Registration Dossier

Administrative data

Description of key information

Skin irritation: non-irritating, OECD TG 404, 2018

Skin irritation: non-irritating (15 minute/42 hour: relative mean viability = 86.9%), OECD TG 439, 2018

Eye irritation: non-rritating, OECD TG 405, 2018

Eye irritation: not-irritating (IVIS = 0.2 and IVIS < 3.0), OECD TG 437, 2018

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-04-2018 to 16-04-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Test system:
human skin model
Remarks:
EPISKIN TM Small Model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 18-EKIN-015
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 10-04-2018 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 11-04-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours. Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. The tissues were then processed for MTT-loading and 3 hour incubation at 37 °C, 5% CO2.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 5.6% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 4.0%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 0.787 and the standard deviation of viability was 11.9%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 5.0%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
EpiSkin RHE Model (Lot no.: 15-EKIN-015). The principle of the assay is 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. to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. As a complimentary endpoint the concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Application of test item and rinsing:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. Following 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl ; Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Concentration (if solution): Tested as supplied (undiluted).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl ; The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
- Concentration (if solution): 5%
Duration of treatment / exposure:
Tissues were treated with the test item for 15 minutes and then washed with DPBS with Ca++ and Mg++ to remove residual test item.
Positive control: SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C. Then 3 hours incubation prior to and/or MTT loading and Formazan extraction.
Number of replicates:
Triplicate (n=3) ; treatment and concurrent negative control and positive control groups
Irritation / corrosion parameter:
other:
Value:
86.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3; SD = 5.0% ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item

OD570 of tissues

Mean OD570 of triplicate tissues

± SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.872

0.787

0.093

110.8

100*

11.9

0.687

87.3

0.803

102.0

Positive Control Item

0.080

0.044

0.031

10.2

5.6

4.0

0.029

3.7

0.024

3.0

Test Item

0.645

0.684

0.040

82.0

86.9

5.0

0.724

92.0

0.682

86.7

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). 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-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 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are 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 86.9% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.0%. The mean OD570 for the negative control treated tissues was 0.787 and the standard deviation value of the viability was 11.9%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 5.6% relative to the negative control treated tissues and the standard deviation value of the viability was 4.0%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-10-2018 to 30-10-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
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
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: recognised animal supplier
- Age at study initiation: 12 - 52 weeks
- Weight at study initiation: 3.010 or 3.090 kg
- Housing: Individually housed in suspended cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod: 12 hours light / 12 hours dark
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Test material was used as supplied.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours (initial observation); additional observations are made on Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).
Number of animals:
2; following the guideline sequential testing approach
Details on study design:
TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive (2.5 cm x 2.5 cm cotton gauze patch secured with surgical adhesive tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema _________________________________________________________________________0
Very slight erythema (barely perceptible) ________________________________________________1
Well-defined erythema ________________________________________________________________2
Moderate to severe erythema __________________________________________________________3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ________________4

Oedema Formation
No oedema __________________________________________________________________________0
Very slight oedema (barely perceptible) _________________________________________________1
Slight oedema (edges of area well-defined by definite raising) _____________________________2
Moderate oedema (raised approximately 1 millimetre) ____________________________________3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) ___4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 1h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: mean score (n=2)
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: mean score (n=2)
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 1h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: mean score (n=2)
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: mean score (n=2)
Irritant / corrosive response data:
Very slight erythema (score = 1) and very slight to slight edema (score = 1 to 2) were noted immediately, at 1 hour observation at treated skin sites. At the 24, 48 and 72 hours observations: well defined erythema (score = 2) and very slight edema (score = 1) were observed. All effects reversed by the 7 day observation.
Other effects:
- Other adverse local effects: None reported.
- Other adverse systemic effects: One female gained bodyweight during the study (+0.142 g) and one female had a body weight loss (-0.030 g). Applicant assessment indicates that the bodyweight loss was very minor and there were no clinical sign correlations reported.

