Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion:

Corrosive to the skin (OECD 431, GLP, K, rel.1).

Eye irritation:

Severe eye damage, top-down approach: ICE: Category 1 (irreversible effects on the eye) (OECD 438, GLP, WoE, rel.1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-07 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 431 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)
Specific details on test material used for the study:
- Storage condition of test material: The test item was stored dry and in the dark in a closed aluminium bag at room temperature (20 ± 10 °C). On 23 February 2017 the aluminium bag was opened and the contents were split into single use aliquots. The test item aliquots were stored in amber jars flushed with nitrogen.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH up and down strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue batch number(s): EpiDermTM Tissues (0.63cm2) lot number 25803
- Production Date: 03 April 2017
- Shipping date: 03 April 2017
- Delivery date: 04 April 2017
- Date received: 04 April 2017
- Date used: 06 April 2017
- Assay Medium lot number: 033017TME
- Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 37 °C, in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing was performed using a wash bottle and was achieved by filling and emptying each tissue under a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test item
- Observable damage in the tissue due to washing: No visible damage to the rinsed tissues was noted following the rinsing step..
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, humidified atmosphere of 5% CO2.
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Filter bandwidth: filter band pass ± 10
- Linear OD range of spectrophotometer: 0.0 to 2.6 nm.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.
- Viability: OD (540-570 nm) = 1.7 ± 0.105 and 1.739 ± 0.134 for viable and non-viable tissues, respectively (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 5.36 hours and 6.71 hours for viable and no-viable tissues, respectively (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Long term antibiotic and antimycotic free culture)
- Reproducibility: For the previous 43 experiments conducted between August 2016 and February 2017 using this test method, the mean OD of the positive control was 0.079 ± 0.025 after 3 minutes of exposure and 0.068 ± 0.025 after 60 minutes of exposure. The mean percentage viability was 4.3 ± 1.0 after 3 minutes of exposure and 3.8 ± 1.0 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤15% relative to the negative control treated tissues after 1 hour exposure). In this same period the mean OD of the negative control was 1.845 ± 0.239 after 3 minutes of exposure and 1.810 ± 0.235 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.8 and ≤2.8 for every exposure time).
The historical control values given were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562nm and 570nm is not considered to impact upon the applicability of the historical comparison for this study.

NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : 2
- Method of calculation used: True viability = mean OD (treated viable tissues) - [OD (treated killed tissues)-OD (untreated killed tissues)]

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PR
EDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- If the test item is corrosive with the viability less than 25% after 3 min exposure: H314; Sub-category 1A
- If the test item is corrosive with the viability greater than or equal to 25% after 3 min exposure: H314; Sub-category 1A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of sterile distilled water was used as supplied.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied.
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Duplicate tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
73.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
4.6%
Remarks on result:
other: corrosive to the skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
4.2%
Remarks on result:
other: corrosive to the skin
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.542 for the 3 Minute exposure period and 1.589 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.2% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Historical data: Furthermore, in order to confirm the integrity of the test system a comparison was made with historical control data for negative and positive controls obtained by the laboratory in the previous six months.
The historical control values were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562 nm and 570 nm is not considered to impact upon the applicability of the historical comparison for this study. All values obtained were within the historical range of the testing laboratory.

Table 7.3.1/1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

Mean OD570 of individual tissues (tvt)

Mean OD570 of duplicate tissues

Corrected OD570 of tissues
(tvt-[tkt-ukt])

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.566

1.542

-

0.035

2.2

100*

1.517

60 Minutes

1.631

1.589

0.059

3.7

1.547

Positive Control

3 Minutes

0.081

0.071

-

0.014

N/A

4.6

0.061

60 Minutes

0.075

0.068

0.011

N/A

4.2

0.060

Test Item

3 Minutes

1.281

1.367

1.128

0.121

8.8

73.1

1.452

60 Minutes

0.507

0.475

0.080

0.046

9.7

5.0

0.442

 

3 minute exposure corrected mean OD570= 0.344 (tkt) – 0.105 (ukt) = 0.239

60 minute exposure corrected mean OD570= 0.521 (tkt) – 0.126 (ukt) = 0.395

3 minute exposure direct MTT reduction relative to the negative control = 15.5%
60 minute exposure direct MTT reduction relative to the negative control = 24.9%

Relative mean viability (%) = (Mean OD570 of test item / Mean OD570 of negative control) x 100

