Registration Dossier

Administrative data

Description of key information

In vitro skin corrosion: Negative
In vitro skin irritation: Negative
Acute dermal irritation in rabbit: Negative
In vitro eye irritation (BCOP): Negative
In vivo eye irritation: Negative

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
Study period:
The study was conducted between 25 February 2014 to 28 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.37 or 2.57 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: The test material was moistened with 0.5 ml of distilled water.
Controls:
no
Amount / concentration applied:
0.5 g of the test item.
Duration of treatment / exposure:
4 hours
Observation period:
1, 24, 48 and 72 hours
Number of animals:
2 rabbits
Details on study design:
On the day before the test two rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

On the day of the test a suitable test site was selected on the back of each rabbit. At each test site a quantity of 0.5 g of the test item, moistened sufficiently with 0.5 mL of distilled water to achieve a paste, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.

Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Evaluation of skin reactions (see table below).

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal #1
Remarks:
73978 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 1hour
Remarks on result:
other: Non-irritant
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal #2
Remarks:
73979 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 1 hour
Remarks on result:
other: Non-irritant
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
73978 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 1 hour
Remarks on result:
other: Non-irritant
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
73979 Male
Time point:
other: Mean score at 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 1 hour
Remarks on result:
other: Non-irritant
Irritant / corrosive response data:
Skin Reactions

The individual scores for erythema/eschar and edema are given in Table 1.
Very slight erythema and very slight edema were noted at both treated skin sites immediately after patch removal.
Both treated skin sites appeared normal one hour after patch removal.
Other effects:
Body Weight

Individual body weights and body weight change are given in Table 2.
Both animals showed expected gain in body weight during the study

Table 1: Individual Skin Reactions

Skin Reaction

Observation Time
(following patch removal)

Individual Scores – Rabbit Number and Sex

Total

73978Male

73979Male

Erythema/Eschar Formation

Immediately

1

1

(2 )

1 Hour

0

0

( 0 )

24 Hours

0

0

0

48 Hours

0

0

( 0 )

72 Hours

0

0

0

Edema Formation

Immediately

1

1

( 2 )

1 Hour

0

0

( 0 )

24 Hours

0

0

0

48 Hours

0

0

( 0 )

72 Hours

0

0

0

Sum of 24 and 72‑Hour Readings (S) :        0

Primary Irritation Index (S/4)              :        0/4 = 0.0

Classification                                       :        NON‑IRRITANT


(   ) =    Total values not used for calculation of primary irritation index

Table 2: Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 3

73978
Male

2.37

2.43

0.06

73979
Male

2.57

2.68

0.11

Interpretation of Results:

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

 The scores for erythema and edema at the 24 and 72‑Hour readings were totaled for the two test rabbits (8 values) and this total was divided by 4 to give the primary irritation index of the test item. The test item was classified according to the following scheme devised byDraize, J.H. (1959):

 

Primary Irritation Index

Classification of Irritancy

0

Non-irritant

> 0 to 2

Mild irritant

> 2 to 5

Moderate irritant

> 5 to 8

Severe irritant

 

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.

 

The results were also interpreted according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a primary irritation index of 0.0 and was classified as non irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit.

 

 Results

A single 4‑Hour, semi‑occluded application of the test item to the intact skin of two rabbits produced very slight erythema and very slight edema immediately after patch removal. Both treated skin sites appeared normal one hour after patch removal.

 

Conclusion

The test item produced a primary irritation index of 0.0 and was classified as non‑irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

 

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

 

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
Study period:
Experimental Starting Date: 09 June 2014; Experimental Completion Date: 26 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd. At the start of the study the animals weighed 2.53 or 3.29 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
A volume of 0.1 mL of the test item, which was found to weigh approximately 93 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye.
Duration of treatment / exposure:
Single application with observation to 72 hours.
Observation period (in vivo):
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment
Number of animals or in vitro replicates:
2
Details on study design:
MEASUREMENT OF pH:
The pH of the test material was determined prior to commencement of the study and found to be as follows:
10% w/w aqueous preparation of the test material: pH 8.66 immediately, pH 8.65 after 10 minutes.

