Isoleucine, N-(1-oxohexadecyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-18 to 2012-08-09

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

There were occasional procedural deviations that were minor and did not impact the integrity or outcome of the study

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:

(2S,3S)-2-(hexadecanoylamino)-3-methylpentanoic acid

Cas Number:

54617-29-7

Molecular formula:

C22H43NO3

IUPAC Name:

(2S,3S)-2-(hexadecanoylamino)-3-methylpentanoic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): L-Isoleucine, N-(1-oxohexadecyl)
- Substance type: Lipoamino acid
- Physical state: White powder with granule
- Purity test date: 2011-06-02
- Purity: 99% dry extract
- Lot/batch No.: 1110400022
- Retest date of the lot/batch: 2014-04-13
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratory, (Bar Harbor, Maine, USA, 04609)

- Age at study initiation: from 10 to 11 weeks

- Weight at study initiation: from 20 to 26 g

- Housing: Animals were group housed in stainless steel rodent cages equipped with an automatic watering system. In the main test, on Day 6, before the 3H-TdR injections, the animals were individually housed in solid bottom cages. Following group assignment, all cages were clearly labeled with a color-coded cage card indicating at least the following: study number and animal number (including gender and group number).
- Diet/ Water(e.g. ad libitum):
A standard certified commercial chow (Harlan Teklad Certified Global Rodent Diet #2018C) was provided to the animals ad libitum. Concentrations of the contaminants (e.g., heavy metals, aflatoxin, organophosphate and chlorinated hydrocarbons) were routinely measured by the manufacturers (batch certificates on file at CiToxLAB North America).
Municipal tap water (which was exposed to ultraviolet light and purified by reverse osmosis) was provided to the animals ad libitum, via an automatic watering system. Periodic analyses of the municipal tap water (purified water) were performed by a CiToxLAB North America designated facility and the results are retained on file at CiToxLAB North America.

- Acclimation period: 7 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3C
- Humidity (%): 50 ± 20%;
- Air changes (per hr): 10 – 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0.5% ; 1% ; 2.5% ; 5% ; 10%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

Dose concentrations for the dose selection phase (preliminary assay) and selected solvent (N, N-dimethylformamide (DMF)) were gave by the sponsor, and were confirmed within the solubility assay performed at CiToxLAB North America.
Main Test Phase:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that were obtained in the preliminary test:
• The vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item, based on data given by the sponsor using N, N-dimethylformamide (DMF) as a solvent and as confirmed within the solubility assay performed at CiToxLAB North America.
• The concentrations were selected from the concentration series 25%, 10%, 5%, 2.5%, 1.0%, 0.5%, etc.
The maximum concentration of the test item was defined on the basis of the results of the preliminary assay and selected to avoid overt systemic toxicity and excessive local skin irritation; the latter being defined by a > 25% increase of the ear thickness

- Lymph node proliferation response:
Lymph node cell proliferative response was measured as described by Kimber and Dearman.
On Day 6 of the main test, all animals of all groups received a single intravenous injection (animal No. 4502 punctured twice) of an adjusted volume of 230 µL (based on the radioactivity concentration analysis results (87.73 µCi/g)) of NaCl 0.9% containing a dose of 20 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
Approximately 5 hours post-3H-TdR administration, the main test animals were euthanized by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes (ALN) were excised bilaterally. The lymph nodes were pooled for each experimental group. The number of ALN sampled for each group was documented. A single cell suspension of ALNCs was prepared by mechanical disaggregation in Petri dishes using the plunger of a syringe. The carcasses were then appropriately disposed of without any further examination.


MAIN STUDY

The test item would be regarded as a skin sensitizer when the SI for a dose group is >= 3 together with consideration of a dose-response relationship. Other relevant criterias such as radioactivity levels, chemical toxicity, and ear thickness was also taken into account to evaluate the data.
Every attempt was made to determined the EC3 value (theoretical concentration resulting in a SI value of 3) as requested by the Ministry. Given sufficient data, the EC3 would be determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation to be used for calculation of EC3 is:
EC3 = c + [(3 – d)/(b – d)] x (a – c)
Where a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.
Categorization of contact allergens on the basis of relative skin sensitization potency, using EC3 values derived from the LLNA are listed in the table below.