Coefficient of variation = Standard deviation / Mean OD570 of duplicate tissues

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

OD = optical density

N/A = not applicable

tvt =    treated viable tissues

tkt =    treated killed tissues

ukt = untreated killed tissues

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Remarks:
combination of category 1B-and-1C
Conclusions:
Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

Duplicate tissues were treated with 25 mg of the undiluted test item wetted with 25 µL of sterile water to increase tissue surface contact for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). 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 73.1 and 5.0 % after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 4.6 and 4.2 % after 3 and 60 minutes exposure, respectively. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to Regulation (EC) No 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin icorrosion endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

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:
04 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.
Qualifier:
according to
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day (4th September 2017).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. All eyes were placed in the superfusion chamber by 9.36 am (4th September 2017). Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None
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 g of test item (undiluted) was applied to the cornea
- For the purpose of this study the test item was used as supplied and was handled under safety lighting.
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32.0 ± 1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
0.03 mL of the negative control item, Sodium chloride 0.9% w/v, was applied to the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye.

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish.
- 0.03 g of test item was applied to the cornea and the test item remained in place for 10 seconds.

OBSERVATION PERIOD
- 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.

REMOVAL OF TEST SUBSTANCE
- The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

METHODS FOR MEASURED ENDPOINTS:
- 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.
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
[(corneal thickness at time (t) / corneal thickness at time = 0) / corneal thickness at time =0] x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
37.09
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Other effects / acceptance of results:
OCULAR REACTIONS
Corneal Opacity Scores: Complete corneal opacity; iris invisible was noted in all positive control treated eyes and all test item treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in all positive control treated eyes.
Fluorescein Retention Scores: Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes and all test item treated eyes. Very minor single staining was noted in both negative control treated eyes.

ACCEPTANCE OF RESULTS:
Acceptance criteria met for negative control: Yes; Maximal mean score for corneal opacity: 0.5; Mean score of Fluorescein Retention: 0.5; Maximal mean corneal swelling compared to time zero: 1.37%
Acceptance criteria met for positive control: Yes; Maximal mean score for corneal opacity: 4.0; Mean score of Fluorescein Retention: 3.0; Maximal mean corneal swelling compared to time zero: 40.47%
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Table 7.3.2/3: Individual Scores and Mean Scores for Corneal Effects – Test Item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3B

0.5

4

4

4

4

4

6B

0.5

4

4

4

4

4

8B

0

4

4

4

4

4

Mean

0.3

4.0

4.0

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

3B

 

3

 

 

 

 

6B

 

3

 

 

 

 

8B

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

3B

0.70

1.00

1.08

1.08

1.06

1.00

6B

0.70

0.78

0.86

0.84

0.86

0.90

8B

0.73

0.94

0.98

0.92

0.84

1.02

Mean

0.71

0.91

0.97

0.95

0.92

0.97

Mean Corneal Swelling (%)

 

27.70

37.09

33.33

29.58

37.09

ICE Class

IV

Corneal Epithelium Condition

3B

N

N

N

N

N

N

6B

N

N

N

N

N

N

8B

N

N

N

N

N

N

ICE Classes Combined:

3 x IV

Classification:

Category 1

N= Normal

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 

The test item was applied, as supplied, at 0.03 g onto the cornea of each of three enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 4.0, corresponding to the ICE class IV;

- mean score of fluorescein retention: 3.0, corresponding to the ICE class IV;

- maximal mean corneal swelling: 37.09% corresponding to the ICE class IV.

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, Imidazole, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye damage endpoint.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation/corrosion:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin corrosion/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?

NO

The substance is not stable when exposed the air of the atmosphere (and maybe to light and aqueous medium) therefore its biological properties are difficult to anticipate.

Existing human data

2

Are there adequate existing human data which provide evidence that the substance is a corrosive
or irritant?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

YES

At the initiation of the dossier, no relevant in vivo study was available. However the substance is considered as irritating to the skin in an EFSA opinion (EFSA Journal 2012;10(7):2786).

Existing data from general toxicity studies via the dermal route and from sensitisation studies

4a

Is the substance classified as acutely toxic by the dermal route (Category 1)?

NO

 

4b

Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?

NO

 

4c

Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated
exposure?

