Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-06-06 to 2007-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(2004)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

EC Number:

264-761-2

EC Name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

Cas Number:

64265-45-8

IUPAC Name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

Test material form:

liquid

Details on test material:

- Analytical purity: 97%
- Physical state: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 22 - 27 g
- Housing: Individual housing in labeled Macrolon cages (Ml type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNlFF® Spezialdiaten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7- 23.0°C
- Humidity (%): 43 - 78%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

5

Details on study design:

PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest. A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the undiluted test substance. The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the ear was cleaned of residual test substance with tap water/vehicle and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Induction (days 1,2 and3): The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
- Treatment (day 6): Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 pCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital (0.2 mL/animal). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING AND MEASUREMENTS:
- Tissue processing for radioactivity (day 6): A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation
through stainless steel gauze (diameter 125 pm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4° C during the night.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM.

Positive control substance(s):

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

Results and discussion

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.5 and 5.5 respectively. An EC3 value of 15.6% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.9

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

6.8

Test group / Remarks:

50%

Results 3

Parameter:

SI

Value:

8.5

Test group / Remarks:

100%

Results 4

Parameter:

EC3

Value:

30.6

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

No irritation was observed in any of the animals examined. The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY

- Skin reactions/Irritation: No skin reactions were observed in any of the animals examined.

- Macroscopy of the auricular lymph nodes and surrounding area: The majority of nodes were considered normal in size, except for enlarged nodes of two animals at 50% and two animals at 100%. No macroscopic abnormalities of the surrounding area were noted.

- Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study

- Toxicity and Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.9, 6.8 and 8.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3 and Amphopropionate C8 is therefore regarded as skin sensitiser. The data showed a dose-response and an EC3 value of 30.6% was calculated.

Executive summary:

In a local lymph node assay according to OECD guideline 429 (2002) and EU Method B.42 (2004) Amphopropionate C8 dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 25, 50 and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.9, 6.8, and 8.5 were determined with the test item at concentrations of 25, 50, and 100% in dimethylformamide, respectively. These results indicate that the test substance could elicit an SI ≥ 3 and Amphopropionate C8 is therefore regarded as skin sensitiser under the conditions of this study. The data showed a dose-response and an EC3 value of 30.6% was calculated.

Based on these results Amphopropionate C8 is classified as Category 1B based on CLP, EU GHS (Regulation (EC) No 1272/2008).