Registration Dossier

Administrative data

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:
2015-05-11 to 2015-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline and GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/Jcr
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley; Indianapolis, IN
- Age at study initiation: 8.5 weeks (Date Born: 10 Apr 15)
- Weight at study initiation: 19.2-23.2 g
- Housing: 1-5 per cage, Polycarbonate boxes with bedding
- Diet: ad libitum, PMI Feeds Inc.™ Formulab #5008
- Water: ad libitum, Municipal water supply analyzed by TCEQ Water Utilities Division
- Acclimation period: 5 days quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-28
- Humidity (%): 49-91
- Air changes (per hr): 10+
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100 %
No. of animals per dose:
5 (female)
Details on study design:
RANGE FINDING TESTS: was not conducted

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: open application
- Criteria used to consider a positive response: EC3

TREATMENT PREPARATION AND ADMINISTRATION:
Five females were selected for each of three Test groups (Groups I - III). On Days 1, 2 and 3, each Test animal in its group received an open application of 25 µL of appropriate dilution (25 or 50 %) of test item in 4:1 v/v acetone:olive oil vehicle, or 100 % test item, to the dorsum of both ears. The Vehicle Control group (5 females) was treated the same way as test animals, but with vehicle alone instead of test item. The Positive Control group (5 females) was treated with 100 % alpha-hexylcinnamaldehyde. All Test and Control animals were given a two-day rest period on Days 4 and 5.
On Day 6 of the study, all Test and Control animals were injected in the tail vein with 250 µL of 0.01 M phosphate-buffered saline (PBS; Sigma, Lot SLBJ110V, Exp Jun 2024), pH 7.4 at 25°C per manufacturer, containing 20 µCi of [methyl-3H] Thymidine (PerkinElmer, Lot 201408, Exp Aug 2015). Five hours after injection, animals were sacrificed with an overdose of CO2, the draining auricular lymph nodes excised and pairs from each individual animal processed.

Observations
Individual body weights were recorded on Day 1 prior to dosing, and Day 6. prior to injection. All Test and Control animals were observed daily for clinical signs of toxicity and any signs of excessive irritation at the test site.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparisons Test was performed on the DPM counts. If the test groups showed a stimulation index (SI) of > 3, then the extrapolated EC3 (estimated concentration required for a 3-fold stimulation index value) was calculated from the stimulation index values at 25% and either the 50% or 100% concentration and the substance was assessed as a weak, moderate- strong or extreme sensitizer.
Parameter:
SI
Remarks on result:
other: Please refer to "Any other information on results".
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Please refer to "Any other information on results".

Body weight

One animal in each of Test Groups I and II lost weight during the study.

Clinical signs

All animals appeared normal for the duration of the study.

Stimulation Index or Test/Vehicle Control Ratio derived for each Test group based on group mean DPM:

Animal

Group

Test Item

Concentration [%]

Average DPM Count

per Mouse

Number of Mice

in Group

Test/Vehicle

Control Ratio (Stimulation Index)

Vehicle Control

NA

534

5

NA

Test Group I

25

599

5

1.1

Test Group II

50

579

5

1.1

Test Group III

100

814

5

1.5

Positive Control

NA

2490

5

4.7*

NA - Not applicable

* - Positive Control used to confirm animal sensitization potential and validate procedures

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance induced a stimulation index of < 3 in all test groups. Therefore it was considered to be not sensitising.
Executive summary:

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test item Cantryl possessed a significant potential to cause skin sensitization. Five females were assigned to each of three groups, designated Groups I - III. Test groups were treated with an appropriate dilution (25 or 50 %) in 4:1 v/v acetone:olive oil, or 100 % test item. Each animal received 25 µL to the dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A Vehicle Control group of five females was run concurrently, treated in the same manner with vehicle only instead of test item or dilution. A Positive Control group of five females was also run concurrently, treated with 100 % alpha-hexylcinnamaldehyde.

The test item produced a stimulation index < 3 in all groups of test animals, and therefore was not a senitizer.

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

A skin sensitization study (key) was conducted on 3 groups of 5 female mice to determine if test item Cantryl possessed a significant potential to cause skin sensitization. Five females were assigned to each of three groups, designated Groups I - III. Test groups were treated with an appropriate dilution (25 or 50 %) in 4:1 v/v acetone:olive oil, or 100 % test item. Each animal received 25 µL to the dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A Vehicle Control group of five females was run concurrently, treated in the same manner with vehicle only instead of test item or dilution. A Positive Control group of five females was also run concurrently, treated with 100 % alpha-hexylcinnamaldehyde. The test item produced a stimulation index < 3 in all groups of test animals, and was therefore not a sensitizer.

In a study (supporting) the potential of the test substance at a concentration of 10 % in dipropylen glycol was assessed to cause skin sensitization. Therefore, ten female albino guinea pigs of the Dunkin Hartley-strain were used. After an intradermal injection of Freund's Complete Adjuvant a topical dose of the test material (0.1 mL at a concentration of 10 %) was applied (day 4 of the experiment). On days 7 and 9 further topical applications were performed to the same test sites. On day 22, another test site was clipped and depilated. On day 23, the animals received a topical application of 2.5 % test material (challenge). Skin reactions were recorded 24 and 48 hours later. As a result, no reactions were observed and therefore the test material was considered to not be a skin sensitizer at the tested concentration.


Migrated from Short description of key information:
The test substance was not sensitising in LLNA test in mice.

Justification for selection of skin sensitisation endpoint:
The study was conducted according to OECD and GLP and the obtained result was supported with another in vivo study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data did not indicate a sensitising property of the test substance to the skin. Therefore the substance is not to be classified for skin sensitisation under Regulation (EC) No 1272/2008 (CLP).