[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Endpoint summary

Description of key information

Skin irritation. Key study. Method according to OECD 439, GLP study. The mean corrected percent viability of the EpiDerm™ RHE treated tissues was 106.1 %, versus 2.2% in the positive control. Therefore, the test item is not irritant to the skin.

Eye irritation. Based on the available data, the test item is not irritating to the eyes.

Key study. Method according to OECD 438, GLP study. According to the overall in vitro classification, no prediction can be made, since the combinations of the 3 endpoints for the test item were 1 x III, 2 x II. However, based on histopathological findings, the test item might have a negative effect on the corneas.

Key study. Method according to OECD TG 492, GLP study. The relative mean viability of the EpiOcular™ tissues treated with the test item was 102%. Based on the results, the test item is non-irritant to Reconstructed human Cornea-like Epithelium. Thus, the test item is considered to be non-irritant to the eyes.

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:

22/11/2017 - 20/02/2018.

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDerm™

Source species:

human

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: human adult donors, not specified.

Source strain:

other: 00267

Justification for test system used:

The EpiDerm™ model has been validated for irritation testing and its use is recommended by the relevant OECD guideline (OECD No. 439); therefore, it was considered suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis (MatTek In Vitro Life Science Laboratories, Bratislava).
- Tissue batch number(s): 25867
- Production date: 18.12.2017
- Delivery date: 19.12.2017
- Date of initiation of testing: 05.12.2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (15-25ºC).
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 mL DPBS, 15 times at 1-min intervals.
- 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: 0.3 mg/mL
- Incubation time: 3h
- Spectrophotometer: FLUOstar Omega (BMG Labtech)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: OD range 0.0 - 3.5.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.965 ± 0.215
- Barrier function: 4.9 h
- Morphology: functional startum corneum, viable basal cell layer and intermediate spinous and granular layers.
- Contamination: no.
- Reproducibility: yes.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 1 hour exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 1 hour exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ± 2.5 mg

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

1 h ± 0.5 min

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD (mean) = 2.183

Positive controls validity:

valid

Remarks:

viability = 2.2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Following exposure with the test item, the mean cell viability was 106.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non irritant to skin. The validity criteria were met.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, no data correction was necessary.
- Colour interference with MTT: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: This proficiency verification with 10 reference chemicals, in the in vitro skin irritation test, in the EpiDerm™ model, used in OECD 439 (2010) demonstrated that the laboratory is proficient to perform this study. 4 non-irritant chemicals all gave a clearly non-irritant response and the 5 irritant chemicals all gave a clearly irritant response.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the negative control tissues must be ≥ 0.8 and ≤ 2.8. The mean OD value was 2.183.
- Acceptance criteria met for positive control: yes, the mean viability of positive control tissues expressed as % of the negative control tissues must be ≤ 20%. The mean viability of positive control tissues was equal to 2.2 %.
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation (SD) within replicates should be ≤ 18%. Standard deviation for tissues treated with negative control, positive control and test item were equal to 11.5 %, 0.1 %, 15 %, respectively.
- Values for negative control and for positive control were within the range of historical data of the test facility.

Table 1. Summary of results.

	Negative control	Positive control	Test item: Piperacillin Acid
Tissue no. 1 viability	85.3	2.1	123.3
Tissue no. 2 viability	108.5	2.3	105.1
Tissue no. 3 viability	106.3	2.1	89.9
Mean Tissue viability	100	2.2	106.1
SD (±)	11.5	0.1	15

The mean OD value for blank was 0.027 ± 0.0004.

Table 2. Blank correlated absorbance values (OD 570 nm).

OD 570 nm		Negative control		Positive control		Test item: Piperacillin Acid
Tissue number	Measurement replicate	OD 570 nm	Mean	OD 570 nm	Mean	OD 570 nm	Mean
1	A	1.873	1.861	0.047	0.046	2.704	2.692
	B	1.849		0.046		2.681
2	A	2.388	2.368	0.051	0.051	2.345	2.294
	B	2.347		0.051		2.243
3	A	2.354	2.32	0.044	0.045	1.989	1.963
	B	2.286		0.046		1.936
Mean of the three tissues		2.183		0.047		2.316

Interpretation of results:

GHS criteria not met

Remarks:

not irritating according to EU criteria.

Conclusions:

The test item is not irritant to skin.