TREATMENT PREPARATION AND ADMINISTRATION:
- Prior to the in-life phase, solubility assays were performed by CiToxLAB North America to verify solubility of the test item at the concentration of 25% (w/v) in N, N¬-dimethylformamide (DMF). As a solution to the naked eye at 25% was obtained, the test item was not tested at lower concentrations in the same vehicle.
- The test item was administered as a solution in the vehicle. As required, the test item was grounded to a fine powder and then mixed by vortex or under magnetic agitation, with the required quantity of vehicle. The test item concentrations were expressed in % (w/v).
-For both phases, on Days 1, 2, and 3, a dose-volume of 25 µL of the positive control, negative/vehicle control, or test item dosage formulations was applied to the dorsal surface of both ears (one concentration/ear in the Preliminary Phase, and one concentration/animal in the Main Test Phase), using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane ((2-chloro-2-(difluoromethoxy)-1, 1, 1-trifluoro-ethane) anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed. On Day 2 of the preliminary test, a small leakage from right ear was observed on animal No. 2502. This was considered without impact on the study as the results for this animal were consistent and dosing is performed for 3 days on the same ears and as such would not significantly affect the results.
The dosing formulations were stirred continuously throughout the dosing procedure. Any dosing formulations remaining after each daily dosing were discarded.

Positive control substance(s):

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

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group
(SI = 7.78). The experiment was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Group SI Negative control (AOO) 1 Negative control (DMF) 1 Positive control (HCA) 25% 7.78 Test item 0.5% 0.42 Test item 1% 0.45 Test item 2.5% 0.84 Test item 5% 1.96 Test item 10% 1.80

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Group nbre of node DPM/group DPM/node Negative control (AOO) 8 221.5 27.69 Negative control (DMF) 8 271.6 33.95 Positive control (HCA) 25% 8 1722.6 215.33 Test item 0.5% 8 113.5 14.19 Test item 1% 8 121.8 15.23 Test item 2.5% 8 228.3 28.54 Test item 5% 8 532.2 66.53 Test item 10% 8 489.1 61.14

Results 3

Parameter:

SI

Value:

ca. 0.42

Test group / Remarks:

4 female / concentration 0.5 %

Results 4

Parameter:

SI

Value:

ca. 0.45

Test group / Remarks:

4 female / concentration 1 %

Results 5

Parameter:

SI

Value:

ca. 0.84

Test group / Remarks:

4 female / concentration 2.5 %

Results 6

Parameter:

SI

Value:

ca. 1.96

Test group / Remarks:

4 female / concentration 5 %

Results 7

Parameter:

SI

Value:

ca. 1.8

Test group / Remarks:

4 female / concentration 10 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Under the experimental conditions of this study, the test item, L-Isoleucine, N-(1-oxohexadecyl), gave a negative results in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Executive summary:

The objective of this study was to evaluate the potential of the test item, L-Isoleucine, N-(1-oxohexadecyl),to induce contact hypersensitivity, using the murine Local Lymph Node Assay (LLNA).

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of 2 female mice received the test item, L-Isoleucine, N-(1-oxohexadecyl) dissolved inN,N-dimethylformamide (DMF), by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 25, 10, 5, or 2.5 % with a dose-volume of 25µL. From Day 1 to Day 3 and then on Day 6, the thickness of both ears of each animal was measured and the local reactions recorded. On completion of the observation period, the animals wereeuthanized per the standard operating procedures and the carcasses appropriately disposed of without post-mortem investigations.

Based on the preliminary test results, the test, negative, and positive control items in the main test, were administered daily from Day 1 to Day 3 inclusive as shown below:

Group	Number of animals	Administration	Concentration (%)
3	4 females	Negative Control (AOO, 4:1 (v/v))	0
4	4 females	Negative Control (DMF)	0
5	4 females	Positive Control Item^a	25% of HCA
6	4 females	Low Dose	0.5^b
7	4 females	Low-mid Dose	1.0^b
8	4 females	Mid Dose	2.5^b
9	4 females	Mid-high Dose	5.0^b
10	4 females	High Dose	10.0^b

a:α-hexyl cinnamaldehyde (HCA) in Acetone:Olive Oil, 4:1 v/v (AOO).

b: Was chosen according to the results obtained in the preliminary test.

DMF:N, N-dimethylformamide

Following the end of the 3-day administration period, main phase animals were euthanized on Day 6 and ear thickness measurements and LLNA was performed.

There were no L-Isoleucine, N-(1-oxohexadecyl)-related mortalities or body weight changes observed.

The SI of the positive control was >3; this experiment was therefore considered valid

A summary of the test and positive control items results are presented in the following table

Main Test Phase Results

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item (in DMF)	0.5	I	0.42
Test item (in DMF)	1.0	I	0.45
Test item (in DMF)	2.5	II	0.84
Test item (in DMF)	5.0	II	1.96
Test item (in DMF)	10.0	II	1.80
HCA (in AOO)	25	I	7.78
Negative control (AOO)	-	I	1.00
Negative control (DMF)	-	I	1.00

No notable lymphoproliferation was noted with the test item at any tested concentrations (0.5 to 10%). No EC₃value could be determined in the absence of a SI ≥ 3. Under the experimental conditions of this study, the test item, L-Isoleucine, N-(1-oxohexadecyl), gave a negative results in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.