PROCEDURE:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.

A volume of 0.1 mL of the test item, which was found to weigh approximately 93 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to a six point scale.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.

SCORING:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation: Draize Scale for Scoring Ocular Irritation

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Any clinical signs of toxicity, if present, were also recorded.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.




Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(74366 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
4
Remarks on result:
other: No corneal effects noted
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(74391 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
4
Remarks on result:
other: No corneal effects noted
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(74366 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(74391 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
2
Remarks on result:
other: No iridial inflammation noted
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Remarks:
(74366 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #2
Remarks:
(74391 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(74366 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(74391 Male)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.

No corneal effects were noted during the study.

Iridial inflammation was noted in one treated eye 1 hour after treatment.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye at the 24 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 48 Hour observation.

Both treated eyes appeared normal at the 72 Hour observation.
Other effects:
Individual body weights and body weight change are given in Table 3.

Both animals showed expected gain in body weight during the study.

Table 1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

74366 Male

74391 Male

IPR= 0

IPR = 0

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

1
Hour

24
Hours

48
Hours

72
Hours

CORNEA

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

D

1

0

0

0

0

0

0

0

Score (D x 5)

5

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

2

1

1

0

2

2

1

0

B = Chemosis

2

1

0

0

2

1

0

0

C = Discharge

1

1

0

0

2

1

0

0

Score (A + B + C) x 2

10

6

2

0

12

8

2

0

Total Score

15

6

2

0

12

8

2

0


IPR=Initial pain reaction

Table 2     Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

74366 Male

15

6

2

0

74391 Male

12

8

2

0

Group Total

27

14

4

0

Group Mean Score

13.5

7.0

2.0

0.0

Table 3     Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 3

74366 Male

3.29

3.35

0.06

74391 Male

2.53

2.59

0.06

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a maximum group mean score of 13.5 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

Conclusion

The test item produced a maximum group mean score of13.5and was classified as amildirritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

 

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/Corrosion:

1) The purpose of this test, (in vitro), is to evaluate the corrosivity potential of the test item using the EPISKINin vitroReconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT- loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Conclusion

The test item was classified as non-corrosive to the skin. The following classification criteria apply:

EU DSD (67/548/EEC): Not classified for corrosivity.

EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.

UN Packing Group: Non-Corrosive.

2) The purpose of the test, (in vitro), was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. 

At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200µL samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 562 nm.

Results

The relative mean viability of the test item treated tissues was 118.5% after the 15‑Minute exposure period and 42 hours post‑exposure incubation period.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

3) The study, (in vivo), was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit.

A single 4‑Hour, semi‑occluded application of the test item to the intact skin of two rabbits produced very slight erythema and very slight edema immediately after patch removal. Both treated skin sites appeared normal one hour after patch removal.

 The test item produced a primary irritation index of 0.0and was classified as non‑irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Eye irritation:

1) A study (in vitro, BCOP), was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea.

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).

A test item that induces an mIn VitroIrritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant.

The In Vitroirritancy scores are summarized as follows:

 

Treatment

In VitroIrritancy Score

Test Item

0.3

Negative Control

2.7

Positive Control

86.0

Conclusion

The test item was considered not to be an ocular corrosive or severe irritant.

2) The study, (in vivo), was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

The test item produced a maximum group mean score of 13.5and was classified as amildirritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

 

 


Justification for selection of skin irritation / corrosion endpoint:
Results from both in vitro studies (in vitro skin corrosion and in vitro skin irritation) are negative.
The in vivo test is also negative.

Justification for selection of eye irritation endpoint:
Results from the in vitro study (BCOP) is negative.
The in vivo test is also negative.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).