Sodium 5-oxo-1-palmitoyl-L-prolinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.August.2002-02.September.2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

The relative humidity recorded in the animal room was sometimes outside of the target ranges specified in the study plan. CIT became CiToxLAB France, data referring to expiry date or stability of the test item were missing to characterize the test item.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

no

Remarks:

No Study Director was assigned from 06 October 2004 to 05 March 2014. The final study plan was not signed by the Study Monitor and the sponsor did not provide expiry data of the test item. These deviations were considered not to be GLP compliance.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 5-oxo-1-palmitoyl-L-prolinate

EC Number:

261-406-3

EC Name:

Sodium 5-oxo-1-palmitoyl-L-prolinate

Cas Number:

58725-33-0

Molecular formula:

C21H38NNaO3.Na

IUPAC Name:

sodium (2S)-1-hexadecanoyl-5-oxopyrrolidine-2-carboxylate

Test material form:

liquid

Remarks:

brown liquid at receipt and then solid

Details on test material:

Test item
Supplier : Société Seppic
Name : LCA02011
Batch number
- Study plan : 1179001
- labeling : none
Description : brown liquid at receipt and then solid
Container : one plastic flask
Date of receipt :05 August 2002
Storage condition : at room temperature

At the finalisation of the study report, no analytical certificate was available. Consequently, characterisation of the test item was not complete since no expiry date and no stability data were given.

Specific details on test material used for the study:

Test item
Supplier : Société Seppic
Name : LCA02011
Batch number
- Study plan : 1179001
- labeling : none
Description : brown liquid at receipt and then solid
Container : one plastic flask
Date of receipt :05 August 2002
Storage condition : at room temperature

At the finalisation of the study report, no analytical certificate was available. Consequently, characterisation of the test item was not complete since no expiry date and no stability data were given.
The batch No." 1179001" which was absent from the label on the container was confirmed by the Study Monitor on a statement dated 22 May 2003.

Vehicle
The vehicle used was a mixture acetonefolive oil (4/1, viv): acetone, batch No. 0126152 (Carlo Erba, Rueil-Malmaison, France) and olive oil, batch No. 050K6072 (Sigma, Saint-Quentin-Fallavier, France).

Reference item
The reference item was O.-hexylcinnamaldehyde (HCA), batch No. 01.016AQ (Aldrich, Saint-Quentin-Fallavier, France), dissolved in a mixture acetonefolive oil (4/1, Vfw) at the concentration of 25% (v/v). The preparation was made freshly on the morning of administration and any unused material was discarded that same day.

Reactive used for the proliferation assay
The reactive used for the proliferation assay was IHI methyl-thymidine (H-TdR). batch No. B464B(Amersham, Les Ulis, France).
Three days before the injections, the required quantity of H-TdR was diluted in 0.9% NaCl
(20 uCi in 250 L of 0.9% NaCl per animal). The obtained solution was stored at +4°C and protected from light before use.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Animals:
Animals Species, strain and Sex: CBAJ mouse, nulliparous and non-pregnant females.
Reason for this choice: species generally accepted by regulatory authorities for this type of study. Females have been chosen since this sex is recommended by regulatory authorities for this type of study.
Breeder:Janvier, Le Genest-Saint-Isle, France.
Number: 20 females.
Age/weight: on the first day of treatment, the animals were approximately 9 weeks old and had a mean body weight I standard deviation of 20.6 g + 0.7 g.
Acclimation: at least 5 days before the beginning of the study,
Allocation:on day 1, animals were assigned to the treatment groups by hand procedure.
Identification: individually by a number on the tail,

Environmental conditions:
The conditions in the animal room were set as follows:
temperature : 22 + 2°C
relative humidity : 30 to 70%,
light/dark cycle : 12h/12 h,
ventilation : approximately 12 cycles/hour of filtered, non-recycled air. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the Supplier for composition and contaminant levels.

Food and water
All animals had free access to A04 C peleted diet (UAR, Villemoisson, Epinay-sur-Orge, France) and tap water (filtered using a 0.22 micron filter) contained in bottles. Each batch of food is analyzed by the supplier for composition and contaminant levels. The diet formula is presented in Appendix 1.
Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by external laboratories. The results of these analyses are archived at CiToxLAB France.
No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

1,3 and 5%

No. of animals per dose:

4 animals

Details on study design:

PRE-SCREEN TESTS:
-Solubility: The test item at the concentration of 5% was freely soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v)
- Irritation: The test item, vehicle or reference item were applied over the ears (25 microl per ear) for three consecutive daus (days 1,2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).
- Ear thickness measurements: days 1,2,3 and 6

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is > 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results. Whenever possible, calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.

