Registration Dossier

Administrative data

Description of key information

Skin irritation, in vitro: Irritating, OECD TG 439, 2016

Eye irritation, in vivo: non-irritating, OECD TG 405, 2016

Supporting information: Eye irritation, in vitro: no prediction can be made, OECD TG 438, 2016

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:
2016
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: June 2015; signature: September 2015
Test system:
human skin model
Remarks:
EPISKIN TM Small Model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin RHE Model (Lot no.: 15-EKIN-050). 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. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post exposure incubation period was also determined, as a complimentary endpoint.
The EPISKIN 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
Duration of treatment / exposure:
Tissues were treated with the test material for 15 minutes and then washed with DPBS with Ca++ and Mg++ to remove residual test material.
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.
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups
Details on study design:
TEST SITE
- Area of exposure: 10 μl of the undiluted test substance was added topically into 12-well plates on top of the skin tissues. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Value:
15.2
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 = 3.3% ; Score in terms of percentage of negative control. (migrated information)
Irritation / corrosion parameter:
other: other: inflammatory mediator IL 1α release
Value:
225.677
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure. Reversibility: no data. Remarks: n = 3; SD = 48.491; Mean Concentration (pg/mL). (migrated information)
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:

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 (n=50): the mean OD of the positive control was 0.103; range 0.049 to 0.243 and the mean percentage viability was 12.2%; range 4.0% to 29.4%. In this same period the mean OD of the negative control was 0.854; range 0.629 – 1.245. The mean OD562 for the negative control treated tissues was imperceptibly outside the lower limit. However, this was within the OECD TG 439 guideline acceptance range and all other values obtained within the test fell within the historical range, therefore the test system performed adequately.
Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test substance

Item

OD562of tissues

Mean OD562 of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.608

0.626

0.022

97.1

100*

3.5

0.650

103.8

0.619

98.9

Positive Control Item

0.081

0.089

0.014

12.9

14.2

2.2

0.082

13.1

0.105

16.8

Test Item

0.100

0.095

0.020

16.0

15.2

3.3

0.113

18.1

0.073

11.7

OD = Optical Density

SD = Standard deviation

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

 

The relative mean tissue viability for the positive control treated tissues was 14.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2%. The mean OD562 for the negative control treated tissues was 0.626 and the standard deviation value of the viability was 3.5%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.3%. All assay acceptance criteria were met.

 

Furthermore, within the Historic Control Data (HCD) (n=50): the mean OD of the positive control was 0.103; range 0.049 to 0.243 and the mean percentage viability was 12.2%; range 4.0% to 29.4%. In this same period the mean OD of the negative control was 0.854; range 0.629 – 1.245. The mean OD562 for the negative control treated tissues was imperceptibly outside the lower limit. However, this was within the OECD TG 439 guideline acceptance range and all other values obtained within the test fell within the historical range, therefore the test system performed adequately.

 

The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 225.677 pg/mL.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test material is considered to be irritating to skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small 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 inflammatory mediator IL-1α 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 562 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 15.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The inflammatory mediator IL 1α in the retained culture medium was measured. The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 225.677 pg/mL. All assay acceptability criteria were met. Under the conditions of this study, the test substance is considered irritating to the skin and would be considered to meet the criteria under Regulation (EC) 1272/2008 for Skin Irritation Category 2: Irritant.

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.1.1 (as amended by Commission Regulation (EU) 2016/863) the in vitro skin corrosion (OECD TG 431) study does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. An available in vitro (OECD TG 439) skin irritation assay indicates that the substance is skin irritating category 2. Available data in other endpoints (such as skin sensitisation and/or acute dermal toxicity) indicates that the substance is not skin corrosive and a definitive conclusion on the classification can be made. Furthermore, 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. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.2, July 2017) the study does not need to be conducted.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
2016
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: June 2015; signature: September 2015
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: 3.85 - 3.89 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 (m/f). Testing was conducted in two and the response in those animals was such that exposure of a third would not affect classification of the test item, therefore, no further testing was needed under guidelines.
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.

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:
0.165
Max. score:
2
Reversibility:
fully reversible within: 48-hours
Remarks on result:
other: mean; n=2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1.5
Max. score:
3
Reversibility:
fully reversible within: 7-days
Remarks on result:
other: mean; n=2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
1.165
Max. score:
4
Reversibility:
fully reversible within: 7-days
Remarks on result:
other: mean; n=2
Irritant / corrosive response data:
No corneal effects were noted. Iridial inflammation was noted in both treated eyes 1 hour after treatment and persisted in one treated eye at the 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. All treated eyes appeared normal at the 7-day observation.

