Methyl 4-(acetylamino)-5-bromo-o-anisate

Endpoint summary

Description of key information

Skin corrosion:

Key study: OECD 431 and EU B.40bis. GLP study. The mean corrected percent viability of the epidermis skins treated with the test item were 112.46% and 102.55% (considered as 100%) after 3 minutes and 1 hour exposure respectively, versus 29.45% and 0.72% in the positive control (potassium hydroxide 8N). Therefore, the test item is non corrosive to the skin.

Skin irritation:

Key study: OECD 439 and EU B.46. GLP study. The mean corrected percent viability of the treated tissues was 92.3 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to the skin.

Eye irritation:

Key study: OECD 438 and EU B.48. GLP study. The combination of the three endpoints for the test item was 3 x I. Therefore, the test item does not require classification for eye irritation and serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Reconstructed Human Epidermis Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 4, 2017 - April 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-039
- Delivery date: April 4, 2017
- Expiration date: April 10, 2017
- Date of initiation of testing: April 4, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: E1x800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.4 (CV = 2.8%) specification OD > 0.7. Historical negative control mean OD range = 0.834 - 1.574.
- Barrier function: 4.8 h (Specification 0h < ET50< 10h)
- Morphology: 5.5 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of test item (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 47 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 92.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean corrected percent viability of the treated tissues was 92.3 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: A yellow nonhomogenous suspension was observed after 3 hours of incubation between 36.9°C and 37.8°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A colorless solution was obtained after 3 hours of incubation between 36.1°C and 37.4°C, 5% CO2.
In isopropanol: A colorless solution was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY:yes, A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.Summary of proficiency chemicals testing according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the standard deviation of the negative control group was 4.3%, the acceptablility criteria is SD ≤ 18%, OD mean of negative control is 1.077 and the acceptability criteria is OD >0.6 x<1.5
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The results were expressed as a viability percentage compared with the negative control:

 

Viability %= OD_{test item}/ OD_{negative control}x 100

 

The OD values obtained for each test simple were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.

 

Table 1. Individual and average values of OD after 42 minutes exposure

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	0.965	1.026	1.077	95.2	100.0	4.3
		1.052
		1.060
	2	1.109	1.117		103.7
		1.222
		1.121
	3	1.090	1.089		101.1
		1.076
		1.102
Positive control	4	0.012	0.012	0.013	1.1	1.2	0.1	Irritant
		0.012
		0.012
	5	0.013	0.013		1.2
		0.013
		0.013
	6	0.013	0.013		1.2
		0.012
		0.013
Test item PH-17/0101	7	0.856	0.910	0.994	84.5	92.3	6.8	Non irritant
		0.950
		0.924
	8	1.044	1.035		96.1
		1.031
		1.029
	9	1.029	1.038		96.3
		1.027
		1.057

# mean of 3 values (triplicate of the same extract)

OD: optival density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD≤18%.

The acceptance criteria were met.

 

Notes:

-         If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

-         If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

 

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 92.3 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to the skin.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). This test was performed according to OECD TG 439 and EU method B.46, following GLP. The test item was applied, as supplied, at the dose of 16 mg to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 47 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.The mean corrected percent viability of the treated tissues was 92.3%, versus 1.2 % in the positive control (5% Sodium Dodecyl Sulfate). The mean corrected percent viability of the treated tissues was 92.3 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 3, 2017 - March 8, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Test system:

human skin model

Remarks:

epiCS model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS, Cell Systems
- Tissue batch number(s): 100-AG0420-1
- Production date: 06/03/ 2017
- Shipping date: 07/03/2017
- Delivery date: 07/03/2017
- Date of initiation of testing: 08/03/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.6ºC-37.9ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed 20 times with 1 mL of DPBD
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: E1x800 absorbance microplate reader BioTek
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: the linearity range of optical density measured is validated for an optical density between 0 and 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control mean OD = 0.901 (specification ≥ 0.8 ≤ 2.8). Historical negative control mean OD range = 0.934 – 1.511
- Barrier function: 58,3% (Specification >50%)
- Morphology: 5 cornified layers, 4 vital cell layers (pass)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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%.