Executive summary:

An in vitro skin irritation test of the test item was performed in an EpiDerm™ reconstructed human epidermis model, according to OECD TG 439 (GLP study). Disks of EpiDerm™ (three units) were treated with the test item and incubated for 1 hour at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2 and 95% RH. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted and quantified spectrophotometrically. Concurrent negative and positive controls were run, and all validity criteria were met. The mean cell viability was 106.1% compared to the negative control, while the viability of the positive control was 2.2%. The obtained value is above the threshold of 50%, therefore, the test item was considered as non irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14/11/2017 - 20/01/2018

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)

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Characteristics of donor animals (e.g. age, sex, weight): healthy approximately 7-week-old chickens used for human consumption; their body weights ranging from 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 40 min.
- indication of any existing defects or lesions in ocular tissue samples: the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. Only eyeballs without damage were analysed (fluorescein retention and opacity profusion scores fell below 0.5). Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were also replaced.

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 mg of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No post-treatment incubation was performed.

Number of animals or in vitro replicates:

Three in vitro replicates.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After a careful excision of the eyelids so as not to damage the cornea, the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were analysed (fluorescein retention and opacity profusion scores fell below 0.5). Only eyeballs without damage were dissected. The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological salt drip.
After the insertion of the eyeballs to the apparatus, another evaluation of corneal opacity and swelling was performed. Eyeballs with the corneal opacity and/or fluorescein retention score higher than 0.5 or some additional signs of damage were replaced. Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were also replaced. The corneal thickness was determinated using the depth measuring device no. 1 on the slit-lamp microscope and an SP-100 pachymeter (TOMEY).

EQUILIBRATION AND BASELINE RECORDINGS
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological salt at constant temperature of 32 ± 1.5°C and in the average volume of 0.10-0.15 mL/min.
After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline (0.03 mL).

POSITIVE CONTROL USED: imidazole (0.03 g).

APPLICATION DOSE AND EXPOSURE TIME: 0.03 mg of test item, negative and positive control applied for 10 seconds each.

OBSERVATION PERIOD
The corneas treated with the test item and the control items were evaluated pretreatment and at approximately 30, 75, 120, 180, and 240 (± 5) minutes after the post-treatment rinse. At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed 30 minutes after the end of the exposure.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The corneal surface were rinsed from the eye with 20 mL of physiological saline at ambient temperature. Additional rinsing was not necessary.
- Indicate any deviation from test procedure in the Guideline: No deviations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: qualitative assessment by assigning appropriate values to opaque areas, the mean corneal opacity value was calculated for all test eyeballs for all observation time points. Based on the highest mean score corneal opacity the final score was given.
- Damage to epithelium based on fluorescein retention: yes. Qualitative assessment of damage to epithe lium based on application of fluorescein to the eye (fluorescein retention) 30 min after end of exposure.
- Swelling: quantitative measurement, measured with optical pachymeter (SP-100, TOMEY) on a slit-lamp microscope (BP 900 LED, HAAG STREIT). The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.
- Macroscopic morphological damage to the surface: qualitative assessment with slit-lamp microscope.
- Others (e.g., histopathology): Histopathology evaluations: the eyeballs were fixed in a 4% solution of formaldehyde for 24h. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The histological preparations were evaluated under a light microscope. The following layers of the cornea were evaluated: the anterior corneal epithelium, the anterior elastic lamina (Bowman’s membrane), the corneal stroma, the posterior elastic lamina (Descemet’s membrane), the posterior corneal epithelium.

SCORING SYSTEM:
- Mean corneal swelling (%):
0 to 5 ICE Class I
>5 to 12 ICE Class II
>12 to 18 (>75 min after treatment) ICE Class II
>12 to 18 ( <75 min after treatment) ICE Class III
>18 to 26 ICE Class III
>26 to 32 ( >75 min after treatment) ICE Class III
>26 to 32 ( <75 min after treatment) ICE Class IV
>32 ICE Class IV

- Mean maximum opacity score:
0.0 – 0.5 ICE Class I
0.6 – 1.5 ICE Class II
1.6 – 2.5 ICE Class III
2.6 – 4.0 ICE Class IV

- Mean fluorescein retention score at 30 minutes post-treatment:
0.0 – 0.5 ICE Class I
0.6 – 1.5 ICE Class II
1.6 – 2.5 ICE Class III
2.6 – 3.0 ICE Class IV

DECISION CRITERIA: The decision criteria indicated in the TG was used.

