3,5,8,10,13,15,18,20,23,25,28,30,33,35,38,40,41,43,45,47,51,53,55,57,59,61,63, 65,67,69,71,73-dotriacontazapentacosacyclo [35.3.3.36,7.311, 12.316,17.321,22.326,27.331,32.22,41.236,43.13,40.15,8.110,13.115,18.120,23.125,28.130,33.135,38.145,51.147,73.153,55.157,59.161,63.165,67.169,71] octacontane-44,46,48,49,50,52,54,56,58,60,62,64,66,68,70,72-hexadecone, hydrochloride hydrate

Endpoint summary

Description of key information

In Vitro Skin Irritation: Not irritating in the EPISKIN model test. 
In Vitro Skin Corrosion: Not corrosive in the EPISKIN model test. 
In Vitro Eye Irritation: Not irritating in the isolated chicken eye test.

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

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

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYI/38593-5/2012

Species:

other: EPISKIN-SM reconstituted human epidermis

Strain:

other: SkinEthic, France, Batch No.:15-EKIN-015, Expiry Date: 20 April 2015

Vehicle:

water

Remarks:

10μL distilled water

Controls:

yes

Duration of treatment / exposure:

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was
terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2

Observation period:

No observation period required for the study type.

Number of animals:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Value:

93.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

ADDITIONAL CONTROLS

As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. The

optical density (measured at 570 nm) of this tissue was 0.069, Non Specific Colour % was calculated as 10.4% (see Table 1). This value was above 5%, therefore additional data calculation was necessary.

 

As colour change (purple precipitate) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in Table 2. Based on these observed mean OD (0.051), the calculated NSMTT is 7.7%.

 

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

 

The mean OD value of the three negative control tissues was in the recommended range (0.659). Standard deviation of the viability results for negative control samples was 5.3.

 

The positive control treated tissues showed 11.9% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 5.3.

 

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 11.1.

 

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, in this in vitro EPISKIN model test with CUCURBIT[8]URIL, the results indicate that the test item is non-irritant to skin.

Executive summary:

The purpose of this study is to classify the skin irritant potential of CUCURBIT[8]URIL. Study was performed to OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (26 July 2013) Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “ In Vitro Skin Irritation Reconstructed Human Epidermis Model Test” EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Following exposure with CUCURBIT[8]URIL, the mean cell viability was 93.5% compared to the negative control (after adjustment for colour and non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test with CUCURBIT[8]URIL, the results indicate that the test item is non-irritant to skin.

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

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 Ocular Corrosives and Severe Irritants)

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYI/38593-5/2012

Species:

other: chicken eye

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

CHICKEN HEADS COLLECTION AND TRANSPORT
Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the
cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with
20 mL physiological saline. Then the fluorescein treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the
head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bentscissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the
eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the
orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on
the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight
pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp
holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel
tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and
examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the
cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage,
were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Duration of treatment / exposure:

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Observation period (in vivo):

Four hour observation period.

Number of animals or in vitro replicates:

The negative control eye was treated with 30 μL of physiological saline. One eye was treated with physiological saline, three eyes with the test item and another three with Imidazole.

Irritation parameter:

cornea opacity score

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

after 240 min

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

after 75 min

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test Item 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.3%	I
Mean maximum corneal swelling at up to 240 min	3.3%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	1.00	II
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	2xI 1xII

 Based on this in vitro eye irritation in the isolated chicken eyes test with CURCURBIT[8]URIL, the test item in not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.

 

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9.6%	II
Mean maximum corneal swelling at up to 240 min	26.7%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	2.83	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.

 

NEGATIVE Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified.

 

 

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.

Executive summary:

The purpose of this study was to provide detailed information about the effects of CUCURBIT[8]URIL on the cornea to see if a classification is required. Study was performed to OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants). After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of physiological saline

(Salsol solution, 0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

No significant corneal swelling was observed during the four hour observation period. No corneal opacity change was observed on three eyes and fluorescein retention change (severity 1) was noted on all test item treated eyes; particles of test item were stuck to the cornea and could not be washed off during the study. The effects were clearly greater than a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo. In conclusion based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for investigation of full classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vitro Skin Irritation

An in vitro skin irritation test of CUCURBIT[8]URIL test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. 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 using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test item. Furthermore, three additional test item treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with CUCURBIT[8]URIL, the mean cell viability was 93.5% compared to the negative control (after adjustment for colour and non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with CUCURBIT[8]URIL, the results indicate that the test item is non-irritant to skin.

In Vitro Skin Corrosion

An in vitro skin corrosivity test of CUCURBIT[8]URIL test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKIN (two units) were treated with CUCURBIT[8]URIL test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution from the test item. Furthermore, two additional test item treated and two negative control treated killed epidermis units were used to determine the MTT interacting potential of the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with CUCURBIT[8]URIL, the mean cell viability was 100.8% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with CUCURBIT[8]URIL, the results indicate that the test item is not corrosive to the skin.

In Vitro Eye Irritation

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (26th July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of physiological saline (Salsol solution, 0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

No significant corneal swelling was observed during the four hour observation period.

No corneal opacity change was observed on three eyes and fluorescein retention change (severity 1) was noted on all test item treated eyes; particles of test item were stuck to the cornea and could not be washed off during the study. The effects were clearly greater that a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo.

Based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.

Justification for selection of skin irritation / corrosion endpoint:
Endpoint conclusion derived from in vitro experimental study in accordance with OECD Guideline 439 and EU Method B46 performed at a GLP accredited laboratory.

Justification for selection of eye irritation endpoint:
Endpoint conclusion derived from in vitro experimental study in accordance to OECD Guideline 438, EU Method B48 and US EPA Procedure OPPTS 870.2400 performed at a GLP accredited laboratory.

Justification for classification or non-classification

In Vitro Skin Irritation and Corrosion

The substance is not classified under CLP as it does not fill the requirements for skin irritation/corrosion classification.

In Vitro Eye Irritation

Based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.