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

7 Days

1 Hour

24 Hours

48 Hours

72 Hours

7 Days

CORNEA

 

 

 

 

 

 

 

 

 

 

Degree of Opacity

0

0

0

0

0

0

0

0

0

0

Mean (24 – 72 h)

 

 

 

0

 

 

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

Area of Cornea Involved

0

0

0

0

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

IRIS

1

1

0

0

0

1

0

0

0

0

Mean (24 – 72 h)

 

 

 

0.33

 

 

 

 

0.00

 

 

 

 

 

 

 

 

 

 

 

 

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

Redness

2

2

2

1

0

2

2

1

1

0

Mean (24 – 72 h)

 

 

 

1.67

 

 

 

 

1.33 

 

 

 

 

 

 

 

 

 

 

 

 

Chemosis

2

2

1

1

0

2

2

1

0

0

Mean (24 – 72 h)

 

 

 

1.33

 

 

 

 

1.00

 

 

 

 

 

 

 

 

 

 

 

 

Discharge

2

2

0

0

0

2

1

0

0

0

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test material 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 material to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test material to the non-irrigated eye of two rabbits produced no corneal effects. Iridial inflammation was noted in both treated eyes 1 hour after treatment and persisted in one treated eye at the 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. Both treated eyes appeared normal at the 7-Day observation. Under the conditions of this study, the test substance 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:
2016
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 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2015; signature: September 2015
Species:
other: chicken
Strain:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
other: negative (0.9% w/v sodium chloride solution) and positive (benzalkonium chloride) controls were used in this study.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.
Observation period (in vivo):
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report.

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.

NEGATIVE CONTROL USED: Yes,.

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 ml ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL isotonic saline. Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical opachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 3.
- Mean maximum opacity score: See tables 1 to 3.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 3.

DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.
Irritation parameter:
cornea opacity score
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Irritation parameter:
fluorescein retention score
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
percent corneal swelling
Value:
17.65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

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: Not applicable. Applicant assessment of the data indicates that although the positive control were in general within the current historic control range (HCD) – the maximum corneal thickness (%) was below the current PC range. However, a clear positive response was seen in all individual PC eyes tested (across all categories: ICE class IV) and the test system functioned adequately.

For further information see table 1 and 2.

Table 1. Ocular reactions

 

Reaction

ICE Prediction

Test Item

 

 

Maximal mean score for corneal opacity

2.0

ICE Class III

Mean score of Fluorescein Retention

1.3

ICE Class II

Maximal corneal swelling

17.65%

ICE Class II

 

 

 

Positive Control Item

 

 

Maximal mean score for corneal opacity

4.0

ICE Class IV

Mean score of Fluorescein Retention

3.0

ICE Class IV

Maximal corneal swelling

32.66%

ICE Class IV

 

 

 

Negative Control Item

 

 

Maximal mean score for corneal opacity

0.0

ICE Class I

Mean score of Fluorescein Retention

0.5

ICE Class 1

Maximal corneal swelling

-1.45%

ICE Class I

 

- Corneal Opacity Scores

Easily discernible translucent corneal opacity was noted in all test item treated eyes. Corneal opacity over the entire area of the cornea was noted in all positive control treated eyes. No opacity of the corneal was noted in the negative control treated eyes. No morphological effects were noted in the test item or negative control item treated eyes and one positive control treated eye. Sloughing was noted in two positive control treated eyes.

- Fluorescein Retention Scores

Single cell staining or focal or confluent fluorescein staining was noted in the test item treated eyes. Very minor single cell fluorescein staining was noted in the negative control treated eyes. Applicant assessment indicates that this was not significant and the negative controls were clearly negative. Confluent large areas of fluorescein staining were noted in all positive control treated eyes.