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

ca. 1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Remarks:

240 min

Run / experiment:

mean

Value:

ca. 1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

240 min

Run / experiment:

maximum

Value:

ca. 9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not observed
- Gross examination: eyeballs treated with the test item did not exhibit any changes of the corneal surface.
- Histopathological evaluation: Corneas treated with the test item revealed: eyeball no. 1 showed slight exfoliation, slight erosions and slight necrosis of the superficial layer of the anterior corneal epithelium, and vacuolation of the corneal stroma; eyeball no. 2 showed slight exfoliation, slight erosions and slight necrosis of the superficial layer of the anterior corneal epithelium, and slight vacuolation of the corneal stroma; and eyeball no.3 showed slight necrosis of the superficial layer of the anterior corneal epithelium, and dissection of the corneal stroma. Based on these results, the test item can have a negative effect on the cornea.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified
- Acceptance criteria met for positive control: The positive control (acetic acid) was classified as severely irritating, UN GHS Classification: Category 1
This experiment was considered to be valid on the basis of the OECD guideline used

Table 1. Fluorescein retention.

Test item	eyeball no.	1	2	3	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	2.0	2.0	1.0	1.7	III
Positive control	eyeball no.	4	5	6	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	3.0	3.0	3.0	3.0	IV
Negative control	eyeball no.	7	8	9	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	0.0	0.0	0.0	0.0	I

Table 2. Corneal opacity.

Test item	eyeball no.	1	2	3	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	1.0	1.0	1.0	1.0	II
	75 minutes	1.0	1.0	1.0	1.0	II
	120 minutes	1.0	1.0	1.0	1.0	II
	180 minutes	1.0	1.0	1.0	1.0	II
	240 minutes	2.0	1.0	1.0	1.3	II
Positive control	eyeball no.	4	5	6	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	4.0	4.0	4.0	4.0	IV
	75 minutes	4.0	4.0	4.0	4.0	IV
	120 minutes	4.0	4.0	4.0	4.0	IV
	180 minutes	4.0	4.0	4.0	4.0	IV
	240 minutes	4.0	4.0	4.0	4.0	IV
Negative control	eyeball no	7	8	9	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	0.0	0.0	0.0	0.0	I
	75 minutes	0.0	0.0	0.0	0.0	I
	120 minutes	0.0	0.0	0.0	0.0	I
	180 minutes	0.0	0.0	0.0	0.0	I
	240 minutes	0.0	0.0	0.0	0.0	I

Table 3. Corneal swelling.

Test item	eyeball no.	1	2	3	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	7.44	12.57	4.46	8.2	II
	75 minutes	8.53	15.67	2.67	9.0	II
	120 minutes	8.17	14.70	1.96	8.3	II
	180 minutes	4.90	14.70	3.74	7.8	II
	240 minutes	2.00	12.96	3.57	6.2	II
Positive control	eyeball no.	4	5	6	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	45.02	47.37	48.98	47.1	IV
	75 minutes	58.86	54.70	54.53	56.0	IV
	120 minutes	66.24	67.67	65.43	66.4	IV
	180 minutes	75.46	72.56	76.71	74.9	IV
	240 minutes	95.39	84.02	84.66	88.0	IV
Negative control	eyeball no	7	8	9	average	ICE class
	0 minutes	0.0	0.0	0.0	0.0	I
	30 minutes	-7.69	-2.51	-5.60	-5.3	I
	75 minutes	-2.35	-3.13	-4.80	-3.4	I
	120 minutes	-1.88	-2.98	-0.80	-1.9	I
	180 minutes	-1.88	-3.13	-1.44	-2.2	I
	240 minutes	-1.41	-3.76	-1.28	-2.2	I

Table 4. Gross evaluation of the treated corneas.

observation after time t (minutes)	Test item			positive control imidazole			negative control physiological saline
	eyeball no.			eyeball no.			eyeball no.
	1	2	3	4	5	6	7	8	9
0	NC	NC	NC	NC	NC	NC	NC	NC	NC
30	TI	TI	TI	RC, TI	RC, TI	RC, TI	NC	NC	NC
75	TI	TI	TI	RC, TI	RC, TI	RC, TI	NC	NC	NC
120	TI	TI	TI	RC, TI	RC, TI	RC, TI	NC	NC	NC
180	TI	TI	TI	RC, TI	RC, TI	RC, TI	NC	NC	NC
240	TI	TI	TI	RC, TI	RC, TI	RC, TI	NC	NC	NC

Table 5. Summary of results.

