 8 point grading scale ranging from "a" (very slight) to "h"(very severe). At the end of the test the reaction grades which were used to calculate the total irritation score per site, a mean irritation score per site per day for each treatment group. A single application of the test chemical produced marginal erythema in a few animals and marginal edema in one animal at 24 hours. By 48 and 72 hours there were no visible macroscopic changes. The response was similar to that produced by Coumarin and Diethyl phthalate, but considerably less than that produced by Geraniol and Cyclamen aldehyde. The mean score per site per day for test chemical was calculated to be 0.34. Based on the scores and observations, the test material can be considered as not irritating to New Zealand White rabbit skin.

 

Another Preliminary irritation study of test chemical was performed in female guinea pigs to determine the suitable concentration for the skin sensitization study.In the preliminary test,8mm filter paper(Whatman 3mm) patches, in 11mm "Fintest" aluminium patch test cups(Epitest Ltd.) were saturated with 10% of test concentrations in acetone and the cups were applied to the shaved flanks of 4 previously untreated guinea pigs of the same sex. The patches were held in place by an adhesive plaster (Poroplast) wound around the trunk. 24 hours later, the patches were removed, the reaction sites being examined 24 and 48 hours subsequently. The reactions were scored for irritation on a scale from 0(no reaction) to +++ (intense erythema and edema). The concentration giving the highest non-irritant concentration was chosen for the flank challenge. Since the test chemical did not induce any cutaneous effects at tested concentrations (10%), the test material chemical was considered to be not irritating to the skin of guinea pigs.

 

The above results were supported by a closed patch test of test chemical performed on human subjects to determine its irritation potential. The test chemical was applied at concentration of 1% in petrolatum on skin of each subject under occlusive condition for 48 hours. None of the subjects showed any cutaneous reaction within the 48 hour. Therefore, the test chemical was considered to be not irritating to the skin of human subjects.

 

Further, a Skin irritation study of test chemical was performed in rabbits to assess the degree of irritancy caused by the chemical.In this test, each rabbit received undiluted test chemical was dermally on intact or abraded skin for 24 hours and later observed for skin reactions. The test chemical caused slight skin irritation to the skin of treated rabbits. Hence the test chemical was considered as slightly irritating to the skin of rabbit.

 

The overall negative results were further supported by the predicted Danish QSAR study. According to Danish QSAR database, skin irritation effects were estimated by using four different models i.e, Battery, Leadscope, SciQSAR and CASE Ultra for test chemical. Based on estimation, no severe skin irritation effects were known when the test chemical was exposed to rabbit skin. Hence, the test chemical can be considered as not irritating to the rabbits skin.

 

Although slight skin reactions were observed in one study but the potential of causing skin irritation was not enough to classify the chemical as skin irritant. Thus on the basis of available data and majority of results obtained in various studies, the test chemical can 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 chemicalhas been investigated for potential for ocular irritationto a greater or lesser extent. The studies are based on in vivo experiments in rabbits fortest chemical that have beensummarized as below;

 

An eye irritation study was conducted in accordance with Modified Federal Hazardous Labelling Act Method by using a single rabbit. The rabbit was inspected with a hand torch before any treatment and those without any serious corneal lesions were taken. Corneal thickness was also measured before the start of the test. The lower eyelid was gently pulled away from the globe and 10 mg of test substance applied on one eye into the cup so formed; the eyelid was then released. Animal was watched during and immediately after the application of the test chemical. 15 minutes after the application of the test chemical the eyes were observed for conjunctival reactions. One day after the application of test material and then at daily intervals for five days per week for three weeks or until the eye is completely clear. Fluorescein was applied to eyes to assist in the detection of corneal lesions. Each day after application of the test chemical, the corneal thickness was measured using a slit lamp. Corneal swelling was expressed as: (Corneal thickness on day of measurement)/(Corneal thickness on before treatment)-1)*100% . The test chemical caused no corneal, conjunctival, or iridial damage 24 hours after treatment at tested concentration of 10 mg. Hence, the test chemical can be considered to be not irritating to New Zealand White rabbit eyes.

 

The same experiment was repeated with 50mg of the test substance applied to the conjunctival sac of 6 New Zealand White rabbits. The test chemical failed to cause any ocular reactions 24 hours after treatment to 50mg of the test chemical. Hence, the test chemical can be considered to be not irritating to New Zealand White rabbit eyes.

 

Based on the available key data and supporting study,it can be concluded thatchemical is unable to cause eye 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''.

 

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 and eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and eye as per CLP.