Iron(1+), chloro[dimethyl 9,9-dihydroxy-3-methyl-2,4-di(2-pyridinyl-kN)-7-[(2-pyridinyl-kN)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate-kN3,kN7]-, chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 April 2004 to 26 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

Dated 24th April 2002

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:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

EC Number:

610-388-9

Cas Number:

478945-46-9

Molecular formula:

C28H31N5O6FeCl2

IUPAC Name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Unilever, batch no. S2539801
- Expiration date of the lot/batch: 1 January 2005

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container at the room temperature (20 deg Celsius ± 3 deg Celsius). Keep in dark.
The supporting data for purity (characterisation), stability and homogeneity of the test item were not made available at the time of issuing this report. However the sponsor is adressing this in a GLP compliant study SEAC Study reference No. AC030449.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation:16-24g
- Housing: Individually in Makrolon type-2 cages
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 ad libitum
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum
- Acclimation period: 6 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 deg C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h with at least 8h music during light period

- IN-LIFE DATES: From: 5 May 2004 To: 17 May 2004

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 0.25, 0.5 and 1% (w/v)

No. of animals per dose:

5 animals were used per conditions

Details on study design:

PRE-SCREEN TESTS:
No pre-screen test was performed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A test item is regarded as a sensitizer if the following criteria are fullfilled :
-Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3 fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
-The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance. The weight/volume dilutions were prepared individually. Test item formulation was made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity was maintained during treatment by use of magnetic stirrer.
Each test group of mice was treated with (epidermal) topical application to the dorsal surface of each lobe (left and right) with the test item. A further three groups of mice were treated with an equal volume of either, the positive control item dilution, the positive control vehicle (AOO) or the negative control material (DMF). The application volume, 25 µL, was spread over the entire dorsal surface (diameter 8 mm) of each lobe once daily for the three consecutive days. A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.
3HTdR was administered five days after the first topical application at 250µLby intravenous injection via a tail vein.

Positive control substance(s):

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

Statistics:

The mean body weights and mean DPM/mouse values for each test and control group were calculated. Standard deviations of the data used to determine these mean values were calculated.
The t-test was conducted for the assessment of significant differences between the positive control item group and its vehicle control group. The Dunnett-test was used for the assessment of the significant differences between the test item groups and the vehicle control group.

Results and discussion

Positive control results:

In this study, a Stimulation Index (SI) of 11.0 was obtained with the positive control item at a concentration of 25% (w/v) in acetone:olive oil , 4:1 (v/v). This SI confirmed that HCA is a skin sensitizer.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 11

Variability:

SD : 2..6

Test group / Remarks:

Positive Control

Results 2

Key result

Parameter:

SI

Value:

ca. 3.4

Variability:

0.5

Test group / Remarks:

Test item at 0.25%

Results 3

Key result

Parameter:

SI

Value:

ca. 3.9

Variability:

SD : 1.3

Test group / Remarks:

Test item at 0.5%

Results 4

Key result

Parameter:

SI

Value:

ca. 4.5

Variability:

1.3

Test group / Remarks:

Test item at 1%

Cellular proliferation data / Observations:

No clinical signs were observed in any animals of the two vehicle control groups or the three test item groups. On the second application day, a slight ear erythema was observed at both dosing sites in all mice of the positive control group, persisting for a total of three days. In addition, on the third application day, a slight ear swelling was observed at both dosing sites in all mice of this group, persisting for a total of two days.
An EC3 value could not be determined because this calculation requires an SI value of less than 3.

Any other information on results incl. tables

Table 1 :Summary of results

Group	DPM/mouse	SI
DMF	598±135
AOO	691±190
HCA 25%	7565±1770	11.0±2,6**
Test item 0.25%	2059±280	3,4±0,5**
Test item 0.5%	2324±803	3,9±1,3**
Testitemt1%	2691±758	4,5±1,3**

 

 

 

 

 

** p <0.01

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions of the study, the test item induced delayed hypersensibility contact for each concentration tested. The test item induced a SI of 3.4, 3.9 and 4.5 for respectively 0.25, 0.5 and 1% dose level. hence, the test item Stainless E-700-2003 was classified as Catergory 1A sensitizer according to CLP regulation.

Executive summary:

The purpose of this GLP-compliant study was to assess the potential sensitising effect of the test item using mice on a Local Lymph Node Assay (LLNA) according OECD guideline 429 method.

CBA/Ca mice were used and were topically treated with 0.25, 0.5 and 1% of the test item. Three control groups were used : one HCA positive control group, Acetone/Olive oil group and Dimethylformamide group.

Each test group of mice was treated with (epidermal) topical application to the dorsal surface of each lobe(left and right) with the test item. A further three groups of mice were treated with an equal volume of either, the positive control item dilution, the positive control vehicle (AOO) or the negative control material (DMF). The application volume, 25 µL, was spread over the entire dorsal surface (diameter 8 mm) of each lobe once daily for the three consecutive days. A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied. 3HTdR was administered five days after the first topical application at 250µLby intravenous injection via a tail vein. Level of incorporated 3HTdR was determined after sacrifice of the mice and the lymph node draining. A scintillation approach was used to quantified the incorporated 3HTdR. Stimulation Index was determined.

Under the experimental conditions of the study, the test item induced delayed contact hypersensibility for each concentration tested. The test item induced a SI of 3.4, 3.9 and 4.5 for respectively 0.25, 0.5 and 1% dose level. Hence, the test item Stainless E-700-2003 was classified as Catergory 1A sensitizer according to CLP regulation.