Table 1. Individual skin reactions

Skin Reaction

Observation Time
(following patch removal)

Individual Scores

Number and Sex

#1 (Female)

#2 (Female)

Erythema/Eschar Formation

Immediately

1

1

1 Hour

1

1

24 Hours

2

2

48 Hours

2

2

72 Hours

2

2

7 Days

0

0

14 Days

-

-

Edema Formation

Immediately

2

1

1 Hour

1

1

24 Hours

1

1

48 Hours

1

1

72 Hours

1

1

7 Days

0

0

14 Days

-

-

 

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 6.0 ; mean score = 2.0

2: total = 6.0; mean score = 2.0

Edema Formation:

1: total = 3.0; mean score = 1.0

2. total = 3.0; mean score = 1.0

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item is not considered to be irritating.
Executive summary:

The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 under GLP to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 and 14 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight to well-defined erythema and very slight to slight edema. Treated skin sites appeared normal at the 7-Day observation. No corrosive effects were noted. No other skin reactions noted. Mean scores for following grading at 24, 48 and 72h were 2.0 in erythema and eschar and 1.0 in edema scoring criteria. At day 7, mean scores were zero for erythema and edema, respectively. Under the conditions of the study, the item is not considered to be a skin 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:
29-10-2018 to 09-11-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese MAFF, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 12-52 weeks old
- Weight at study initiation: 2.990 - 3.052 kg
- Housing: individually housed in suspended metal cages; with environment enrichment
- Diet: certified rabbit food ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
A volume of 0.1 mL of the test material, 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 material, and then released. The left eye remained untreated and was used for control purposes.
Observation period (in vivo):
Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test substance, according to numerical evaluation.
Number of animals or in vitro replicates:
2 (1 m / 1 f). Testing was conducted sequentially. Number of replcates in accordance with the relevant OECD guideline.
Details on study design:
The study was performed in a stepwise manner and was started by treatment of a single rabbit. The other animals were treated in a similar manner after considering the degree of eye irritation observed in the first and/or second animal. As applicable.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The irritation was assessed according to Draize (1977) numerical scoring system. At each observation period, the highest scores given were recorded. Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: mean; n=2
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
other: mean; n=2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72-hours
Remarks on result:
other: mean; n=2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 0.3 - <= 0.7
Max. score:
4
Reversibility:
fully reversible within: 72-hours
Remarks on result:
other: mean; n=2
Irritant / corrosive response data:
No corneal effects were noted. No iridial inflammation was noted. Moderate conjunctival irritation was noted in treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations. All treated eyes appeared normal at the 72 hours.
Other effects:
- Lesions and clinical observations: None reported.
- Ophthalmoscopic findings: Not applicable.
- Histopathological findings: Not applicable.
- Effects of rinsing or washing: Not applicable.
- Other observations: All females gained bodyweight during the study.

Table 1. Individual scores and mean scores for 24, 48 and 72 hours

Organism number

1

2

Time After Treatment

1 Hour

24 Hours

48 Hours

72 Hours

1 Hour

24 Hours

48 Hours

72 Hours

CORNEA

 

 

 

 

 

 

 

 

Degree of Opacity

0

0

0

0

0

0

0

0

Mean (24 – 72 h)

 

 

 

0.0

 

 

 

0.0

 

 

 

 

 

 

 

 

 

IRIS

0

0

0

0

0

0

0

0

Mean (24 – 72 h)

 

 

 

0.0

 

 

 

0.0

 

 

 

 

 

 

 

 

 

CONJUNCTIVAE

 

 

 

 

 

 

 

 

Redness

2

1

1

0

2

1

1

0

Mean (24 – 72 h)

 

 

 

0.7

 

 

 

0.7

 

 

 

 

 

 

 

 

 

Chemosis

1

1

1

0

1

1

0

0

Mean (24 – 72 h)

 

 

 

0.7

 

 

 

0.3

 

 

 

 

 

 

 

 

 

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not irritating to the eye.
Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test item to the non-irrigated eye of two rabbits produced no corneal and iridial effects. Moderate conjunctival irritation was noted in treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations. All treated eyes appeared normal at the 72 hours. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-09-2018 to 11-02-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier.
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Post-excision, placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics over ice packs.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL during transport.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for ca. 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: 0.9% w/v Sodium chloride solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol; > 99.8% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. Furthermore, following the final 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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD492 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: 1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean of for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 65.1. ACTUAL: PC IVIS = 38.8
2. sodium chloride 0.9% w/v solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤2.30 and for permeability ≤0.41. ACTUAL: NC IVIS = 2.0, opacity ≤ 2.0 and permeability ≤ 0.003.