TREATMENT PREPARATION AND ADMINISTRATION:
Treatment preparation
The vehicle used was a mixture acetonefolive oil (4/1, viv): acetone, batch No. 0126152 (Carlo Erba, Rueil-Malmaison, France) and olive oil, batch No. 050K6072 (Sigma, Saint-Quentin-Fallavier, France).

The test item was prepared in the vehicle at the chosen concentrations. The concentrations were expressed in % (wiv). All dosage form preparations were made freshly on the morning of administration and stored under nitrogen gas (on days 2 and 3). Any unused material was discarded that same day.

The reference item was O.-hexylcinnamaldehyde (HCA), batch No. 01.016AQ (Aldrich, Saint-Quentin-Fallavier, France), dissolved in a mixture acetonefolive oil (4/1, Vfw) at the concentration of 25% (v/v). The preparation was made freshly on the morning of administration and any unused material was discarded that same day.

The reactive used for the proliferation assay was IHI methyl-thymidine (H-TdR). batch No. B464B(Amersham, Les Ulis, France).
Three days before the injections, the required quantity of H-TdR was diluted in 0.9% NaCl
(20 uCi in 250 L of 0.9% NaCl per animal). The obtained solution was stored at +4°C and protected from light before use.

Administration
On days 1, 2 and 3, a dose-volume of 25 L of the control or dosage form preparations was applied to the dorsal surface of both cars, 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 anesthezia during the administration, No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Statistics:

no data

Results and discussion

Positive control results:

In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI= 13.53) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 13.53

Test group / Remarks:

positive control / concentration 25%

Results 2

Key result

Parameter:

SI

Value:

ca. 1.22

Test group / Remarks:

treated group / concentration 1%

Results 3

Key result

Parameter:

SI

Value:

ca. 1.44

Test group / Remarks:

treated group / concentration 3%

Results 4

Key result

Parameter:

SI

Value:

ca. 1.55

Test group / Remarks:

treated group / concentration 5%

Results 5

Key result

Parameter:

EC3

Value:

ca. 22.2

Test group / Remarks:

The EC3 value (%) of the test item calculated from the SI values obtained at three tested concentrations

Cellular proliferation data / Observations:

Proliferation Assay:
The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of H-TdR. The cell viability was higher than 80% in each group.
In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI it 3.53) were noted. The study was therefore considered valid.
In the treated groups, a very slight dose-related increase in the stimulation index but which never reached the threshold positive value of 3 was noted. Therefore, under our experimental conditions, the test item at concentrations a 5% does not induce delayed contact hyperSensitivity in the murine Local Lymph Node Assay.

Determination of the EC3 value:
The EC3 value of the test item calculated from the SI values obtained at the three testedi concentrations was equal to 22.2% (see Appendix 2). However, as the maximum concentration tested in this study was 5%, further investigations at concentrations higher than 5% may be necessary to confirm this EC3 value.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under our experimental conditions, the test item LCA02011 should not be considered as a skin sensitizer.

Executive summary:

The aim of this study was to evaluate the potential of the test item ICA02011 to induce delayed contact hypersensitivity using the murine Local Lymph Node ASSay (LLNA).

Methods

At the request of the Sponsor, twenty female CBAJ mice were allocated to five groups of four animals each:

• three treated groups receiving the test item LCA02011 at the concentrations of 1, 3 and 5%,


• one negative control group of four animals receiving the vehicle (mixture acetonefolive oil (471)),


• one positive control group receiving the reference item, O.-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

The test item, vehicle or reference item were applied over the ears (25 u per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days i. 2, 3 and 6.

Results

At the request of the Sponsor, the test item was tested at the maximum concentration of 5%. The test item at the concentration of 5% was freely soluble in the first recommended vehicle, acetonefolive oil (4/1, viv).

Systemic clinical signs and mortality

No mortality and no clinical signs were observed during the study.

Local irritation

No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups.

Proliferation assay

In the treated groups, a very slight dose-related increase in the stimulation index but which never reached the threshold positive value of 3 was noted. Therefore, under our experimental conditions, the test item LCA02011 at concentrations < 5% did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

The EC value of the test item calculated from the SI values obtained at the three tested concentrations was equal to 22.2% (see Appendix 2). However, as the maximum concentration tested in this study was 5%, further investigations at concentrations higher than 5% may be necessary to confirm this EC3 value.

Conclusion

Under our experimental conditions, the test item LCA02011 should not be considered as a skin Sensitizer.