	Maximal ICE Class			Conclusion
	Fluorescein retention	Corneal opacity	Corneal swelling	UN GHS Classification
Piperacillin Acid	III	II	II	No prediction can be made
imidazole	IV	IV	IV	Category 1
physiological saline	I	I	I	No Category

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on this in vitro eye irritation study, a prediction cannot be made. However, based on the histopathological evaluation, the test item might have a negative effect on the cornea.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD 438 / EU B.48 method (Isolated Chicken Eye), under GLP. Eyeballs were isolated from
chickens killed for human consumption, the test system was equilibrated and zero reference measurements were taken. Three groups of three eyeballs each were exposed to either 0.03 g test item, 0.03 g imidazole (positive control) or 0.03 mL physiological saline (negative control). After 10 seconds of exposure, all eyeballs were rinsed with 20 mL of physiological salt at ambient temperature and examined. Fluorescein retention was measured 30 min after, while corneal opacity and corneal swelling were evaluated 30, 75, 120, 180, and 240 minutes after. Then, the results of each endpoint were assigned to ICE classes according to OECD guideline recommendations, and histopathological evaluation of the corneal layers was conducted.

The results for the test item treated eyes showed a mean fluorescein retention of 1.7 (ICE class III), mean corneal opacity from 0.0 to 1.3 (ICE class II), and a maximal mean corneal swelling of 9.0 % (ICE class II). Concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations did not exhibit any changes of the treated test item corneal surface. Histopathological examinations of the corneas treated with the test item revealed: exfoliation (eyeball no. 1), slight exfoliation (eyeball no. 2), erosions (eyeball no. 1), slight erosions (eyeball no. 2), necrosis (eyeball no. 1), slight necrosis of the superficial layer of the anterior corneal epithelium (eyeballs no. 1, no. 2, no. 3), vacuolation (eyeball no. 1), slight vacuolation of the corneal stroma (eyeball no. 2), dissection of the corneal stroma (eyeball no. 3). Based on these results, no prediction can be made since the combination of the 3 endpoints was 1 x III, 2 x II. However, according to the histopathological findings, the test item can have negative effects on the chicken cornea.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18/10/2018 - 26/10/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ model and the corresponding test method have been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The test was performed using EpiOcular™ Reconstructed Human Corneum Epithelium (RhCE) from MatTek Corporation. The system consists of normal, human-derived corneal keratinocytes which have been cultured in an air-liquid interphase to form a multilayered highly differentiated model of the human ocular epithelium. Ultrastructurally, the EpiOcular system closely parallels human cornea, thus providing a useful in vitro means to assess ocular toxicology. The certificate of Analysis of the test system is included in the report. The tissue viability and barrier function tests are within the acceptable ranges and indicate the appropiate formation of the mucosal barrier and a viable basal cell layer. Tissues were used within 72 h of their production.
The cells used to produce EpiOcular (TM) tissue are screened for potential biological contaminants:
- HIV-1 virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis B virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis C virus - oligonucleotide-directed amplification: Not detected.
- Bacteria, yeast and other fungi - long term antibiotic, antimycotic free culture: Not detected.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: 2 additional controls for the non-specific OD

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ± 2 mg

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 min)

Number of animals or in vitro replicates:

2 tissues per test substance and for each control were used.

Details on study design:

- Details of the test procedure used: Two tissues (0.6 cm2) were pre-incubated for 1h at 37ºC with PBS, and then dosed topically with 50 mg test item and exposed for 6 hours (± 15 min) at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours (± 15 min) at 37ºC, 5% CO2, ≥ 95% RH.
The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

- RhCE tissue construct used, including batch number: RhCE EpiOcular™, Source: MatTek Corporation (Mylnské Nivy 73, 82105 Bratislava, Slovakia.), Tissue batch number(s): 27076, Production date: 23/10/2018. The EpiOcular tissues were used within 72 hours of their production.
- Doses of test chemical and control substances used: 50 mg test item, 50 µL in all controls.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure 6 h at 37ºC, 5% CO2; post-exposure immersion 25 min at room temperature; post-exposure incubation 18 h at 37ºC, 5% CO2.
- Number of tissue replicates used per test chemical and controls: 2 tissues per test substance and for each control were used.
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: Wavelength: 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to OECD 492, the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Therefore, the test item is identified as not requiring classification if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. Otherwise, the test item is identified as potentially requiring classification and labelling.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: the full internal validation dossier is available for review in the laboratory GLP archive.

Irritation parameter:

other: cell viability (%)

Run / experiment:

mean

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD = 2%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 1.92; SD = 0.08%)
- Acceptance criteria met for positive control: yes (mean cell viability = 0.417 %; SD = 0.1%)
- Range of historical values if different from the ones specified in the test guideline: no.

Table 1. Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls.

