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Diss Factsheets

Administrative data

Description of key information

Skin irritation, in vitro: non-irritating (mean relative viability 59.4%), OECD TG 439, 2015

Supporting study: Skin Irritation, in vivo (rat): mild transient irritation, OECD TG 402, 2015

Eye irritation, in vitro: non-irritating (ICE predictions: 2xI, 1xII combination 3 endpoints), OECD TG 438, 2015

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:
04-12-2014 to 09-03-2015
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2014; signature: May 2014
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:
EpiSkin RHE Model (Lot no.: 15-EKIN-009). 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
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.
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 item 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 item was classified according to remaining cell viability following exposure of the test item.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Value:
59.4
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 = 3.4% ; 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:

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: Concurrent controls within the OECD TG 439 guideline acceptance ranges. Therefore the test system performed adequately.

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

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.798

0.819

0.03

97.4

100*

3.3

0.810

98.9

0.849

103.7

Positive Control Item

0.049

0.051

0.004

6.0

6.2

0.5

0.056

6.8

0.048

5.9

Test Item

0.462

0.486

0.03

56.4

59.4

3.4

0.481

58.7

0.516

63.0

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 6.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.5%. The mean OD562 for the negative control treated tissues was 0.819 and the standard deviation value of the viability was 3.3%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.4%. All assay acceptance criteria were met.

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 considered to be not 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 item in accordance with GLP 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 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 59.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation between three replicates was 3.4%. The concurrent positive control relative mean viability was 6.2% and the standard deviation was 0.5%. The standard deviation in the negative control tissues was 3.3%. All assay acceptability criteria were met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08-07-2015 to 12-11-2015
Reliability:
2 (reliable with restrictions)
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'.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
note that for local effects only: the reshaving at day 8 is a deviation is suggestive of effects that are non-test item related, which limits the reliability for assessment.
Qualifier:
according to guideline
Guideline:
other: EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
yes
Remarks:
See above
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: June 2015; signature: September 2015
Species:
rat
Strain:
Wistar
Remarks:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: Males: 234 - 260 g ; Females: 201 - 212 g; the weight variation did not exceed ±20% of the mean weight for each sex.
- Fasting period before study: Not applicable
- Housing: suspended solid floor polypropylene cages furnished with woodflakes; the initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24 Hour exposure period and in groups of four, by sex, for the remainder of the study.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES:08-07-2015 to 18-08-2015
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg
- Concentration (if solution): Not applicable.
- Constant volume or concentration used: Dose volume was ca. 2.16 mL/kg. Dose level was adjusted by bodyweight.
Duration of treatment / exposure:
24 hours
Observation period:
Once daily during days 1-14. All abnormalities were recorded.
Number of animals:
5 per sex per dose (5 male/5 female)
Details on study design:
TEST SITE
- Area of exposure: the day before treatment the back and flanks were clipped free of hair. Dorsal area application.
- % coverage: Approximately 10% of total body surface
- Type of wrap if used: The area of application was covered piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self-adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item
- Time after start of exposure: 24 h

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : Clinical observations and mortality checks were conducted at approximately 0.5, 1, 3, and 5 hours and subsequently twice daily for days 2 to 15. Local effects were examined once daily days 1 to 14 after the completion of the 24-hour exposure period. Individual bodyweights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.

SCORING SYSTEM:
- Method of calculation: Full details on the scoring and criteria (appears consistent with Draize (1977) for Erythema and Edema formation) are given in the full study report.
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal #1
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #1
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal #2
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #2
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal #3
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #3
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal #4
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #4
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal #5
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks:
crust formation seen days 2 to 5
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #5
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks:
crust formation seen days 2 to 5
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal: #1
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #1
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal: #2
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #2
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal: #3
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #3
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal: #4
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #4
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
erythema score
Remarks:
mean
Basis:
animal: #5
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritation parameter:
edema score
Remarks:
mean
Basis:
animal #5
Remarks:
female
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 48/72/96h (24h exposure)
Irritant / corrosive response data:
- Dermal reactions: All indicated minimal (score = 0) erythema and edema from day 1 to day 14. Crust formation was seen in a single male at day 2 to 5'. All effects reversed by day 7. See 'other information on results incl. tables' for further information.
Other effects:
- Other adverse local effects: None. See 'other information on results incl. tables' for further information.
- Other adverse systemic effects: None.

