Registration Dossier

Administrative data

Description of key information

The skin irritation and corrosion potential of the test substance was assessed in two different in vitro and one in vivo studies.  Based on the results of these studies, the test substance was considered to be neither a corrosive nor an irritant.
The eye irritation potential of the test substance was assessed in an in vivo Bovine Corneal Opacity and Permeability study as well as in an in vivo study using rabbits. Based on the results of these studies, the test substance was not considered to be an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 15 December 2015 and 18 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Animals and Animal Husbandry
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.57 or 3.52 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 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 Envigo RMS (UK) Limited, 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 12 hours continuous light and 12 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.

Justification
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, no further testing was needed.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 g of the test item moistened sufficiently with 0.5 mL of distilled water.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
Two
Details on study design:
Procedure
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, 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.

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:
erythema score
Remarks:
75285 Male
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: No corrosive or irritant effects were observed
Irritation parameter:
erythema score
Remarks:
75286 Male
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: No corrosive or irritant effects were observed
Irritation parameter:
edema score
Remarks:
75285 Male
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: No corrosive or irritant effects were observed
Irritation parameter:
edema score
Remarks:
75286 Male
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: No corrosive or irritant effects were observed
Irritant / corrosive response data:
Dark blue colored staining, not preventing evaluation of skin responses, was noted at both treated skin sites at all observations. No evidence of skin irritation was noted during the study.
Other effects:
Body Weight
Both animals showed expected gain in body weight during the study.

Individual Skin Reactions

Skin Reaction

Observation Time
(following patch removal)

Individual Scores

Total

Rabbit Number and Sex

75285Male

75286Male

Erythema/Eschar Formation

Immediately

0STA

0STA

(0 )

1 Hour

0STA

0STA

( 0 )

24 Hours

0STA

0STA

0

48 Hours

0STA

0STA

( 0 )

72 Hours

0STA

0STA

0

Edema Formation

Immediately

0

0

( 0 )

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

STA =    Dark blue colored staining

Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 3

75285
Male

2.57

2.60

0.03

75286
Male

3.52

3.64

0.12

Interpretation of results:
GHS criteria not met
Conclusions:
The test item can be considered to be non-irritant.
Executive summary:

A skin irritation study was conducted according to OECD Guideline 404 and EU Method B.4. Two New Zealand White rabbits were exposed to a single 4‑hour, semi‑occluded application of the test item to the intact skin. There was no evidence of skin irritation recorded. The test item produced a primary irritation index of 0. Hence the test item can be considered to be non-irritant.

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 04 November 2014 and 06 November 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Test system:
human skin model
Remarks:
EPIDERM™ Reconstructed Human Epidermis Model
Source species:
human
Details on test system:
Supplier: MatTek Corporation, Ashland, MA, USA
Date received: 04 November 2014
EpiDermTM Tissues (0.5cm2) lot number: 19693
Assay Medium lot number: 103014 ZSD
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator overnight.
Control samples:
other: Yes
Duration of treatment / exposure:
3 minute and 60 minute exposure periods.
Duration of post-treatment incubation (if applicable):
3 hours
Controls:
yes
Irritation / corrosion parameter:
other: Relative Mean Viability %
Run / experiment:
Duration: 3 minutes
Value:
81.8
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not corrosive
Irritation / corrosion parameter:
other: Relative Mean Viability%
Run / experiment:
Duration 60 minutes
Value:
74.1
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not corrosive
Other effects / acceptance of results:
Direct MTT Reduction
The test item proved positive in the direct MTT reduction test (MTT solution turned blue). However the intrinsic color of the test item was blue and therefore the blue MTT solution could be due to direct MTT reduction or due to the coloration of the test item. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT or cause color interference and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues to determine any possible direct MTT or color interference.

