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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2010 to 01 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline (the observed deviation is not considered to have compromised the reliability of the study); adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
during the acclimation period, the relative humidity recorded in the animal room was sometimes outside of the target ranges (down to 15 % relative humidity)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
as cited above
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of barium chromate, copper dichromium tetraoxide and copper oxide
EC Number:
701-287-1
Molecular formula:
CuO, CuCr2O4, BaCrO4
IUPAC Name:
Reaction mass of barium chromate, copper dichromium tetraoxide and copper oxide
Details on test material:
- Name of test material (as cited in study report): Chromite de cuivre
- Substance type: multiconstituents
- Physical state: black powder
- Composition of test material, percentage of components: 34% of copper, 8.5% of baryum, 31% of chrome
- Purity test date: 11 January 2010
- Lot/batch No.: 09/0177
- Expiration date of the lot/batch: 17 August 2014
- Storage condition of test material: in darkness at room temperature

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: animals of the preliminary test were approximately 11-12 weeks old and the animals of the main test were approximately 9 weeks old
- Weight at study initiation: mean body weight ± standard deviation of 20.9 ± 1.3 g
- Housing: mice were housed individually in disposable crystal polystyrene cages
- Diet (e.g. ad libitum): 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°C +/-2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From 16 March 2010 to 29 March 2010

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 2.5, 5, 10, 25 and 50%
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test substance was not soluble in the first recommended vehicle (acetone/olive oil (4/1, v/v)). A homogeneous suspension was obtained at the maximum tested concentration of 50%. in dimethylformamide.
- Irritation: the test item was non-irritant in the preliminary test, whatever the concentration

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (based on the design adopted by ICCVAM (Interagency Coordination Committee on the Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard Identification and Risk Assessment, September 2000), with the addition of the evaluation of local irritation)
- Criteria used to consider a positive response: stimulation indice (SI) ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared at the chosen concentration in DMF by successive dilutions. The dosage form preparations were homogenized by vortex.
On days 1, 2 and 3, 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:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 6.59) were noted.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Value:
5.79
Test group / Remarks:
%
Parameter:
SI
Value:
1.57
Test group / Remarks:
2.5 %
Parameter:
SI
Value:
2.53
Test group / Remarks:
10 %
Parameter:
SI
Value:
5.51
Test group / Remarks:
25 %

Any other information on results incl. tables

A dose-related increase in the SI was recorded at all the concentrations, and the threshold positive value of 3 was exceeded at the concentration of 10%.

In the absence of excessive local irritation at the concentrations of 10 and 25%, the significant lymphoproliferative responses observed at these concentrations were attributed to delayed contact hypersensitivity.

The EC3 value for the test item Chromite de cuivre is equal to 5.79%.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item Chromite de cuivre induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained in this experiment, the test item Chromite de cuivre should be considered as a moderate sensitizer.
Executive summary:

Methods:

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

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

• five treated groups of four animals receiving the test item Chromite de cuivre at the concentration of 2.5, 5, 10, 25 or 50% in dimethylformamide (vehicle),

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

• one positive control group of four animals receiving the reference item, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

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:

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), dimethylformamide was chosen from the other proposed vehicles. A homogeneous suspension was obtained at the maximum tested concentration of 50%.

Consequently, the concentrations selected for the preliminary test were 5, 10, 25 and 50%.

Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (50%).

Systemic clinical signs and mortality

Neither mortality nor clinical signs were observed during the study.

Local irritation

A black coloration of the skin of the ears was noted on days 2 and 3 in all animals treated with the test item and on day 6 in all animals treated at the concentrations higher or equal to 5%. This coloration masked the evaluation of the cutaneous reactions on days 2 and 6 in animals treated at the concentrations higher or equal to 10% and on day 3 in animals treated at the concentrations higher or equal to 5%.

A significant increase in ear thickness (> 30%) was observed in the animals treated at the concentration of 50%.

Proliferation assay

A significant lymphoproliferation was noted in the positive control group given HCA at 25%, the study was therefore considered valid.

A dose-related increase in the SI was recorded at all the concentrations, and the threshold positive value of 3 was exceeded at the concentration of 10%.

The results are presented in the following table: 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

2.5

non-irritant

1.57

Test item

5

non-irritant

2.53

Test item

10

slightly irritant

5.51

Test item

25

non-irritant

8.42

Test item

50

irritant

20.40

HCA

25

-

6.59

 In the absence of excessive local irritation at the concentrations of 10 and 25%, the significant lymphoproliferative responses observed at these concentrations were attributed to delayed contact hypersensitivity.

The EC3 value for the test item Chromite de cuivre is equal to 5.79%.

Conclusion:

Under the experimental conditions of this study, the test item Chromite de cuivre induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.

According to the EC3 value obtained in this experiment, the test item Chromite de cuivre should be considered as a moderate sensitizer.

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