NO

LLNA available: tested only up to 40%. No sign of irritation or
corrosion leading ot C&L.
NB: In general, irritation data from the Local Lymph Node Assay are not usable. The test substance is applied to the dorsum of the ear by open topical application

Existing/new (Q)SAR data and read
-across

5a

Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion
potential of the substance?

NO

No alert using Toxtree (v2.6.6)

5b

Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?

NO

 

Existing in vitro data

6a

Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met

NO

(at the initiation of the dossier, no test was available)

6b

Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted
in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are
met.

NO

(at the initiation of the dossier, no test was available)

6c

Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?

NO

 

Weight-of- Evidence analysis

7

The “elements” described above may be arranged as appropriate. Taking all available existing and
relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?

NO

Due to the inconsistency of the data as well as the instability of the substance when exposed the air of the atmosphere (and maybe to light and aqueous medium), a new in vitro test is required. A top-down approach has been chosen based on the data set.

New in vitro test for corrosivity

8

Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

YES

Following the top-down approach, an in vitro test for corrosion (OECD TG 431) was performed. Mean tissues viability = 73.1% after 3minutes, 5.0 % after 1 hour <=> Corrosive to skin.
No further test needed to conclude.

New in vitro test for irritation

9

Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

 

New in vivo test for corrosion/irritation

10

To be used only as a last resort

NO

 

The in vitro skin corrosion study (Envigo, 2017, Rel.1) was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model. The relative mean viability of the test item treated tissues was 73.1 and 5.0% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 4.6% and 4.2% after 3 and 60 minutes exposure, respectively. The quality criteria required for acceptance of results in the test were satisfied. With a tissue viability < 15% after 60 minutes of exposure, the test material was considered to be corrosive to skin.

Eye irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the eye damage/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Conclusion of the information strategy on skin corrosion/irritation

0

Is the substance classified as a skin corrosive?

YES

New in vitro test for corrosion (OECD TG 431) <=> Corrosive to skin. But a false positive result was suspected therefore more data were needed to conclude.

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?

NO

The substance is not stable when exposed the air of the atmosphere (and maybe to light and aqueous medium) therefore its biological properties are difficult to anticipate.

Existing human data

2

Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

At the initiation of the dossier, no relevant in vivo study was available. However the substance is considered as irritating to the eyes in an EFSA opinion (EFSA Journal 2012;10(7):2786).

Existing/new (Q)SAR data and read-across

4

Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?

NO

No alert using Toxtree (v2.6.6)

Existing in vitro data

5a

Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

(at the initiation of the dossier, no test was available)

5b

Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?

NO

 

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?

NO

Due to the inconsistency of the data as well as the instability of the substance when exposed the air of the atmosphere (and maybe to lgiht and aqueous medium), a new in vitro test is required. A top-down approach has been chosen based on the data set.

New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)

7a

Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.

YES

Following the top-down approach, an in vitro test for corrosion (OECD TG 438) was performed. Combination of the 3 endpoints is 3xIV <=> Corrosive to eyes.

7b

Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?

NO

 

New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)

8b

Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?

NO

 

The substance was shown to be corrosive to the skin in the new in vitro skin corrosion EpiDerm test, therefore the substance was expected to be able to induce serious eye damage. However, based on available data, a false positive results was suspected for the EpiDerm test. Therefore, a new ICE assay (Envigo, 2017, Rel.1) was conducted according to the OECD guideline No. 438 and in compliance with GLP. The ocular reactions observed in eyes treated with the test item were: maximal mean score of corneal opacity: 4.0, corresponding to the ICE class IV; mean score of fluorescein retention: 3.0, corresponding to the ICE class IV; maximal mean corneal swelling: 37.09% corresponding to the ICE class IV. The combination of the three endpoints for the test item was 3 x IV. The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, Imidazole, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

The test item was therefore considered as "Corrosive/Severe irritant" to the eye.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the substance should be classified as skin corrosive sub-category 1B (H314 “Causes severe skin burns and eye damage) and eye damage Category 1 (H318: "Causes serious eye damage) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available regarding respiratory irritation. In general, a classification for corrosivity is considered to implicitly cover the potential to cause respiratory tract irritation and so the additional Category 3 is considered to be superfluous. However, such substance has to be supplementary labelled with EUH071 if there is a possibility of exposure via inhalation taking into consideration the saturated vapour concentration and the possibility of exposure to particles or droplets of inhalable size. It is also strongly recommended to apply the precautionary statement P260: "Do not breathe dust/fume/gas/mist/vapours/spray."