The direct reduction or color interference caused by the test item relative to the negative control value:

3-Minutes; 0.141 (tkt) – 0.164 (ukt) / 2.061 (mean of negative control) = 0.0%

60-Minutes; 0.106 (tkt) – 0.161 (ukt) / 2.360 (mean of negative control) = 0.0%

tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues

After the 3 and 60 minute exposures the test item was considered to have caused no, 0.0%, interference due to direct MTT reduction or color interference. There was no reported presence of staining of the tissues following the test item exposure period either.

Test Item, Positive Control Item and Negative Control Item

Mean OD562 values and viabilities for the negative control, positive control and test item are given in Table 1.

 

The relative mean viability of the test item treated tissues was as follows:

 

60 minutes exposure

:

74.1 %

3 minutes exposure

:

81.8 %

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7.1% relative to the negative control treated tissues following the 3‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562 for the negative control treated tissues was 2.061 for the 3-Minute exposure period and 2.360 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

 

Exposure Period

(minutes)

OD562of individual tissues

Mean OD562of duplicate tissues (tvt)

 

Water-killed tissues

True Viability
tvt-(tkt-ukt)

Relative Mean % Viability

 

tkt

 

ukt

tkt-ukt

 

Negative Control

 

3 min

1.941

 

2.061

 

 

 

 

 

 

 

 

 

 

 

100*

 

2.180

 

 

60 min

2.258

 

2.360

 

 

 

 

 

100*

 

2.461

 

 

 

Positive Control

 

3 min

0.166

0.147

 

 

 

 

 

 

 

 

7.1

 

0.127

 

 

60 min

0.050

 

0.051

 

 

 

 

2.2

 

0.051

 

 

Test Item

 

3 min

1.572

 

1.685

 

 

 

0.141

 

0.164

 

0.000

 

 

1.685

81.8

 

1.798

 

60 min

1.418

 

1.748

 

0.106

 

0.161

 

0.000

 

1.748

74.1

 

2.078

* =     The mean viability of the negative control tissues is set at 100%

tvt =   treated viable tissues

tkt =   treated killed tissues

ukt =   untreated killed tissues

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

An in vitro skin corrosion study was conducted to evaluate the corrosivity potential of the test item using the EPIDERM™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item proved positive in the direct MTT reduction test (MTT solution turned blue). However the intrinsic color of the test item was blue and therefore the blue MTT solution could be due to direct MTT reduction or due to the coloration of the test item. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT or cause color interference and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues to determine any possible direct MTT or color interference. 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 each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data was presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues after 60 minutes exposure was 74.1% while it was found to be 81.8% after 3 minutes exposure. Hence, the test item was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 18 February 2015 and 23 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Test system:
human skin model
Remarks:
EPISKIN™ Reconstructed Human Epidermis Model
Source species:
human
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 17 February 2015
EpiSkinTM Tissues (0.38cm2) lot number: 15-EKIN-007
Maintenance Medium lot number: 15-MAIN3-007
Assay Medium lot number: 15-ESSC-007
Amount/concentration applied:
10 mg (26.3 mg/cm2)
Duration of treatment / exposure:
15 minutes
Number of replicates:
triplicates
Irritation / corrosion parameter:
other: other: Relative Mean Viability
Value:
92
Vehicle controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 Minutes. Remarks: The relative mean viability of the test item treated tissues was 92.0% after a 15 Minute exposure period and 42 hours post exposure incubation period.
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was 92.0% after a 15‑Minute exposure period and 42 hours post‑exposure incubation period. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

Direct MTT Reduction

The direct MTT reduction test was inconclusive due to the dark blue color of the test item. Therefore, an additional procedure using water-killed tissues was performed to assess for the possibility of direct MTT reduction. An additional procedure was also performed using viable tissues to assess for the possibility of color interference. The results of the additional procedures showed a negligible degree of interference due to possible direct reduction of MTT or color interference. It was therefore considered unnecessary to use the results of the additional procedures for quantitative correction of results and reporting purposes.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 12.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.0%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562for the negative control treated tissues was 0.987 and the standard deviation value of the viability was 20.0%. 

