[1,1'-Biphenyl]-4-pentanoic acid, .gamma.-[(3-carboxy-1-oxopropyl)amino]-.alpha.-methyl-, 4-ethyl ester, calcium salt (2:1), (.alpha.R,.gamma.S)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9-23 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

AHU377

Cas Number:

1369773-39-6

IUPAC Name:

AHU377

Details on test material:

Molecular formula
C48H56CaN2O10

Molecular weight
861.04

Description
White to off-white powder

Batch
0722008

Content of drug
98.9%

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Animal species and strain:
Mouse, Balb/c strain, inbred, SPF-Quality
Breeder/supplier:
Charles River France, L’Arbresle Cedex, France
Number of animals
30 females.
Age:
Approximately 8 weeks (at initiation of treatment).
Body weight range:
18 to 22 gram (at initiation of treatment).
Health Check:
A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Animal quarters/husbandry
Location:
Specific Pathogen Free area: animal rooms no. 18 and 19.
Room temperature:
18.2 to 22.5° C.
Room relative humidity:
40 to 71%. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Air changes:
Approximately 15 per hour.
Lighting cycle:
Fluorescent light for a 12-hour light/12-hour dark cycle.
Animal caging:
Group housing in Macrolon cages (MIII type; height 18 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Animals were inadvertently individually housed in labeled Macrolon cages (MI type; height 12.5 cm) during the dosing period from Day 1 to Day 3.
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
Cage enrichment:
Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment during group housing.
Analysis of paper:
Results of analysis for each batch of paper did not reveal any findings that were considered to have affected the study integrity.
Food:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.
Analysis of food:
Results of analysis for each batch of diet (nutrients and contaminants) were assessed and did not reveal any findings that were considered to have affected the study integrity.
Water:
Free access to tap-water.
Analysis of water:
Certificates of analysis were examined and archived. Analysis of water did not reveal any findings that were considered to have affected study integrity.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Test item concentrations selected for the main study were based on the results of a preliminary study.
In the main study, six Balb/c female mice per group received topical test and control items on the dorsum of both ears once a day on 3 consecutive days. Auricular lymph nodes were taken 24 hours after the last application. Endpoints: visual examination ears and sizes ear-draining lymph nodes, body weight at treatment start and day of necropsy, ear weight (skin irritation), ear-draining lymph node weights and cell counts (LN hyperplasia). Concentrations: vehicle control Dimethyl formamide, positive control DNCB (1-Chloro-2,4-Dinitrobenzene): 0.5% (w/w), and AHU377: 50%, 5%, 0.5% (w/w).

No. of animals per dose:

6

Details on study design:

Induction - Days 1, 2 and 3
For the experimental animals, the dorsal surface of both ears was epidermally treated (25 µl/ear) with the test item concentration, approximately the same time each day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated as described for the experimental animals, except that, instead of the test substance, the vehicle or positive control item was administered.

Weighing of ear punches and lymph nodes - Day 4
Approximately 24 hours after the last treatment, all animals were sacrificed by intra-peritoneal injection with pentobarbital (0.2 ml/animal Euthesate®; Sanofi Sante B.V., Maassluis, The Netherlands).
Both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 8 mm => 0.5 cm2). For each animal both punches were immediately weighed pooled per animal using an analytical balance after which the punches were discarded.
Both auricular draining lymph nodes (left and right) of mice were excised. The relative sizes of the nodes (as compared to normal) were estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. For each animal both lymph nodes were pooled and immediately weight using an analytical balance.

Determination of total cell-counts of lymph nodes – Day 4
Following excision and weighing of the nodes, single cell suspensions of lymph node cells (LNC) were prepared in phosphate buffered saline (PBS) by gentle separation through stainless steel gauze (diameter 200 m mesh). LNC were collected in approximately 0.7 ml of PBS in a 24 wells plate that was kept on ice as much as possible.
Cell counts were determined using a Coulter Counter Z1 Dual (Beckman Coulter, The Netherlands).

Positive control substance(s):

other: 1-CHLORO-2,4-DINITROBENZENE

Statistics:

Calculations were performed in MS EXCEL and statistical analysis was performed with GraphPad Prism 4 (Kruskal-Wallis test, followed by the Mann Whitney test).

Results and discussion

Positive control results:

The positive control item DNCB elicited a reaction pattern with increased LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

0.5% AHU377

Results 2

Key result

Parameter:

SI

Value:

1.39

Test group / Remarks:

5% AHU377

Results 3

Key result

Parameter:

SI

Value:

2.07

Test group / Remarks:

50% AHU377

Results 4

Parameter:

other: Ear weight index

Value:

0.92

Test group / Remarks:

0.5% AHU377

Results 5

Parameter:

other: Ear weight index

Value:

0.98

Test group / Remarks:

5% AHU377

Results 6

Parameter:

other: Ear weight index

Value:

1.49

Test group / Remarks:

50% AHU377

Results 7

Parameter:

other: LN weight index

Value:

1.05

Test group / Remarks:

0.5% AHU377

Results 8

Parameter:

other: LN weight index

Value:

1.22

Test group / Remarks:

5% AHU377

Results 9

Parameter:

other: LN weight index

Value:

1.58

Test group / Remarks:

50% AHU377

Results 10

Key result

Parameter:

other: Cell count index

Value:

1.03

Test group / Remarks:

0.5% AHU377

Results 11

Key result

Parameter:

other: Cell count index

Value:

1.39

Test group / Remarks:

5% AHU377

Results 12

Key result

Parameter:

other: Cell count index

Value:

2.07

Test group / Remarks:

50% AHU377

Cellular proliferation data / Observations:

No irritation of the ears was noted after visual examination in the majority of animals, except for the very slight irritation on one ear of one animal treated at 50%. Visual examination of the nodes revealed that all the nodes of the experimental and positive control animals were enlarged when compared to the vehicle control group. No macroscopic abnormalities of the surrounding areas were noted in any of the animals.
Statistically significant body weight loss was noted in the 0.5% group in comparison to the vehicle control. There were no clinical observations attributable to treatment with AHU377.
The positive control item DNCB elicited a reaction pattern with increased LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.
AHU377 did show a statistically significant decrease in ear weight at 0.5% and a statistically significant increase at 50% concentration.
AHU377 did cause dose related increases in LN weights and cell counts when compared to the vehicle control.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, AHU377 appeared to be a weak sensitizer with weak irritating potential in the murine LLNA TIER 1. However the criteria for classification are not met as a 3 fold increase in cell proliferation was not observed at any of the doses tested in the LLNA assay.