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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

700-970-1

Molecular formula:

C48N32H48O16xH2O

IUPAC Name:

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

Test material form:

solid

Test animals

Species:

other: EPISKIN-SM reconstituted human epidermis

Strain:

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

Test system

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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

93.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.