The standard deviation for the viability results of the negative control treated tissues exceeded the ≤18% assay acceptance criterion. The standard deviation for the three identically treated negative control treated tissues was 20%. The study was not repeated as there were no borderline results in the negative control, positive control or test item treated groups.

 

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.5%. The test item acceptance criterion was therefore satisfied.

Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562of tissues

Mean OD562of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.206

0.987

0.20

122.2

100*

20.0

0.822

83.3

0.933

94.5

Positive Control Item

0.104

0.122

0.05

10.5

12.3

5.0

0.084

8.5

0.177

17.9

Test Item

0.901

0.908

0.03

91.3

92.0

3.5

0.878

89.0

0.946

95.8

SD = Standard deviation

OD = Optical Density

* = The mean viability of the negative control tissues is set at 100 %

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was classified as non-irritant.
Executive summary:

In an in vitro study conducted according to OECD Guideline 439 and EU Method B.46, the skin irritation potential of the test item was evaluated using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 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‑labeled 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 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm. Data was presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 92.0% after the 15‑Minute exposure period and 42 hours post‑exposure incubation period. Hence, the test item was classified as non-irritant. 

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 19 January 2015 and 29 January 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
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., Leicestershire, UK. At the start of the study the animals weighed 2.54 or 3.01 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.

Justification
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, therefore, no further testing was needed.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
A volume of 0.1 mL of the test item, which was found to weigh approximately 92 mg (as measured by gently compacting the required volume into an adapted syringe) was used.
Duration of treatment / exposure:
The test item was not removed.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Two
Details on study design:
Test Item Formulation and Experimental Preparation
For the purpose of the study the test item was used as supplied. The absorption of the test item was not determined.

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 92 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.

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.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977). 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.


Interpretation of Results
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

The results were also interpreted according to the Globally Harmonized System of Classification and Labelling of Chemicals
Irritation parameter:
cornea opacity score
Remarks:
Male 74909
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: Not classified
Irritation parameter:
cornea opacity score
Remarks:
Male 74917
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: Not classified
Irritation parameter:
iris score
Remarks:
Male 74909
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Remarks on result:
other: Not classified
Irritation parameter:
iris score
Remarks:
Male 74917
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Remarks on result:
other: Not classified
Irritation parameter:
conjunctivae score
Remarks:
Male 74909
Basis:
mean
Remarks:
Redness
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Not classified
Irritation parameter:
conjunctivae score
Remarks:
Male 74917
Basis:
mean
Remarks:
Redness
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Not classified
Irritation parameter:
chemosis score
Remarks:
Male 74909
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0.66
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Not classified
Irritation parameter:
chemosis score
Remarks:
Male 74917
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0.66
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Not classified
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 1 hour
Score:
10
Max. score:
110
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 24 hours
Score:
9
Max. score:
110
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 48 hours
Score:
4
Max. score:
110
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Classed as a mild irritant according to a modified Kay and Calandra classification system; not classified according to the Globally Harmonized System of Classification and Labelling of Chemicals.
Irritant / corrosive response data:
Blue colored staining of the fur around the treated eye was noted in both animals during the study.

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 Hour observation.

Both treated eyes appeared normal at the 72 Hour observation.
Other effects:
Body Weight
No body weight gain was noted in one animal and the other animal showed expected gain in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

74909Male

74917Male

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

0

0

0

0

0

0

0

0

Score (D x 5)

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

2

2

1

0

2

2

1

0

B = Chemosis

1

1

1

0

1

1

1

0

C = Discharge

2Sf

1

0

0Sf

2Sf

2Sf

0Sf

0Sf

Score (A + B + C) x 2

10

8

4

0

10

10

4

0

Total Score

10

8

4

0

10

10

4

0

IPR=Initial pain reaction

Sf = Blue colored staining of the fur around the treated eye

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

74909Male

10

8

4

0

74917Male

10

10

4

0

Group Total

20

18

8

0

Group Mean Score

10.0

9.0

4.0

0.0

 

Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 3

74909 Male

3.01

3.01

0.00

74917 Male

2.54

2.56

0.02

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item can be considered as not-irritating to the eyes of rabbits.
Executive summary:

The irritancy potential of the test item to the eye of the New Zealand White rabbit was evaluated in a study conducted according to OECD Guideline 405 and EU Method B.5. A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation. The test item produced a maximum group mean score of 10.0 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. However, the test item does not meet the criteria for classification according to the CLP (Regulation 1272/2008) criteria. Hence, can be considered as non-irritating to the eyes of rabbits. 

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 10 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Species:
other: Eyes from adult cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
other: 0.9% w/v sodium chloride solution
Controls:
yes
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
90 minutes
Number of animals or in vitro replicates:
Three corneas were used
Details on study design:
Test Item Formulation and Experimental Preparation
For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preparation of Negative and Positive Control Items
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.


Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM.

A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.


Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken for the negative and positive controls and each cornea was visually observed.

For the test item treated corneas, after the normal rinsing step and refilling of the anterior chamber, some of the blue coloured test item was observed to be diffusing out of the cornea into the MEM when cornea 18 was being monitored for opacity. Therefore, after all the test item treated corneas had been rinsed they were placed into the incubator for 1 hour post rinsing to increase the chance of obtaining a correct opacity value with little interference from blue staining. After the additional 1 hour incubation, the MEM in the anterior and posterior chamber was replaced with fresh MEM. The opacity was measured and the condition of the corneas visually observed. The corneas commenced the permeability phase of the study once the opacity was recorded.


Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.


Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.

360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.


Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.


Evaluation of Results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.


Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.


Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.


In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.


Visual Observation
The condition of the cornea was visually assessed immediately after rinsing.
Irritation parameter:
cornea opacity score
Run / experiment:
mean of three experiments
Value:
10.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
In Vitro Irritancy Score
The in vitro irritancy scores are summarized as follows:

Test item in vitro irritancy score = 10.3
Negative control in vitro irritancy score = 2.4
Positive control in vitro irritancy score = 77.3

Blue staining of the test item treated corneas partly contributed to the IVIS score of 10.3. However, any interference caused by the presence of staining was considered not to have affected the final classification according to the prediction model (see discussion of report).
Other effects:
None specified

Corneal Epithelium Condition

The corneas treated with the test item were clear, however there was an area of staining on the cornea. The majority of the affected corneas were clear but the areas of blue staining were likely to be elevating the individual opacity scores.No staining was noted on cornea 18.

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 73.8 – 103.6. The positive control acceptance criterion was therefore satisfied.

 

The negative control gave opacity of ≤4.7 and permeability ≤0.085. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements Inclusive of Stained Test Item Treated Corneas

Treatment

Cornea Number

Opacity

Permeability (OD)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

1

4

3

 

0.068

 

 

9

1

2

1

 

0.045

 

 

17

1

2

1

 

0.043

 

 

 

 

 

1.7*

 

0.052¨

 

2.4

Positive Control

3

0

51

51

49.3

1.796

1.744

 

12

2

64

62

60.3

1.759

1.707

 

16

0

46

46

44.3

1.785

1.733

 

 

 

 

 

51.3·

 

1.728·

77.3

Test Item

18

1

5

4

2.3

0.178

0.126

 

19

4

14

10

8.3

0.186

0.134

 

20

1

15

14

12.3

0.320

0.268

 

 

 

 

 

7.7·

 

0.176·

10.3

OD= Optical density            * = Mean of the post-treatment -pre‑treatment values            ¨= Mean permeability                     ·= Mean corrected value

Individual and Mean Corneal Opacity and Permeability Measurements Discounting the Stained Test Item Treated Corneas

