(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September to 18 November 2003

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

EC Number:

615-206-1

Cas Number:

709031-45-8

Molecular formula:

C6H10N2O.CH4O3S

IUPAC Name:

(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

Test material form:

solid: particulate/powder

Details on test material:

Batch no: AAQ0195UN

Specific details on test material used for the study:

Idenitity: BMS 482204-03
Appearance: Powder
Storage conditions: Room temperature in the dark
Batch Number PRF02-19-3
Expiry: 11 March 2004

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Animals were in the weight range 17.8 - 22.4 g and approximately eight to twelve weeks of age prior to dosing on Day I. All the mice were acclimatised to the experimental environment for 6 days prior to the start of the study. The mice were allocated without conscious bias to cages within the treatment groups. They were housed individually in polycarbonate cages with woodflake bedding in Building F21, Room 12. The mice were also given Nestlets and untreated wood blocks for environmental enrichment.
A standard laboratory rodent diet (Special Diet Services RMI (E) SQC) and drinking water were provided ad libitum.
The batches of the diet used for the study were analysed by the supplier for nutrients, possible contaminants and micro-organisms, likely to be present in the diet, and which, if in excess, may have had an undesirable effect on the test system. The certificates of analyses were lodged in Huntingdon Life Sciences Ltd. Archives. Water is supplied in conformity with UK Water Act 1989 and subsequent amendments. The certificates of analyses were lodged in Huntingdon Life Sciences Ltd. Archives.

Animal room environmental controls were set to maintain temperature within the range 21 ± 2°C and relative humidity within 40 to 70%. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. These environmental parameters were continuously recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (0600 - 1800 hours GMT) in each 24 hour period.

Each cage was identified by a coloured label displaying the dose level, study number, animal mark and the initials of the Study Director and Home Office licensee. Mice were identified individually within each group by tail marking.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

The maximum practical concentration for pinna dosing was 25% w/v in Acetone: Olive Oil (4:1 v/v). Based on this information the following concentrations were selected: 5, 10 and 25 % w/v

No. of animals per dose:

Five mice per group

Details on study design:

The test substance formulations were prepared in Acetone:Olive Oil (4:1 v/v) at the required concentrations freshly on each day of dosing. The absorption of the test substance was not determined.
Chemical analysis of the homogeneity, stability and purity of the test substance was not undertaken as part of this study and remains the responsibility of the Sponsor

Groups of five mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µI of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. In addition, a group of five mice was similarly dosed with the vehicle alone in the same manner.

Administration of 3H-methyl Thymidine:
Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 µI of phosphate buffered saline containing 3H-methyl Thymidine1 fHTdR: 80 µCi/ml) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle (26 gauge) after the mouse had been heated in a warming chamber.

Termination
Five hours following the administration of 3HTdR on Day 6 all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised for each experimental animal. 1.0 ml of phosphate buffered saline was added to the lymph nodes for each animal. The animals were then discarded and no further investigations were carried out.

Preparation of single cell suspensions
A single cell suspension of lymph node cells (LNC} was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding JO ml phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 ml trichloroacetic acid (TCA: 5%) following the final wash.

Determination of incorporated 3H-methyl Thymidine
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 ml 5% TCA and transferred to 10 ml Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by !3-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for vehicle control nodes (test/control ratio).

Positive control substance(s):

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

Statistics:

Results for each treatment group were expressed as the stimulation index. This was obtained by comparing the proliferation in the vehicle treated control group with the values from the three test groups as follows: the ratio of3HTdR incorporation into lymph node cells, expressed as dpm, relative to that recorded for control lymph nodes is derived for each test group based on the group mean dpm per node. When the stimulation index d, the test substance is regarded as a skin sensitizer with a consideration given to dose response and statistical significance.
Analysis of variance was carried out on the data, from Groups 1, 2, 3 and 4, with treatment group as the factor of interest. If Bartlett's test for homogeneity of variance was significant at the 1 % level, the data were logarithmically transformed prior to analysis in order to stabilise the variances (Bartlett 1937). Comparisons were made between the control and BMS 482204-03 treated groups using Dunnett's test. To investigate the nature of the dose response curve the linear contrast was isolated.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.4

Test group / Remarks:

5%

Results 2

Parameter:

SI

Value:

1.8

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

1.5

Test group / Remarks:

25%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

BMS 482204-03 is not regarded as a potential skin sensitizer.

Executive summary:

The study was performed to assess the skin sensitization potential of BMS 482204-03 (MSA salt) using the murine local lymph node assay (LLNA). The study was performed in compliance with:

OECD Guideline for Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay". (24 April 2002).

EPA Health Effects Test Guidelines OPPTS  870.2600 Skin Sensitization EPA 712-C-03-197. March 2003.

 

ICCVAM (Interagency Co-ordinating Committee on the Validation of Alternative Methods).  (1999). The murine local lymph node assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds.  NIH Publication No. 99-4494, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA. The procedure followed was based on the method described in:

KIMBER I., HILTON J. and WEISENBERGER C. (1989). The murine local lymph node assay for identification of contact allergens:   A preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis 21, 215-220.

BASKETTER D.A. and SCHOLES E.W. (1992). Comparison of the local lymph node assay with the guinea-pig maximisation test for the detection of a range of contact allergens.   Food and Chemical Toxicology 30, 65-69

In this study, four groups of five mice were treated as follows: Control (Acetone:Olive Oil 4:1 v/v), 5%, 10% and 25 w/v

Each group of mice was treated by daily application of 25µl of each of the respective test or control (negative) material, to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine by fl-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC oftest nodes relative to that recorded for control nodes (test/control ratio). The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3HTdR incorporation compared to the vehicle control values. In this assay the test/control ratios obtained for 5, 10 and 25 % w/v were 1.4, 1.8 and 1.5 respectively which indicates that BMS 482204-03 (MSA salt) did not show the potential to induce skin sensitization (delayed contact hypersensitivity).

 Responses to the positive control substance hexyl cinnamic aldehyde (HCA), in a contemporaneous study (addendum) demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.