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

3 minutes at RT + 1 hour at 37ºC

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

ca. 112.46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

ca. 102.55

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non corrosive

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes. Demonstrated with some of the substances fro the updated list of the 2015 revision of TG OECD 431 (the remaining proficiency chemicals will be evaluated progressively). The rest of the proficiency substances are from the list of the 2004 version of TG OECD 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD of the tissues replicates is between 0.8 and 2.08 in both times of exposure.
- Acceptance criteria met for positive control: Yes, the mean viability of the tissue replicates exposed for 3 minutes or 1 hour is < 20%.
- Acceptance criteria met for variability between replicate measurements: Yes, is in the range 20-100% viability, the OD is ≥ 0.3 and the difference of viability between the two tissue replicates is <30%.

The results were expressed as a viability percentage compared with the negative control:

 

Viability %= OD_{test item}/ OD_{negative control}x 100

 

For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, were reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

 

Table 1. Individual and average values of OD after 3 minutes exposure

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	1	0.909	0.909	0.875	103.95	100.0	7.9
		0.897
		0.920
	2	0.820	0.840		96.05
		0.836
		0.862
Positive control	3	0.230	0.240	0.258	27.44	29.45	4.0
		0.238
		0.250
	4	0.270	0.275		31.45
		0.275
		0.279
Test item PH-17/0101	9	1.018	0.968	0.984	110.69	112.46	3.5
		0.932
		0.954
	10	1.005	0.999		114.24
		0.996
		0.995

# mean of 3 values

OD: optical density

The optical density was measured after a 1:3 dilution of the formazan extracts in isopropanol.

Acceptability criteria:

-         Negative control: mean OD of the tissue replicates should be≥0.8 and≤2.8 for epiCS® model, for every exposure time.

-         Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be <20% for epiCS model.

-         Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

 

  

Table 2. Individual and average values of OD after 1 hour exposure

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	11	0.822	0.826	0.901	91.68	100.0	16.6
		0.825
		0.830
	12	0.983	0.976		108.32
		0.966
		0.979
Positive control	13	0.004	0.004	0.007	0.44	0.72	0.6
		0.004
		0.004
	14	0.005	0.009		1.00
		0.007
		0.014
Test item PH-17/0101	19	0.836	0.827	0.924	91.79	102.55	21.5
		0.811
		0.832
	20	1.041	1.021		113.32
		1.011
		1.009

# mean of 3 values

OD: optical density

The optical density was measured after a 1:3 dilution of the formazan extracts in isopropanol.

Acceptability criteria:

-         Negative control: mean OD of the tissue replicates should be≥0.8 and≤2.8 for epiCS® model, for every exposure time.

-         Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be <20% for epiCS model.

-         Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

 

 

Table 3. Conclusion after 3 minutes and after 1 hour exposure.

 

	Mean viability (%)
	3 min exposure	1 hour exposure	Conclusion
Positive control	29.45	0.72	Corrosive sub-category 1A
Test item PH-17/0101	112.46	102.55	Non Corrosive

Note

The item is to be classified as non-corrosive:

-         If the viability after 3 minutes exposure is grater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

The item is to be classified as corrosive:

-         If the viability after 3 minutes exposure is strictly 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 strictly less than 15%.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the epidermis skins treated with the test item were 112.46% and 102.55% (considered as 100%) after 3 minutes and 1 hour exposure respectively, versus 29.45% and 0.72% in the positive control (potassium hydroxide 8N). Therefore, the test item is non corrosive to the skin.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical application on in vitro human reconstructed epidermis (epiCS®, CellSystems). This test was performed according to OECD TG 431 and EU method B.40bis, following GLP. The test item was applied, as supplied, at the dose of 25 mg to 2 Human skin model surfaces previously moistered with 25 µL of DPBS during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the epidermis skins treated with the test item were 112.46% and 102.55% (considered as 100%) after 3 minutes and 1 hour exposure respectively, versus 29.45% and 0.72% in the positive control (potassium hydroxide 8N). Therefore, the test item has to be considered as Non-corrosive to skin, corresponding to UN GHS No Category.