Treatment

Cornea Number

Opacity

Permeability (OD)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

1

4

3

 

0.068

 

 

9

1

2

1

 

0.045

 

 

17

1

2

1

 

0.043

 

 

 

 

 

1.7*

 

0.052¨

 

2.4

Positive Control

3

0

51

51

49.3

1.796

1.744

 

12

2

64

62

60.3

1.759

1.707

 

16

0

46

46

44.3

1.785

1.733

 

 

 

 

 

51.3·

 

1.728·

77.3

Test Item

18

1

5

4

2.3

0.178

0.126

 

19

4

Discounted value

-4

0.0

0.186

0.134

 

20

1

Discounted value

-1

0.0

0.320

0.268

 

 

 

 

 

0.8·

 

0.176·

3.4

OD= Optical density            * = Mean of the post-treatment -pre‑treatment values            ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment

Cornea Number

Observation

Post Treatment

Negative Control

1

Clear

9

Clear

17

Clear

Positive Control

3

Cloudy

12

Cloudy

16

Cloudy

Test Item

18

Clear

19

Clear*

20

Clear*

* = Area of staining on the cornea. The majority of the affected corneas were clear but the areas of blue staining were likely to be elevating the individual opacity score. No staining was noted on cornea 18.

 

Interpretation of results:
other: No prediction of eye irritation can be made.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.
Executive summary:

A Bovine Corneal Opacity and Permeability (BCOP) test was performed to evaluate the potential of the test item to cause eye damage. 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 Vitro Irritancy Score (IVIS).  The In Vitro irritancy scores for test item was calculated to be 10.3. Blue staining of the test item treated corneas partly contributed to the IVIS score of 10.3. However, any interference caused by the presence of staining was considered not to have affected the final classification according to the prediction model. Based on these findings, no prediction of eye irritation can be made. The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion/Irritation

The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. To this end, an in vitro skin corrosion study was conducted to evaluate the corrosivity potential of the test item using the EPIDERM™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item proved positive in the direct MTT reduction test (MTT solution turned blue). However the intrinsic colour of the test item was blue and therefore the blue MTT solution could be due to direct MTT reduction or due to the coloration of the test item. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT or cause colour interference and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues to determine any possible direct MTT or colour interference. 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 each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data was presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues after 60 minutes exposure was 74.1% while it was found to be 81.8% after 3 minutes exposure. Hence, the test item was considered to be non-corrosive to the skin.

 

Further to this, the skin irritation potential of the test item was assessed using the EPISKINreconstructed human epidermis model after a treatment period of 15 minutes followed by a postexposure incubation period of 42 hours. Classification of irritation potential is based upon relative mean tissue viability. The relative mean viability of the test item treated tissues was 92.0 %. Hence, the substance was considered to be non-irritant to the skin.

The skin irritation potential of the test item was also assessed using an in vivo method performed according to OECD Guideline 404. The test item produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin.

Based on the above studies, the substance was determined to be non-irritant to the skin.

Eye Irritation

Initially, a Bovine Corneal Opacity and Permeability (BCOP) test was performed to evaluate the potential of the test item to cause eye damage. 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 an In Vitro Irritancy Score (IVIS).  The In Vitro irritancy scores for test item was calculated to be 10.3. Blue staining of the test item treated corneas partly contributed to the IVIS score of 10.3. However, any interference caused by the presence of staining was considered not to have affected the final classification according to the prediction model. Based on these findings, no prediction of eye irritation can be made. The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

 

Further to this, the eye irritation potential of the test item was assessed according to OECD Guideline 405 using an in vivo method. A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation. The test item produced a maximum group mean score of 10.0 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. However, the test item does not meet the criteria for classification according to the CLP (Regulation 1272/2008) criteria. Hence, can be considered as non-irritating to the eyes of rabbits. 

Based on the above results, the substance can be considered not-irritating to the eyes.

Justification for classification or non-classification

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