[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

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

[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

EC Number:

262-811-8

EC Name:

[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

Cas Number:

61477-96-1

Molecular formula:

C23H27N5O7S

IUPAC Name:

[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

Test material form:

solid

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.