Octanoic acid, compound with dicyclohexylamine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-23 - 2017-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Octanoic acid, compound with dicyclohexylamine (1:1)

EC Number:

239-914-1

EC Name:

Octanoic acid, compound with dicyclohexylamine (1:1)

Cas Number:

15816-71-4

Molecular formula:

C20H39NO2

IUPAC Name:

octanoic acid, compound with dicyclohexylamine (1:1)

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species: Mice CBA/Ca
Source: AnLab Prague, Czech Republic
Number and Sex of Animals: 29 female, non-pregnant and nulliparous animals were used
Age at First Dose: 8-11 weeks
Animal Health: The health condition of animals was examined by a veterinarian
before initiation of the study
Acclimation: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment. The acclimation was according to standard operation procedures.
Housing Condition: The animals were housed in polypropylene cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle The sanitation was performed according to standard operation procedures.
Diet: A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time. The certificate of analysis is included in the raw data.
Water: The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodically monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10 %, 25 % and 50 % (w/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any sign of irritation. Erythema scores were given following Table 1. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 %.
MAIN STUDY
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25 µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250 µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5 M Ffluorodeoxyuridine wereas injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised in PBS and weighted.
Preparation of cell suspensions
Cell suspension of lymph node cells from individual animals was prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged by 600g at 4C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4C for 18-20h. Pellets were centrifuged by 2000g at 4C for 5 min., re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.
Determination of cellular proliferation (incorporated radioactivity)
Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/animal. DPM were calculated according to formula:
CPM
DPM= ------------------------------
absolute detector efficiency
(Absolute detector efficiency for Gamma Counter Wizard2 2470 = 66.73%)
Clinical Observation
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.
Body Weight
The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals.


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The SI (stimulation index) was obtained by dividing the individual DPM for each treatment group by the incorporation of the individual DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, was determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration
d – SI of lower concentration


TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

5.44

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

5.25

Test group / Remarks:

50%

Results 4

Key result

Parameter:

EC3

Value:

13.33

Any other information on results incl. tables

The mean Lymph node weight, DPM, SI, EC3 values

	Lymph node	Number of
	weight (g)	lymph nodes	DPM	SI	EC3
Control	0.0263	10	995	-	13.33
Positive Control	0.0570	10	9076	6.37
Dicyclohexylammoniumoctanoat 10%	0.0373	10	2292	2.30
Dicyclohexylammoniumoctanoat 25%	0.0466	10	5414	5.44
Dicyclohexylammoniumoctanoat 50%	0.0425	10	5224	5.25

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The skin sensitizing potential of Octanoic acid, compound with dicyclohexylamine (1:1) was assessed using the murine local lymph node assay (LLNA), according to OECD Guideline No. 429.
Based on the results of this study, Octanoic acid, compound with dicyclohexylamine (1:1) is considered a skin sensitizer under the condition of this study.

Executive summary:

The skin sensitization potential of Octanoic acid, compound with dicyclohexylamine (1:1)D was evaluated by LLNA method, which was developed on the basis of scientific understanding of the immunological mechanism of skin sensitization.

Because of signs of toxicity in preliminary test, the main study started at concentration of 50%. In our experiments, the test item was applied over three consecutive days, at three concentrations. In the main study all animals survived throughout the test period without showing any clinical signs of toxicity orlocal irritation.In comparison with the control group, the increase inlymph node weight was observedin all treated groups.A similar trend was registered in the evaluation of DPM of the lymph nodes. Calculated SI values in the treated groups were higher than 3 at the concentrations of 25%. The EC3 value is calculated to be 13.33%. As the SI for some treatment dose group is ≥3 and a dose response is observed, the test item is regarded as a potential skin sensitizer.

These results demonstrate that Octanoic acid, compound with dicyclohexylamine (1:1) is a potential skin sensitizer in the Local Lymph Node Assay under the test conditions of this study.