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:

March 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Etablissement Brun, 33820 Etauliers, France), the chickens were killed for human consumption.
- Characteristics of donor animals: spring chickens (approximately 7 weeks old, 1.5-2.5 kg)
- Storage, temperature and transport conditions of ocular tissue: Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The head collection was at 8:15 am and arrived at 9:45 am at the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: No
- 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): 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the eyes: the eyelids were carefully excised, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
Selection of the eyes: The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3ºC and 32.6ºC. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with, (i), a fluorescein retention score of > 0.5, (ii) corneal opacity < 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
The eyes examined and approved were incubated between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e. time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 µL Physiological saline (Dutscher Batch No. 3011922) (1 replicate)

POSITIVE CONTROL USED: 30 mg of Sodium hydroxide (Sigma-Batch No. MKBP7805V) (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied, as supplied, for 10 seconds

OBSERVATION PERIOD
Pretreatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was measured using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. It was calculated using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: It was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: It was measured using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I, the slit-width was set at 9 1/2, equalling 0.095 mm. Themean percentage of corneal swelling was calculated for all observation time points.
- Macroscopic morphological damage to the surface: Include "pitting" of the corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t - corneal thickness at time=0/ corneal thickness at time=0) x 100
- Mean maximum opacity score: 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 slighly 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: 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: please specify if the decision criteria as indicated in the TG was used.
All endpoints were evaluated separately to generate an ICE class for each endpoint and then combined to generate an Irritancy Classification for the test item. The ICE classes were assigned based on a predetermined range.
ICE classification criteria for corneal thickness: 0 to 5 = class I; >5 to 12 = class II; >12 to 18 (>75 min after treatment)= class II; >12 to 18 (≤75 min after treatment)= class III; >18 to 26= class III; >26 to 32 (>75 min after treatment)= class III; >26 to 32 (≤75 min after treatment)= class IV; >32 = class IV.
ICE classification criteria for opacy: 0.0-0.5 0 class I; 0.6-1.5= class II; 1.6-2.5= class III; 2.6-3.0= class IV.
ICE classification criteria for mean fluorescein retention: 0.0-0.5 = clas I; 0.6-1.5= class II; 1.6-2.5=class III; 2.6-3.0= class IV.

Overall in vitro classifications:
No category= 3 x I / 2 x I, 1 x II
No prediction can be made= Other combinations
Category 1 (Corrosive/Severe Irritant)= 3 x IV / 2 x IV, 1 x III / 2 x IV, 1 x II / 2 x IV, 1 x I / Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) / Corneal opacity = 4 at any time point (in at least 2 eyes) / Severe loosening of the epithelium (in at least 1 eye)

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal mean

Value:

ca. 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

Value:

ca. 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:

Maximal mean

Value:

ca. 1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Other effects / acceptance of results:

OTHER EFFECTS:
- No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, 12 of the 13 chemicals tested obtained the same category as OECD 438. There was a borderline chemical (DMSO) over-classified ("No prediction can be made" vs. "No Category") in three separated tests. It should be tested with other adequately validated in vitro tests.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the combination of the three endpoints was 3 x I and classified as "No Category".
- Acceptance criteria met for positive control: Yes, the combination of the three endpoints was 3 x IV ans classified as "Corrosive/Severe Irritant".

Table 1. Individual and average values for evaluation of corneal lesions after treatment with the negative control (Physiological saline)

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	13	0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	13	0.5	0.5	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	13	0.62	0.62	0.62	0.62	0.62	0.62
Corneal swelling (%)	13	-	0	0	0	0	0
ICE class		I
Combination of the 3 endpoints		3 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

 

 

Table 2. Individual and average values for evaluation of corneal lesions after treatment with the positive control (Sodium hydroxyde)