Group	Exposure duration	Tissue No.	OD570 nmmeasurements		Mean blank	cOD570 nmmeasurements		Mean cOD570 nm	Viability (%)
			1^st	2^nd		1^st	2^nd
Negative control	6 h	1	1.909	1.887	0.039	1.870	1.848	1.859	97
		2	2.015	2.008		1.976	1.969	1.973	103
Positive control	6 h	1	0.532	0.525	0.039	0.493	0.486	0.490	26
		2	0.382	0.384		0.343	0.345	0.344	18
Test item	6 h	1	1.983	1.958	0.040	1.943	1.918	1.931	101
		2	2.035	2.031		1.995	1.991	1.993	104
Test item treated (viable tissue) not incubated with MTT	6 h	1	0.044	0.043	0.040	0.004	0.003	0.004	0
		2	0.042	0.042		0.002	0.002	0.002	0

OD: optical density; cOD: blank corrected optical density; MTT: 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide

Table 2. Mean tissue viability and standard deviations for the test item, the negative and positive controls.

Group	Exposure duration	cOD570 nmmeasurements		Viability (%)			NSC (%)
		Mean	SD	Mean	SD	Difference (%)	Mean
Negative control	6 h	1.916	0.080	100	4	6	-
Positive control	6 h	0.417	0.103	22	5	8
Test item	6 h	1.962	0.044	102	2	3
Test item treated (viable tissue) not incubated with MTT	6 h	0.03	0.001	0	0	0	0
Test item (true MTT metabolic conversion)	6 h	1.959	-	102	-	-	-

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results (mean percent tissue viability > 60%.), the test item was deemed non-irritant to the eyes.

Executive summary:

An in vitro EIT study according to OECD 492 was conducted under GLP conditions to assess the irritation potential of the test item. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Based on the preliminary tests, duplicate tissues (0.6 cm²) were pre-incubated for 1 hour at 37ºC in 1 ml pre-warmed assay medium, incubated overnight in fresh assay medium, and then dosed topically with 50 mg test item and exposed for 6 hours ± 15 minutes at 37ºC, 5% CO₂, ≥ 95% RH. After exposure, the tissues were rinsed with D-PBS, transferred to fresh assay medium, and incubated for 25 minutes. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO₂, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO₂, ≥ 95% RH. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine cell viability. Concurrent positive, negative and colour interference controls were run in parallel. All validity criteria were met. The relative mean viability of the EpiOcular™ tissues treated with the test item was 102%. Based on the results (mean percent tissue viability after exposure and postexposure incubation is > 60%.), the test item is non-irritant to Reconstructed human Cornea-like Epithelium. Thus, the test item is considered to be non-irritant to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation: Key study. An in vitro skin irritation test of the test item was performed in an EpiDerm™ reconstructed human epidermis model, according to OECD TG 439 (GLP study). Disks of EpiDerm™ (three units) were treated with the test item and incubated for 1 hour at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2 and 95% RH. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted and quantified spectrophotometrically. Concurrent negative and positive controls were run, and all validity criteria were met. The mean cell viability was 106.1% compared to the negative control. This is above the threshold of 50%, therefore, the test item was considered as not irritant to the skin.

Eye Irritation: Based on the available data, the test item does not cause eye irritation or serious eye damage.

Key study. An in vitro (ex vivo) study was conducted according to OECD TG 438 (GLP study). Three eyeballs per group were isolated from chickens and, after appropriate preparation, were exposed to either 30 mg of the test item, 30 mg of imidazole (positive control) or 30 µL of physiological saline (negative control). According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 1 x III, 2 x II. However, based on histopathological findings, the test item might have a negative effect on the corneas.

Key study. An in vitro study was conducted according to OECD TG 492 (GLP study). Based on the results of preliminary tests, duplicate tissues were pre-incubated and then dosed topically with 50 mg test item and exposed for 6 hours ± 15 minutes at 37ºC, 5% CO₂, ≥ 95% RH. After exposure, the tissues were rinsed with D-PBS, transferred to fresh assay medium, and incubated for 25 minutes. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO₂, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO₂, ≥ 95% RH. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine cell viability. Concurrent positive, negative and colour interference controls were run in parallel. All validity criteria were met. The relative mean viability of the tissues treated with the test item was 102%. Based on the results, the test item is non-irritant to Reconstructed human Cornea-like Epithelium. Thus, the test item is considered to be non-irritant to the eyes.

Justification for classification or non-classification

Skin irritation: Based on the available information (106.1 % tissue viability), the test item is not classified for skin irritation/corrosion, in accordance with CLP Regulation (EU) No. 1272/2008.

Eye irritation: Based on the available information (combination of endpoints 1 x III, 2 x II in OECD 438, 1012% tissue viability in OECD 492), the substance does not cause irritation or serious eye damage according to CLP Regulation (EC) No. 1272/2008.