Registration Dossier

Administrative data

Description of key information

The substance was found to be a non-sensitiser in chemico (direct peptide reactivity assay according to OECD test guideline 442C, GLP study). Applying the integrated approach recommended in the OECD IATA guideline on skin sensitisation, this result was combined with other complementary information in a Weight-of-Evidence approach. As a result, there was sufficient evidence to conclude on the absence of sensitising properties of this multi-constituent substance (“reaction mass of cucurbit[6]uril, cucurbit[7]uril and cucurbit[8]uril”). This conclusion is based on read-across information from cucurbit[8]uril hydrochloride hydrate, together with other supporting evidence. The analogue substance cucurbit[8]uril hydrochloride hydrate was not a skin sensitiser in a GLP guideline study according to OECD TG 429. The analogue substance has a similar structure containing identical monomer units and functional groups, has a similar toxicity profile and similar physico-chemical properties (cf. data matrix). In addition, the following lines of evidence support the read-across:

-     the physico-chemical properties of “reaction mass of cucurbit[6]uril, cucurbit[7]uril and cucurbit[8]uril” suggest a lack of penetration into viable epidermis;

-     the lack of structural alerts and the prediction of “non-sensitiser” with the VEGA CAESAR QSAR model, 2018 version);

-     the lack of protein reactivity in the OECD 442C assay;

-     the lack of activity in in vitro skin irritation and genotoxicity assays; and

-     the lack of formaldehyde release in artificial sweat experiments.


Given that none of several different lines of evidence gives any indication of a sensitising potential, the overall confidence level in the assessment is high. The adaptation of information requirements (here: a battery of in vitro sensitisation tests) according to Annex XI (REACH) provisions (based on weight of evidence, including read-across) is therefore considered adequate.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2014 to 25 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OGYI/38593-5/2012
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting*: 9 weeks old (age-matched, within one week)
Body weight range at starting*: 21.5-22.1 grams (the weight variation in animals in the study did not exceed 20% of the mean weight)
Acclimatization time: at least 5 days
*Note: In the preliminary experiment, mice of 12 weeks of age (21.3-23.4 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.0-25.0°C
Relative humidity: 33-68 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 242/8 (preliminary experiment)
Room/Cabinet (non-radioactive phase): 244/5 (main experiment)
Room/Cabinet (radioactive phase): 139 – 140

Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable
Complete diet for rats/mice” (Batch number: 190 1786 / 680 2237,, Expiry date: January 2015 / March 2015) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably
be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottle, ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36. Hungary).
Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study.
Lignocel 3/4-FASERN Hygienic
Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study. Nest building material was also provided for animals (DC Dental Central Grosshandelges. mbH, Carl-Zeiss Str. 2, D-22946 Trittau, Germany).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB
Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and
individual animal number.
The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25% CUCURBIT[8]URIL in Acetone : Olive oil 4:1 (v/v) mixture
10% CUCURBIT[8]URIL in Acetone : Olive oil 4:1 (v/v) mixture
5% CUCURBIT[8]URIL in Acetone : Olive oil 4:1 (v/v) mixture
Positive control - 25% (w/v) a-Hexylcinnamaldehyde (dissolved in AOO).
Negative control - Acetone : Olive oil 4:1 (v/v) mixture
No. of animals per dose:
Four mice per group.
Details on study design:
Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed at least twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Ear Thickness Measurements
In the preliminary experiment, both ears of each mouse were observed for erythema and scored using Table 3 [3]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was also performed by ear punch weight determination after the euthanasia of the experimental animals.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 oC. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step wasrepeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules.

After overnight (approximately 18 hours) incubation at 2-8 oC, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation indexof 3 or greater is an indication of a positive result.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None specifified
Positive control results:
The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (AOO) using CBA/J Rj mice. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 9.0) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
1
Remarks on result:
other: 5% (w/v)
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
2
Remarks on result:
other: 10% (w/v)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
3
Remarks on result:
other: 25% (w/v)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. The DPM values were 2634.5, 3407.5 and 2983.5 at concentrations of 25, 10 and 5% (w/v), respectively.

Individual Body Weights for all Animals with Group Means

Identity Number

Animal Number

Test group name

Initial body weight (g)

Terminal body weight (g)

Change (%)#

7467

1

Negative vehicle control (AOO)

22.0

22.1

0.5

7539

2

21.5

21.5

0.0

7484

3

21.9

22.4

2.3

7508

4

21.6

22.3

3.2

 

 

Mean

21.8

22.1

1.5

7469

5

CUCURBIT[8]URIL 25% (w/v) in AOO

22.1

21.6

-2.3

7546

6

21.7

21.0

-3.2

7490

7

21.7

22.5

3.7

7516

8

21.9

21.7

-0.9

 

 

Mean

21.9

21.7

-0.7

7475

9

CUCURBIT[8]URIL 10% (w/v) in AOO

22.0

21.3

-3.2

7550

10

21.7

22.9

5.5

7494

11

21.6

22.3

3.2

7525

12

21.9

22.2

1.4

 

 

Mean

21.8

22.2

1.7

7480

13

CUCURBIT[8]URIL 5% (w/v) in AOO

22.1

23.3

5.4

7554

14

21.9

20.7

-5.5

7498

15

21.5

22.6

5.1

7532

16

21.7

21.7

0.0

 

 

Mean

21.8

22.1

1.3

7472

17

Positive control (25% (w/v) HCA in AOO)

22.0

22.4

1.8

7493

18

21.9

22.3

1.8

7512

19

21.7

23.0

6.0

7537

20

21.5

22.2

3.3

 

 

Mean

21.8

22.5

3.2

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM / group

DPM

Number of Lymph Nodes

DPN

Stimulation Index

Background (5% (w/v) TCA)

31

28

-

-

-

-

Negative vehicle control (AOO)

2638

2608.5

8

326.1

1.0

CUCURBIT[8]URIL 25% (w/v) in AOO

2664

2634.5

8

329.3

1.0

CUCURBIT[8]URIL 10% (w/v) in AOO

3437

3407.5

8

425.9

1.3

CUCURBIT[8]URIL 5% (w/v) in AOO

3013

2983.5

8

372.9

1.1

Positive control (25% (w/v) HCA in AOO)

23468

23438.5

8

2929.8

9.0

 

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay, CUCURBIT[8]URIL, tested in a suitable vehicle, was shown to have no sensitisation potential
(non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The purpose of this study was to determine the skin sensitisation potential of CUCURBIT[8]URIL test item following dermal exposure in the Local Lymph Node Assay. Study performed to OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay”(22 July 2010) Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) as amended by Commission Regulation (EU) No 640/2012 of 6 July 2012 (Official Journal L 193, 20/07/2012).

In conclusion, under the conditions of the present assay, CUCURBIT[8]URIL, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not fill the requirements for skin sensitisation classification.