 

Table 2. Individual scoring - test item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3A

0.5

1

2

2

2

2

6A

0

1

2

2

2

2

8A

0

2

2

2

2

2

Mean

0.2

1.3

2.0

2.0

2.0

2.0

ICE Class

III

Fluorescein Retention

3A

 

1

 

 

 

 

6A

 

1

 

 

 

 

8A

 

2

 

 

 

 

Mean

 

1.3

 

 

 

 

ICE Class

II

Corneal Thickness

3A

0.66

0.68

0.70

0.72

0.76

0.76

6A

0.68

0.68

0.72

0.72

0.76

0.80

8A

0.70

0.73

0.75

0.72

0.78

0.84

Mean

0.68

0.70

0.72

0.72

0.77

0.80

Mean Corneal Swelling (%)

 

2.45

6.37

5.88

12.75

17.65

ICE Class

II

ICE Classes Combined:

III, II, II

Classification:

No prediction for eye irritation can be made

 

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

 

Applicant assessment of the data indicates that although the positive control were in general within the current historic control range (HCD) – the maximum corneal thickness (%) was below the current PC range. However, a clear positive response was seen in all individual PC eyes tested (across all categories: ICE class IV) and the test system functioned adequately.

Interpretation of results:
other: no prediction can be made
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.
Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test material in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. Also to identify substances not requiring UN GHS classification. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 2.0 (ICE Class III). The mean fluorescein retention was 1.3 (ICE Class II) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class II with maximal corneal swelling 17.5%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

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

Respiratory irritation

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

Additional information

Skin Irritation:

Key study: in vitro, OECD TG 439, 2016 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small 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-labelled micro tubes and stored in a freezer for inflammatory mediator IL-1α 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 562 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 15.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The inflammatory mediator IL 1α in the retained culture medium was measured. The mean concentration of inflammatory mediator IL 1α in the culture medium retained from the test item treated tissues was 225.677 pg/mL. All assay acceptability criteria were met. Under the conditions of this study, the test substance is considered irritating to the skin and would be considered to meet the criteria under Regulation (EC) 1272/2008 for Skin Irritation Category 2: Irritant.

 

Eye Irritation:

Key study: In vivo, OECD TG 405, 2016 : The study was performed to OECD TG 405 and EU Method B.5 under GLP to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test material to the non-irrigated eye of two rabbits produced no corneal effects. Iridial inflammation was noted in both treated eyes 1 hour after treatment and persisted in one treated eye at the 24 Hour observation. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 and 72 Hour observations. Both treated eyes appeared normal at the 7-Day observation. Under the conditions of this study, the test substance is not considered to be irritating to the eye.

 

Key study: In vitro, OECD TG 438, 2016 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test material in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes remained untreated for control purposes. The mean corneal opacity was 2.0 (ICE Class III). The mean fluorescein retention was 1.3 (ICE Class II) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class II with maximal corneal swelling 17.5%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV) across all categories signifying that the test system performed adequately. Under the conditions of this study, no prediction for eye irritation can be made following assessment of the data for all endpoints.

 

Respiratory Irritation:

Key study: INHALATION: OECD TG 403, 2018 : The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. Following a sighting test, one group of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period. The mean achieved atmosphere concentrations was Group 1: 5.14 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 3.48 μm and 83.6%. The Geometric Standard Deviation was 2.43. There was no mortalities in Group 1. Common abnormalities noted during the study included decreased respiratory rate, ataxia, lethargy, noisy respiration, hunched posture, pilo-erection, sneezing, wet fur and fur stained by clear sticky test item. All animals recovered to appear normal from Days 8 to 11 post-exposure. All animals exhibited body weight losses on the first day post-exposure. Except for three males and one female which exhibited body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period. Dark and/or pale patches on the lungs and/or pale lungs were noted amongst eight animals at necropsy. No macroscopic abnormalities were detected in two males. Under the conditions of this study, the 4-hour inhalation LC50 (male/female) was greater than 5.14 mg/L within the RCCHan WIST rat. Under the conditions of this study, there were no indications of respiratory irritation.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for dermal irritation category 2: H315 

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. An available in vitro (OECD TG 439) skin irritation assay indicates that the substance is skin irritating category 2. Available data in other endpoints (such as skin sensitisation and/or acute dermal toxicity) indicates that the substance is not skin corrosive and a definitive conclusion on the classification can be made. Furthermore, 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.

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification based on the applicants recalculation of the mean scoring and evaluation of the results in three organisms demonstrating that the EU criteria had not been met. Effects in vivo on corneal opacity are non-existent and iritis and conjunctival effects are very low which fully reversed within 72 hours; the overall evidence is indicative of transient and reversible effects on the eye. The available in vitro assay (ICE, OECD TG 438) was unable to provide a prediction in relation to the potential for classification and labelling.

 

References:

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