Applicant assessment indicates: within local effects the test item following a 24 hour exposure to the skin (neat) at 2000 mg/kg bw dose level and semi-occlusive dressing caused only minimal irritation (score = 0) and isolated crust formation on days 2 to 5. All effects had reversed by day 7. The test item had minimal irritation to the skin.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study and under the Globally Harmonized Classification System of Classification and Labelling of Chemicals (GHS), the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight. The test item caused only minimal irritation (score = 0) on days 2 to 5 and all effects had fully reversed by day 7. The test item was not considered to be skin irritating.
Executive summary:

The study was performed according to OECD TG 402 and EU Method B.3 in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There were no signs of system toxicity. There was no mortality during the study. No signs of dermal irritation were noted. Crust formation was noted at the test site of one male 2 to 5 days after dosing. Animals showed expected gains in body weight, except for two females which showed no gain in body weight during the first week but expected gain in body weight during the second week. All animals gained weight during the study. There was no abnormalities on necropsy. Under the conditions of this study, the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight. The test item was not considered to be skin irritating.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-02-2015 to 17-04-2015
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
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2014; signature: May 2014
Species:
other: chicken
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): ca. 7 weeks , 1.5 to 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Extraction time and placing eyes in the superfusion chamber following enucleation was minimized (typically within 2 hours). Intact heads were stored at ambient temperature in perspex containers, humidified by towels moistened with isotonic saline during transport. For use on the same day. All eyes have to fall within the acceptance criteria used in the ICE guideline.
- Time interval prior to initiating testing: Same day (< 24 hours)
- indication of any existing defects or lesions in ocular tissue samples: No. Eyes that had a high baseline fluorescein staining score (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
- Indication of any antibiotics used: No.
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): 0.03 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.
Number of animals or in vitro replicates:
Triplicate (n=3) for test item and reference item ; duplicate (n=2) for negative control
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: sodium chloride solution 0.9% w/v

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 pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable. Samples were taken for possible histopathology where relevant.

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 item 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
Run / experiment:
mean (n=3)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean (n=3)
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
mean (n=3)
Value:
>= 4.24 - <= 6.74
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: Proficiency test item results are reported in the full study report.

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.

Table 1. Ocular reactions

 

Reaction

ICE Prediction

Test Item

 

 

Maximal mean score for corneal opacity

0.00

ICE Class I

Mean score of Fluorescein Retention

0.50

ICE Class I

Maximal corneal swelling

6.74%

ICE Class II

 

 

 

Positive Control Item

 

 

Maximal mean score for corneal opacity

3.00

ICE Class IV

Mean score of Fluorescein Retention

2.67

ICE Class IV

Maximal corneal swelling

71.41%

ICE Class IV

 

 

 

Negative Control Item

 

 

Maximal mean score for corneal opacity

0.25

ICE Class I

Mean score of Fluorescein Retention

0.00

ICE Class 1

Maximal corneal swelling

10.40%

ICE Class II

 

- Corneal Opacity Scores

No opacity or cloudiness of the cornea was noted in any replicate. Severe corneal opacity was noted in all positive control treated eyes. Slight opacity of the corneal was noted in one replicate the negative control treated eyes. No morphological effects were noted in the test item or negative control item treated eyes. Severe morphological effects (sloughing) were seen in all positive control eyes.

- Fluorescein Retention Scores

Slight fluorescein adhering was noted in all the test item treated eyes. Large areas of dense fluorescein staining over most of the cornea were noted in two positive control treated eyes. One positive control eye showed a dense layer of fluorescein staining in small areas of the cornea. No fluorescein retention was noted in the negative 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

1A

0

0

0

0

0

0

2A

0

0

0

0

0

0

3A

0

0

0

0

0

0

Mean

0.0

0.0

0.0

0.0

0.0

0.0

ICE Class

I

Fluorescein Retention

1A

 

0.5

 

 

 

 

2A

 

0.5

 

 

 

 

3A

 

0.5

 

 

 

 

Mean

 

0.5

 

 

 

 

ICE Class

I

Corneal Thickness (micro-meters)

1A

269

272

307

276

279

291

2A

283

300

301

310

306

268

3A

249

263

246

269

264

267

Mean

267.00

278.33

284.67

285.00

283.00

275.33

Mean Corneal Swelling (%)

 

4.24

6.62

6.74

5.99

3.12

ICE Class

II

ICE Classes Combined:

I, I, II

Classification:

UN GHS - No category

 

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.

Interpretation of results:
GHS criteria not met
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 item 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 0.0 (ICE Class I). The mean fluorescein retention was 0.5 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class II with maximal corneal swelling 6.74%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class 2xI, 1xII) 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, the test item is considered to not be irritating to the eye.

The applicant recalculated the scoring in accordance with the OECD TG 438 guideline whereby 3xI or 2xI, 1xII or 2xII, 1xI results in a UN GHS classification - no category prediction. Rather than a "non-ocular corrosive / severe irritant" conclusion indicated in previous editions of the OECD TG 438 guideline. The test item responses were within the range of the concurrent negative controls with the exception of Fluorescein Retention, which was consistently low level (score = 0.5) between all replicates. No replicate gave a positive indication of eye irritation. All acceptability criteria were met within the assay.

