Reaction mass of sodium sulphate and disodium dodecanedioate and Dodecanoic acid, 12-amino-, sodium salt (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 July 2010 to ..............................

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (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:

Reaction mass of sodium sulfate, sodium amino-12-dodecanoate and sodium dodecanoediate

IUPAC Name:

Reaction mass of sodium sulfate, sodium amino-12-dodecanoate and sodium dodecanoediate

Details on test material:

- Name of test material (as cited in study report): Reaction mass of sodium sulfate, sodium amino-12-dodecanoate and sodium dodecanoediate
- Substance type: UVCB (substance of unknown or variable composition, complex reaction products or biological materials)
- Physical state: brown liquid
- Purity test date: 26 march 2009
- Lot/batch No.: T710/712
- Expiration date of the lot/batch: 20 September 2010
- Storage conditions of test material: at room temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary test were approximately 8 weeks old and the animals
of the main test were approximately 9 weeks old
- Weight at study initiation: 23.1 +/- 0.9 g
- Housing: housed individually in disposable crystal polystyrene cages. Each cage contained (except for the 5 hours following the 3H TdR injections) autoclaved sawdust
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (7:00 - 19:00).

IN-LIFE DATES: From: 13 July 2010 To: 26 July 2010

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25, 50 and 100%.

No. of animals per dose:

4 animals per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)),
dimethylformamide was chosen from the other proposed vehicles.
A homogeneous emulsion was obtained at the maximum tested concentration of 50%.
- Irritation: scabs were observed at the concentration of 100%. An increase in ear thickness was recorded at the concentration of 100%, showing the irritant potential of the test item at this concentration. The highest concentration retained for the main test was therefore 50%.
- Lymph node proliferation response: not examined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: SI >= 3

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared at the chosen concentrations in DMF by successive dilutions. The dosage form preparations were homogenized by vortex. The reference item was dissolved in AOO at the concentration of 25% (v/v).
All dosage form preparations were made freshly on the morning of administration and stored at room temperature prior to use.
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.

Positive control substance(s):

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

Statistics:

no

Results and discussion

Positive control results:

A significant lymphoproliferation was noted in the positive control group given HCA at 25%. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Group 2: test item 2.5%: 1.36 Group 3: test item 5%: 1.81 Group 4: test item 10%: 1.70 Group 5: test item 25%: 1.88 Group 6: test item 50%: 1.74

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

DPM per group: Group 1: Vehiche: 574.00 Group 2: test item 2.5%: 783.00 Group 3: test item 5%: 1041.00 Group 4: test item 10%: 977.00 Group 5: test item 25%: 1077.00 Group 6: test item 50%: 1001.00 DPM per node: Group 1: Vehiche: 71.75 Group 2: test item 2.5%: 97.88 Group 3: test item 5%: 130.13 Group 4: test item 10%: 122.13 Group 5: test item 25%: 134.63 Group 6: test item 50%: 125.13

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Reaction mass of sodium sulfate, sodium amino-12-dodecanoate and sodium dodecanoedioate (batch No. T710/712) did not induce
delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

Methods  

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, twenty eight female CBA/J mice were allocated to seven groups:

.           five treated groups of four animals receiving the test item Reaction mass of sodium sulfate, sodium amino-12-dodecanoate at the concentration of 2.5, 5, 10, 25 or 50% in dimethylformamide (vehicle),

.           one negative control group of four animals receiving the vehicle,

.           one positive control group of four animals receiving the reference item,a‑hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes 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 1, 2, 3 and 6.

Results

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), dimethylformamide was chosen from the other proposed vehicles. A homogeneous emulsion was obtained at the maximum tested concentration of 50%. Consequently, the concentrations selected for the preliminary test were 10, 25, 50 and 100%. Since the test item was irritant in the preliminary test at the concentration of 100%, the highest tested concentration retained for the main test was 50%.

Clinical signs and mortality : Neither mortality nor clinical signs were observed during the study.

Local irritation : On day 6, dryness of the skin of the ears was noted in 1/4 females given the concentration of 5%. No notable increase in ear thickness was observed in the animals of the treated groups.

Proliferation assay : A significant lymphoproliferation was noted in the positive control group given HCA at 25%. The study was therefore considered valid. No notable lymphoproliferation was noted at any tested concentrations.

The results are presented in the following table:

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	2.5	non-irritant	1.36
Test item	5	non-irritant	1.81
Test item	10	non-irritant	1.70
Test item	25	non-irritant	1.88
Test item	50	non-irritant	1.74
HCA	25	-	11.56

 

Conclusion

Under the experimental conditions of this study, the test item Reaction mass of sodium sulfate, sodium amino-12-dodecanoate and sodium dodecanoedioate (batch No. T710/712) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.