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Description of key information

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:
from 11 May 2012 to 24 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized guidelines
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/JN
Sex:
female
Details on test animals and environmental conditions:
Species and strain : Mice, CBA/JN
Sex : Females (nulliparous and non-pregnant)
Age and weight range
(at order): 5 to 7 weeks old, 15 to 20 grams
Supplier : Charles River Italia S.p.A., Calco (Lecco), Italy.
Breeder : Charles River France Laboratories, Iffa Credo, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
Date of arrival : 12 April 2012
Weight range at arrival : 15.5 to 17.7 grams
Acclimatisation period : At least 5 days
Veterinary health check : After arrival

Caging:
No. of animals/cage: 1/cage during the study; up to 5 during acclimatisation
Housing: Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
Cage tray control: Daily inspected and changed as necessary (at least twice/week)

Water and diet:
Water : Drinking water supplied to each cage via a water bottle
Water supply : Ad libitum
Diet : 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply : Ad libitum throughout the study
Records of analyses of water and diet are kept on file at RTC. Components present in the drinking water or diet are not at a level likely to interfere with the purpose or conduct of the study.

Housing conditions (parameters set):
Room lighting : Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes : Approximately 15 to 20 air changes per hour
Temperature range : 22°C ± 2°C
Relative humidity range : 55% ± 15%
Actual conditions were monitored and recorded and records retained. No relevant deviations occurred.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary phase:
25, 10, 5, 2.5, 1% w/w in acetone:olive oil 4:1 (v/v)
No toxicity signs (clinical signs or toxicologically relevant body weight losses) were observed at any concentration tested.
According to the results of the irritation screening, the concentration judged as minimally irritant was 10% w/w.

Main assay:
In the main assay, the test item was topically administrered at the concentrations of 10, 5 and 2.5% w/w, in acetone:olive oil 4:1 (v/v).
No. of animals per dose:
group of 7 animals for each concentration.
Details on study design:
A preliminary irritancy test was carried out to select three concentrations to be used in a main assay, according to the criteria described in the relevant guideline for this test.
A main phase was then carried out to fully evaluate lymph node cell reaction.

Main test:
Treatment schedule of animals is summarised as follows.
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
D D D - BrdU C

D : Dermal application of test item, positive control or vehicle(s) 25 µL/ear (50 µL/animal)
BrdU : Intraperitoneal administration of BrdU
C : Sacrifice, processing of lymph nodes and determination of Cell Proliferation

Dosing with the test or control items

Frequency of treatment : Three consecutive days (Days 1, 2, 3) with the test or control item formulations.
Dose calculation : Dose volume of 25 µL/ear/day (50 µL/animal/day) for each animal.
Dosing method : Topical application of the formulations to the dorsal surface of each ear, using a Gilson micropipette.



Dosing with 5-Bromo-2-deoxyuridine (BrdU)

Frequency of treatment : Once only, on Day 5.
Dose calculation : A solution of BrdU, at a concentration of 10 mg/mL in physiological saline (NaCl 0.9%) was administered at the dose volume of 0.5 mL/animal.
Dosing method : Intraperitoneal injection, using 25 G needle and a plastic graded syringe of a suitable volume.

In life observations

Mortality and morbidity: Twice daily.
Clinical signs : Before dosing commenced and daily up to sacrifice (approximately 1 hour after each dosing, in the days of treatment).
Body weight : Allocation (Day 1) and sacrifice (Day 6).

Terminal studies

Termination : Day 6, approximately 24 hours after BrdU injection.
Euthanasia method: Carbon dioxide narcosis.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Positive control results:
In the group treated with the positive control item, a stimulation index of 6.97 was calculated. As it was greater than 2, the test system was regarded as valid.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In a first experiment the calculated stimulation indices were 1.80, 1.91 and 1.24 respectively at low, mid and high dose groups. Since these results were considered borderline, a second experiment was repeated to confirm them. In the second experiment, increases in cell proliferation of draining lymph nodes were observed in all test item treated groups being the calculated stimulation index equal to 2.22, 2.82 and 1.94 respectively at low, mid and high dose level. The proliferative responses at all dose levels were higher than the threshold value of 1.9 for clear positive result. In this experiment, the observed increases were statistically significant at the low and mid- dose level (Groups 2 and 3) but not in the high dose level (Group 4). No dose response relationship was observed.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential of the test item, DICYCLOHEXYLPHTHALATE, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

In the main sensitisation assay, the test item was administered at the concentrations of 10, 5 and 2.5% w/w.

Increases in cell proliferation were observed in all test item treated groups being the calculated stimulation index equal to 2.22, 2.82 and 1.94 respectively in low, mid- and high dose level.

The stimulation index (SI) values at the three dose levels were higher than the threshold of 1.9 for a clear positive results, established in the relevant guideline. Therefore, the results obtained in this study indicate that the test item elicits a mild sensitisation response in mice following dermal exposure.
Executive summary:

The potential of the test item, DICYCLOHEXYLPHTHALATE, to cause skin sensitisation reactions following topical application to the skin of CBA/JN(CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

Preliminary phase

Five concentrations (25, 10, 5, 2.5, 1% w/w in acetone:olive oil 4:1 (v/v), the highest being selected on the basis of a preliminary solubility test) were selected to be used in the preliminary phase, to identify a non toxic and minimally irritant concentration in order to avoid false positive results.

No toxicity signs (clinical signs or toxicologically relevant body weight losses) were observed at any concentration tested. According to the results of the irritation screening, the concentration judged as minimally irritant was 10% w/w.

Main assay

In the main assay, the test item was topically administrered at the concentrations of 10, 5 and 2.5% w/w, in acetone:olive oil 4:1 (v/v).No mortality or toxicologically relevant clinical signs were recorded in animals treated at all dose levels. Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Increases in cell proliferation of draining lymph nodes were observed in the three test item treated groups. The calculated stimulation indices were equal to 2.22, 2.82 and 1.94 respectively at low, mid- and high dose level.

The variations were statistically significant at the low and mid- dose levels but not at the high dose level.

 No dose response relationship was observed.          

The stimulation index (SI) values at the three concentrations were slightly over the threshold of 1.9 for a positive result as established in the relevant guideline. Therefore, the results obtained in this study indicate that the test item
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The potential of the test item, DICYCLOHEXYLPHTHALATE, to cause skin sensitisation reactions following topical application to the skin of CBA/JN(CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b. The results obtained in this study indicate that the test item elicits a mild sensitisation response in mice following dermal exposure.


Migrated from Short description of key information:
Skin sensitization (LLNA:BrdU-ELISA method, mice) according to OECD 442b: the test item elicits a mild sensitisation response in mice following dermal exposure.

Justification for selection of skin sensitisation endpoint:
GLP study in compliance with international recognized guidelines.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the European Directives (Council Regulation (EC) No. 1272/2008 and subsequent revisions) the substance is classified as skin sensitizer (Category I).

No information is available for respiratory sensitization, thus the notifiable is not classified as a respiratory sensitizer as data are lacking