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

2

4

2

5

1

-

0.003

-

-

3

6

4

9

3

-

0.000

-

-

4

3

2

5

2

-

0.003

-

-

-

-

-

-

2.0 #1

-

0.002 #2

-

2.0

Positive Control

5

4

30

31

27

25.0

0.776

0.774

-

6

5

34

33

28

26.0

0.570

0.568

-

7

3

35

37

34

32.0

0.877

0.875

-

-

-

-

-

-

27.7 #3

-

0.739 #3

38.8

Test Item

9

6

6

7

1

0.0

0.035

0.033

-

10

3

3

4

1

0.0

0.009

0.007

-

11

3

5

4

1

0.0

0.007

0.005

-

-

-

-

-

-

0.0 #3

-

0.015 #3

0.2

OD = Optical Density

#1 = Mean of post-incubation – pre-treatment values

#2 = Mean permeability

#3 = Mean corrected value

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model.
Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 under GLP to assess the eye irritancy potential of the test item in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test item was tested through topical application for 10 ± 1 minutes and post-exposure incubation for 120 minutes. The negative control response for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 38.8 and was within the historical positive control data range (29.6 to 65.1). The test item resulted in a mean in vitro irritancy score (IVIS) of 0.2 after 10 minutes of treatment. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model. Under the conditions of this study the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

Key study : In vitro, OECD TG 439, 2018 : The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). 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-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 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are 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 86.9% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.0%. The mean OD570 for the negative control treated tissues was 0.787 and the standard deviation value of the viability was 11.9%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 5.6% relative to the negative control treated tissues and the standard deviation value of the viability was 4.0%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

 

Key study : In vivo, OECD TG 404, 2018 : The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 under GLP to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, 72 hours and 7 and 14 days, respectively. A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight to well-defined erythema and very slight to slight edema. Treated skin sites appeared normal at the 7-Day observation. No corrosive effects were noted. No other skin reactions noted. Mean scores for following grading at 24, 48 and 72h were 2.0 in erythema and eschar and 1.0 in edema scoring criteria. At day 7, mean scores were zero for erythema and edema, respectively. Under the conditions of the study, the item is not considered to be a skin irritant.

 

Eye Irritation:

Key study : In vitro, OECD TG 437, 2018 : The study was performed according to OECD TG 437 and EU Method B.47 under GLP to assess the eye irritancy potential of the test item in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test item was tested through topical application for 10 ± 1 minutes and post-exposure incubation for 120 minutes. The negative control response for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol), was 38.8 and was within the historical positive control data range (29.6 to 65.1). The test item resulted in a mean in vitro irritancy score (IVIS) of 0.2 after 10 minutes of treatment. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model. Under the conditions of this study the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model.

Key study : In vivo, OECD TG 405, 2018 : The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. A single application of the test item to the non-irrigated eye of two rabbits produced no corneal and iridial effects. Moderate conjunctival irritation was noted in treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations. All treated eyes appeared normal at the 72 hours. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation

 

For skin irritation, further in vitro skin corrosion testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. In accordance with section 1.2 of REACH Regulation (EC) No. 1907/2006 Annex XI the weight of evidence indicates that the substance is not skin corrosive and therefore in vitro testing may be omitted. An available in vitro (OECD TG 439, 2018) skin irritation assay indicates that the substance is not irritating to the skin (based on > 60% MTT viability). An available in vivo irritation study (rabbit: OECD TG 404, 2018) indicates the erythema and edema scores were not sufficient for classification (< 2.3 mean scoring 24 to 72 hours in 2/3 organisms) and are suggestive of Globally Harmonised System (revision 4): Skin Irritation category 3 - mild irritant.

 

For eye irritation, the available in vitro assay (BCOP, OECD TG 437, 2018) was suggestive of a not irritating to the eye (IVIS < 3.0). Based on an available in vivo study (OECD TG 405, 2018) where the mean scoring and evaluation of the results where two of three organisms demonstrated that the EU criteria had not been met: the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification. Effects in vivo on corneal opacity and iritis are non-existent and conjunctival effects are low to moderate which fully reversed within 72 hours; the overall evidence is indicative of transient and reversible effects on the eye.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)