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	1	0	4	4	4	4	4
	2	0	4	4	4	4	4
	3	0	4	4	4	4	4
Mean		0.0	4.0	4.0	4.0	4.0	4.0
ICE class		IV
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
Corneal thickness	1	0.60	-	-	-	-	-
	2	0.62	-	-	-	-	-
	3	0.54	-	-	-	-	-
Corneal swelling (%)	1	(-)	(-)	(-)	(-)	(-)	(-)
	2	(-)	(-)	(-)	(-)	(-)	(-)
	3	(-)	(-)	(-)	(-)	(-)	(-)
Mean		-	-	-	-	-	-
ICE class		IV
Combination of the 3 endpoints		3 x IV
CLASSIFICATION		Category 1: Corrosive/Severe irritant

Note:

 (-): evaluation of corneal swelling not possible (Corneal opacity=4 at each examination time, leading to a marked refraction of the light preventing from the evaluation of the corneal swelling with the biomicroscope).

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, 2 & 3.

 

 

Table 3. Individual and average values for evaluation of corneal lesions after treatment with the test item.

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	7	0	0	0	0	0	0
	8	0	0	0	0	0	0
	9	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	7	0.5	0.5	-	-	-	-
	8	0.5	0.5	-	-	-	-
	9	0.5	0.5	-	-	-	-
Mean		0.5	0.5	-	-	-	-
ICE class			III	-	-	-	-
Corneal thickness	7	0.60	0.62	0.62	0.62	0.62	0.62
	8	0.62	0.62	0.62	0.62	0.62	0.62
	9	0.62	0.62	0.62	0.62	0.62	0.62
Corneal swelling (%)	7	-	3	3	3	3	3
	8	-	0	0	0	0	0
	9	-	0	0	0	0	0
Mean		-	1	1	1	1	1
ICE class		I
Combination of the 3 endpoints		3 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

GHS criteria not met

Conclusions:

The combination of the three endpoints for the test item was 3 x I. Therefore, the test item does not require classification for eye irritation and serious eye damage.

Executive summary:

The eye irritation of the test item has been evaluated using the tests Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, following GLP. The test item was applied, as supplied, at the dose of 30 mg to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control (Sodium Hydroxide) and one eye with a negative control (physiological saline). Damages by the tests item were assessed by determination of corneal swelling, opacity, and fluorescein retention were evaluated pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The combination of the three endpoints for the test item was 3 x I. Therefore, the test item does not require classification for eye irritation and serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

Key study: The aim of the study was to evaluate the possible corrosive effects of the test item after topical application on in vitro human reconstructed epidermis (epiCS®, CellSystems). This test was performed according to OECD TG 431 and EU method B.40bis, following GLP. The test item was applied, as supplied, at the dose of 25 mg to 2 Human skin model surfaces previously moistered with 25 µL of DPBS during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the epidermis skins treated with the test item were 112.46% and 102.55% (considered as 100%) after 3 minutes and 1 hour exposure, versus 29.45% and 0.72% in the positive control (potassium hydroxide 8N). Therefore, the test item has to be considered as Non-corrosive to skin, corresponding to UN GHS No Category.

Due to the negative outcome from the skin corrosion study, a skin irritation in vitro study was performed.

Skin irritation:

Key study: The aim of the study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). This test was performed according to OECD TG 439 and EU method B.46, following GLP. The test item was applied, as supplied, at the dose of 16 mg to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 47 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.The mean corrected percent viability of the treated tissues was 92.3%, versus 1.2 % in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to the skin.

Eye irritation.

Key study: The eye irritation of the test item has been evaluated using the tests Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, following GLP. The test item was applied, as supplied, at the dose of 30 mg to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control (Sodium Hydroxide) and one eye with a negative control (physiological saline). Damages by the tests item were assessed by determination of corneal swelling, opacity, and fluorescein retention were evaluated pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The combination of the three endpoints for the test item was 3 x I. Therefore, the test item does not require classification for eye irritation and serious eye damage.

Justification for classification or non-classification

Skin irritation: Based on the available information, mean tissue viability > 50% in the OECD 439 test, the substance is non-irritant to the skin according to CLP Regulation (EC) no. 1272/2008.

Eye irritation: Based on the available information, the combination of the three endpoints was 3 x I in the OECD 438 test, the substance does not cause serious eye damage according to CLP Regulation (EC) no. 1272/2008.