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, 2015 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP 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 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 59.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation between three replicates was 3.4%. The concurrent positive control relative mean viability was 6.2% and the standard deviation was 0.5%. The standard deviation in the negative control tissues was 3.3%. All assay acceptability criteria were met. Under the conditions of this study, the test item is considered to be not irritating to the skin.

Supporting study : In vivo, OECD TG 402, 2015 : The study was performed according to OECD TG 402 and EU Method B.3 in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There were no signs of system toxicity. There was no mortality during the study. No signs of dermal irritation were noted. Crust formation was noted at the test site of one male 2 to 5 days after dosing. Animals showed expected gains in body weight, except for two females which showed no gain in body weight during the first week but expected gain in body weight during the second week. All animals gained weight during the study. There was no abnormalities on necropsy. Under the conditions of this study, the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight. The test item was not considered to be skin irritating.

Applicant assessment indicates: within local effects the test item following a 24 hour exposure to the skin (neat) at 2000 mg/kg bw dose level and semi-occlusive dressing caused only minimal irritation (score = 0) and isolated crust formation on days 2 to 5. All effects had reversed by day 7. The test item had minimal irritation to the skin.

Eye Irritation:

Key study : In vivo, OECD TG 438, 2015 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item 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 0.0 (ICE Class I). The mean fluorescein retention was 0.5 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class II with maximal corneal swelling 6.74%. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class 2xI, 1xII) 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, the test item is considered to not be irritating to the eye.

The applicant recalculated the scoring in accordance with the OECD TG 438 guideline whereby 3xI or 2xI, 1xII or 2xII, 1xI results in a UN GHS classification - no category prediction. Rather than a "non-ocular corrosive / severe irritant" conclusion indicated in previous editions of the OECD TG 438 guideline. The test item responses were within the range of the concurrent negative controls with the exception of Fluorescein Retention, which was consistently low level (score = 0.5) between all replicates. No replicate gave a positive indication of eye irritation. All acceptability criteria were additionally met within the assay.

Respiratory Irritation:

Key study : In vivo, OECD TG 403, 2015 : 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. Two groups 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 were as follows: Group 1: 2.14 mg/L and Group 2: 6.43 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 2.41 μm and 77.7% and Group 2: 2.86 μm and 66.3%. The Geometric Standard Deviation was Group 1: 1.96 and Group 2: 2.23, respectively. There were no male or female mortalities in Group 1 or Group 2. In Group 1, all males and four female animals exhibited body weight losses on Day 1 post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. One female animal showed no body weight gain from Days 1 to 3 post-exposure, however, body weight gains were noted in all females during the remainder of the recovery period.. Within Group 2, all males and females exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted in all males/females during the remainder of the recovery period. In Group 1, One day after exposure, all animals exhibited increased respiratory rate, hunched posture and pilo-erection and one female animal also exhibited sneezing, which persisted for three days. All animals subsequently appeared normal on Day 5 post-exposure. In Group 2, on removal from the chamber all animals exhibited noisy respiration, there were frequent instances of increased respiratory rate and isolated occurrences of decreased respiratory rate. One hour post-exposure, all animals exhibited noisy respiration, all male animals exhibited increased respiratory rate and all females exhibited decreased respiratory rate. One day after exposure, all animals exhibited hunched posture and pilo-erection and there were frequent instances of increased respiratory rate. One male animal also exhibited decreased respiratory rate and noisy respiration. All males and females appeared normal from days 5 to 6 post-exposure. During necropsy in Group 1, dark patches on the lungs were noted in two males (out of five) and one female (out of five). In Group 2, abnormally red lungs or dark patches on the lungs were noted in two males (out of five) and two females (out of five) at necropsy. Under the conditions of this study, the inhalation LC50 (male/female) was > 6.43 mg/L within the RCCHan WIST rat.

Applicant assessment indicates that necropsy findings in the lower respiratory tract were observed in the minority of males/females. There were additionally no correlates in the upper respiratory tract. All males/females were gaining bodyweight and there were no clinical signs at the end of the recovery period, which together indicates that there is no evidence of 'functional impairment'. Therefore, GHS STOT-SE: category 3 respiratory irritant does not apply.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

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 and/or skin irritation testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. The substance does not demonstrate significant skin irritation potential necessary for classification and labelling within an available skin irritation in vitro assay (OECD TG 439) and supporting acute dermal toxicity study (OECD TG 402) at 2000 mg/kg bw dose level. The substance can cause transient mild irritation but which is not sufficient for classification and labelling.

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient irritating effects to the eye but which is not sufficient for classification. Based on the available ICE assay (OECD TG 438) predicting no irritation and/or on the basis of absence of significant skin irritation potential from available in vitro assays and dermal studies. The overall evidence is indicative of mild transient and reversible effects on the eye are to be expected, which are not sufficient for classification.

 

References:

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

2. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.2.7, July 2017)