N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Endpoint summary

Description of key information

In vitro eye irritation

Key study: OECD 438. GLP study. The combination of the three endpoints for the test item was 2 x IV, 1 x III. Therefore, the test item has to be classified in Category 1 "Irreversible effects on the eye".

In vitro skin irritation

Key study: OECD 439. GLP study. The mean corrected percent viability of the treated tissues was 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.

Based on the worst-case scenario and taking into consideration the effects on eye, the erythema observed on skin during the acute dermal toxicity test, and the severe skin reaction observed during the preliminary skin sensitization test applied undiluted (100%), the results of the in vitro skin irritation test can be considered a false negative and the test item should be considered as skin irritant, Category 2

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 3, 2016 - November 24, 2016

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.

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

Details on animal used as source of test system:

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): 16-RHE-122.
- Delivery date: 22 November 2016.
- Expiration date: 28 November 2016.
- Date of initiation of testing: 22 November 2016.

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 at 37ºC, 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek).
- Wavelength: 570 nm.
- Linear OD range of spectrophotometer: The linearity range of the optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.2 (CV=4.6%) (Specification OD>0.7).
- Barrier function: 5.2 h (Specification 4 h- Morphology: 6 cell layers, absence of significant histological abnormalities, well differenciated 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 and 42 hours of post-treatment incubation is less or equal than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation 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 µL

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

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 15 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 58.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.0%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: A yellow solution was observed after 3 hours of incubation between 36.4ºC and 37.8ºC, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT: In isopropanol: a colorless solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank substracted) was 0.001 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, a full demonstration of proficiency was performed with Episkin-SM model, plus a reduced validation with SkinEthic™ RHE model. Adequate results were obtained for the evaluated chemicals. summary of proficiency chemicals tested 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 21.3%, instead of ≤18 as initially scheduled. This is due to a mean optical density (OD) vallue of the replicate. No. 3 treated with the negative control which is slightly above the maximal OD value of 1.5. The higher value obtained in the negative control group could lead to an over-classification of the test item. Considering the results obtained (the cell viability of the three treated epidermises is under 50% with a standard deviation of 4.5%), this deviation is considered as without impact on the conclusion of the study (the test item is not irritant for the skin).
- 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	0.943	1.019	1.309	77.8	100.0	21.3
		1.039
		1.075
	2	1.308	1.334		101.9
		1.352
		1.343
	3	1.512	1.575		120.3
		1.557
		1.657
Positive control	1	0.012	0.013	0.013	1.0	1.0	0.1	Irritant
		0.013
		0.013
	2	0.011	0.011		0.8
		0.012
		0.011
	3	0.013	0.014		1.1
		0.014
		0.014
Test item PH-16/0505	1	0.799	0.836	0.768	63.8	58.7	4.5	Non irritant
		0.756
		0.730
	2	0.738	0.741		56.6
		0.756
		0.730
	3	0.738	0.728		55.6
		0.761
		0.686

# 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:

-         Negative control: OD values of the 3 replicates in the range≥ 0.8 and ≤ 3.0 for SkinEthic RHE model; and SD value of the % viability ≤18%. As the optical density was measured after a 1:2 dilution of the extracts, OD values of the 3 replicates should be in the range≥0.4 and≤1.5.

-         Positive control: classified as irritant, and SD value of the % viability≤18%.

-         Test item: same classification for the 3 replicates, and SD value of the % viability≤18%.

 

Notes:

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

-         If the viability obtained for the test item is less tan 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 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.

Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis ( SkinEthic™ RHE model) in accordance with OECD 439, following GLP. The test item was applied, as supplied, at a dose of 16 µL to 3 living skin models during 42 minutes at room temperature, followed by a rinse with 25 mL of DPBS and a 41 h and 15 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan sant that was quantitatively measured after extraction from tissues. Negative and positive controls were run in parallel. The mean corrected percent viability of the treated tissues was 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). No other effects were observed. In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as non-irritant to skin, corresponding UN GHS No category.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

November 7, 2016

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.

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 8:20 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 µL

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 µL of 5% Benzalkonium chloride (Sigma-Batch No. BCBQ4374V) (3 replicates).

APPLICATION DOSE AND EXPOSURE TIME
30 µL of the test item was applied 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. 3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

ca. 3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

ca. 30

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: A severe loosening of the epithelium was also noted in one treated eye from 30 minutes post-dose.

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	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	16	0.5	0.5	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	16	0.54	0.54	0.54	0.55	0.55	0.55
Corneal swelling (%)	16	-	0	0	2	2	2
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 (5% Benzalkonium chloride)

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	1	0	3	3	3	3	3
	2	0	3	3	3	3	3
	3	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
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	0.54	0.63	0.98	1.06	1.20	1.20
	2	0.60	0.78	0.89	0.99	0.99	0.99
	3	0.56	0.72	0.72	0.88	0.88	0.90
Corneal swelling (%)	1	-	17	81	96	122	122
	2	-	30	48	65	65	65
	3	-	29	29	57	57	61
Mean		-	25	53	73	81	83
ICE class		IV
Combination of the 3 endpoints		3 x IV
CLASSIFICATION		Category 1: Corrosive/Severe irritant

Note:

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

(-): 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).

 

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	3	3	3	3	3
	8	0	3	3	3	3	3
	9	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class		IV
Fluorescein retention	7	0.5	3	-	-	-	-
	8	0.5	3	-	-	-	-
	9	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
Corneal thickness	7	0.54	0.72	0.72	0.74	0.76	0.77
	8	0.62	0.65	0.67	0.67	0.68	0.74
	9	0.60	0.67	0.67	0.76	0.76	0.76
Corneal swelling (%)	7	-	33	33	37	41	43
	8	-	5	8	8	10	19
	9	-	12	12	27	27	27
Mean		-	17	18	24	26	30
ICE class		III
Combination of the 3 endpoints		2 x IV, I x III
CLASSIFICATION		Category 1: Corrosive/Severe irritant

Note: Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eye No. 7.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The combination of the three endpoints for the test item was 2 x IV, 1 x III. Therefore, the test item has to be classified in Category 1 "Irreversible effects on the eye".

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 µL 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 ( 5% Benzalkonium chloride) 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. Maximal mean score of corneal opacity was 3.0 corresponding to ICE class IV; mean score of fluorescein retention was 3.0, corresponding to ICE class IV; and maximal mean corneal swelling was 30%, corresponding to ICE class III. The combination of the three endpoints for the test item was 2 x IV, 1 x III. Therefore, according to CLP regulation (EC 1272/2008) the test item has to be classified in Category 1 "Irreversible effects on the eye".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Skin irritation

Key study: The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis ( SkinEthic™RHE model) in accordance with OECD 439, following GLP. The test item was applied, as supplied, at a dose of 16 µL to 3 living skin models during 42 minutes at room temperature, followed by a rinse with 25 mL of DPBS and a 41 h and 15 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan sant that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the treated tissues was 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as Non-irritant to skin, corresponding UN GHS No category.

In vitro 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 µL 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 ( 5% Benzalkonium chloride) 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 2 x IV, 1 x III. Therefore, according to CLP regulation (EC 1272/2008) the test item has to be classified in Category 1 "Irreversible effects on the eye".

Justification for classification or non-classification

Based on the available information, the test item has to be considered as irritant to skin, Category 2, H315: Causes skin irritation, in accordance with the CLP Regulation EC No. 1272/2008.

On the other hand, the test item has to be classified in Category 1, H318: Causes serious eye damage, in accordance with the CLP Regulation EC No. 1272/2008.