Copper, .beta.-resorcylate salicylate lead complexes

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2010 - 26 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Evaluation of skin sensitisation potential in mice using Local Lymph Node Assay (LLNA)

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:

yes

Remarks:

on day 6, the lymph nodes of group 6 were pooled with those of another study, consequently, data obtained after lymph node samples can not be exploited

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

idem above

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:

Copper Lead Resorcylate Salicylate Complex

EC Number:

614-455-3

Cas Number:

68411-07-4

Molecular formula:

C7H5O4-, C7H5O3- Cu , Pb

IUPAC Name:

Copper Lead Resorcylate Salicylate Complex

Details on test material:

- Name of test material (as cited in study report): Lead-Cooper-Resorcylate-Salicylate (LC 12-15)
- Substance type: multicontituent
- Physical state: slightly pale green powder
- Composition of test material, percentage of components: copper: 11.9%, lead: 35.7%, resorcylate: 14.1%, salicylate: 35.7%
- Lot/batch No.: 09/201
- Expiration date of the lot/batch: 04 November 2011
- Storage conditions of test material: at room temperature.

Specific details on test material used for the study:

Name of test material (as cited in study report): Lead-Cooper-Resorcylate-Salicylate (LC 12-15)
- Substance type: multicontituent

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: approximately 11 weeks old
- Weight at study initiation: 22.4 ± 1.2 g in the first experiment and 22.0 ± 0.9 g in the second experiment
- Housing: housed individually in disposable crystal polystyrene cages
- 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

IN-LIFE DATES: From: 01 June 2010 To: 24 August 2010.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0.05, 0.10, 0.25, 0.5 and 1%

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, methylethylketone and propylene glycol, dimethylsulfoxide was chosen from the other proposed vehicles. A
homogeneous
suspension was obtained at the maximum concentration of 25%.
- Irritation: an increase in ear thickness was recorded at the concentrations of 2.5% to 25%, showing the irritant potential of the
test item at these
concentrations. The highest concentration retained for the main test was therefore 1%.
- Lymph node proliferation response: not performed.
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Stimulation Index (SI) >= 3 (wih exclusion of excessive irritation).
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared at the chosen concentrations in DMSO 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).
On days 1, 2 and 3 of each experiment, 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)

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 3.74 in the first experiment and = 6.42 in the second experiment).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

6.42

Results 2

Key result

Parameter:

SI

Value:

3.74

Results 3

Key result

Parameter:

SI

Value:

3

Variability:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 3.74 in the first experiment and = 6.42 in the second experimen

Cellular proliferation data / Observations:

Disintegrations per minute (DPM)
DPM per group:
Group 1: Vehicle: 5798.00 (first experiment)
Group 2: test item 0.05%: 4947.00 (first experiment)
Group 3: test item 0.10%: 6677.00 (first experiment)
Group 4: test item 0.25%: 8897.00 (first experiment)
Group 5: test item 0.50%: 12858.00 (first experiment)
Group 7: HCA 25%: 21699.00 (first experiment)
Group 8: vehicle: 1150.00 (second experiment)
Group 9: test 1%: 2350.00 (second experiment)
Group 10: HCA 25%: 7382.00 (second experiment)

Stimulation index
Group 2: test item 0.05%: 0.85
Group 3: test item 0.10%: 1.15
Group 4: test item 0.25%: 1.53
Group 5: test item 0.50%: 2.22
Group 9: test item 1%: 2.04

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Lead-Copper-Resorcylate-Salicylate (LC 12-15) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The aim of this study was to evaluate the potential of the test item, Lead‑Copper‑Resorcylate‑Salicylate (LC 12-15), to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice.

 

Methods

 

Preliminary tests were first performed in order to define the concentrations of test item to be used in the main test.

In the main test, 28 female CBA/J mice were allocated to seven groups:

.           five treated groups of four animals receiving the test item at the concentration of 0.05, 0.1, 0.25, 0.5 or 1% in dimethylsulfoxide (DMSO; 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) (AOO).

 

Due to technical problem, data obtained at the concentration of 1% can not be exploited. Consequently, a complementary experiment was performed as follows:

Twelve female CBA/J mice were allocated to three groups:

.           one treated group of four animals receiving the test item at the concentration of 1% in DMSO,

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

.           one positive control group of four animals receiving the reference item at the concentration of 25% in AOO.

 

In each experiment, 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

During the solubility assay, dimethylsulfoxide was chosen from the other proposed vehicles. A homogeneous suspension was obtained at the maximum concentration of 25%.

 

Consequently, the concentrations selected for the preliminary test were, 2.5, 5, 10 and 25%. Since the test item was irritant at the concentrations of 2.5 to 25%, another assay was performed at the concentrations of 0.1, 0.25, 0.5 and 1%.

Since the test item was non-irritant at these concentrations, the highest concentration retained for the main test was 1%.

 

Neither mortality nor clinical signs were observed during the study.

The body weight change of treated animals was similar to that of control animals.

Dryness of the skin was noted on day 6 in 1/4 females given 0.5% and 2/4 females given 1%, as well as in 1/8 females given the vehicle.

No excessive increase in ear thickness was observed at any of the tested concentrations.

 

As all acceptance criteria were met, both experiments were therefore considered valid.

 

The results are presented in the following table:

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.05	slightly irritant	0.85
Test item	0.1	slightly irritant	1.15
Test item	0.25	non-irritant	1.53
Test item	0.5	slightly irritant	2.22
HCA	25	-	3.74
Test item	1	slightly irritant	2.04
HCA	25	-	6.42

 

No notable lymphoproliferation was noted with the test item at any tested concentrations.

 

Conclusion

The test item, Lead-Copper-Resorcylate-Salicylate (LC 12-15), did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.