Resin acids and Rosin acids, cobalt salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-04-11 to 2006-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002-04-24

Deviations:

no

Principles of method if other than guideline:

In addition to the Stimulation indices, the EC3 value (theoretical concentration resulting in a SI value of 3) was determined by linear interpolation of points on the dose-response curve, immediatly above and below the 3-fold threshold. The equation used for calculation of EC3 was: EC3 = c+ [(3-d)/(b-d)] x (a-c). According to a calculated EC value the relative skin sensitisation potency was determined (see table 2 for categorization of contact allergens; see "Any other information on materials and methods incl. tables").
Where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

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:

Resin acids and Rosin acids, cobalt salts

EC Number:

273-321-9

EC Name:

Resin acids and Rosin acids, cobalt salts

Cas Number:

68956-82-1

IUPAC Name:

λ²-cobalt(2+) bis((1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylate)

Details on test material:

- Name of test material (as cited in study report): Produit Y
- Name of test material (as cited in the EC inventory): Resin acids and Rosin acids, cobalt salts
- Physical state: Purple powder
- Storage condition of test material: At room temperature, protected from light and humidity and under argon gas.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: On the first day of the treatment period, the animals were approx. 9 weeks old.
- Weight at study initiation: On the first day of the treatment period, the animals had a mean body weight +/- standardd deviation of 21.2 +/- 1.2 g.
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22.00 cm X 8.50 cm X 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: Free access to SsniffR/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Relative humidity: 30 to 70 %
- Ventilation: approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12
No further information on the test material was stated.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

First preliminary test: 1, 2.5, 5 and 10 % (w/v)
Since the test item was irritant at all the first chosen concentrations, a second prelimiary test was performed.
Second preliminary test: 0.05, 0.1, 0.25 and 0.5 % (w/v)
Main test: 0.025, 0.05, 0.1, 0.25, 0.5 % (w/v)

No. of animals per dose:

2 females per concentration (preliminary test); 4 females per concentration (main test)

Details on study design:

RANGE FINDING TESTS:
Alopecia around the ears was noted at the concentration of 5 and 10 %.
A noteworthy increase in ear thickness was recorded at concentrations ≥ 1 %, showing the irritant potential of the test item at these concentrations.
The highest concentration retained for the main test was therefore 0.5 %.

CHOICE OF THE VEHICLE:
The test item was poorly soluble in the first recommended vehicel, acetone/olive oil (4/1, v/v), the limit of solubility being approximately 10 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the Stimulation Indices (SI) for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results. According to a calculated EC value the relative skin sensitisation potency was determined (see table 2 for categorization of contact allergens; see "Any other information on materials and methods incl. tables").

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared in the vehicle at the chosen concentration.
On days 1, 2 and 3, a dose-volume of 25µL 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. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9 % NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9 % NaCl and pellets obtained were re-suspended in exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima Gold scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.

CLINICAL SIGNS, MORBIDITY AND MORTALITY:
The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.

BODY WEIGHT:
The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).

EAR THICKNESS MEASUREMENTS AND RECORDING OF LOCAL REACTIONS:
Ear thickness measurements and recording of local reactions were performed in order to access any possible irritant effect of the test item, as possible irritancy may be involved in false psoitive lymphoproliferative responses.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of group 1 to 6 was measured using a micrometer.
No measurement of ear thickness was perfomred for the animals of the positive control group.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (colouration, presence of residual test item, ....) was noted.
The irritation level of the test item was determined according to the table 1 (see "Any other information on materials and methods incl. tables")

No further information on details on study design (LLNA) was stated.

Positive control substance(s):

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

Statistics:

The results were expressed as disintegration's/mn (dpm) per group and per node. Stimulation Indices (SI) were calculated according to the following fromula: SI = disintegration's/mn of treated group / disintegration's/mn of control group.
The EC3 value (theoretical concentration resulting in a SI value of 3) was determined by linear interpolation of points on the dose-response curve, immediatly above and below the 3-fold threshold. The equation used for calculation of EC3 was: EC3 = c+ [(3-d)/(b-d)] x (a-c)
Where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

Results and discussion

Positive control results:

In the positive control group given α-hexyl cinnamic aldehyde at the concentration of 25 %, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 9.57) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.15

Test group / Remarks:

0.025%

Results 2

Parameter:

SI

Value:

0.74

Test group / Remarks:

0.05%

Results 3

Parameter:

SI

Value:

1.92

Test group / Remarks:

0.1%

Results 4

Parameter:

SI

Value:

3.64

Test group / Remarks:

0.25%

Results 5

Parameter:

SI

Value:

12.76

Test group / Remarks:

0.5%

Cellular proliferation data / Observations:

0.025 % (w/v): 517.17 (dpm per group); 64.65 (dpm per node) 0.05 % (w/v): 332.05 (dpm per group); 41.51 (dpm per node) 0.1 % (w/v): 864.84 (dpm per group); 108.11 (dpm per node) 0.25 % (w/v): 1639.09 (dpm per group); 204.89 (dpm per node) 0.5 % (w/v): 5754.00 (dpm per group); 719.25 (dpm per node) Vehicle group: 450.82 (dpm per group); 56.35 (dpm per node) Positive control group: 4312.25 (dpm per group); 539.03 (dpm per node)

Any other information on results incl. tables

No mortality and no clinical signs were observed during the study.

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

No cutaneous reactions and no noteworthy increase in ear thickness were observed at any of the tested concentrations.

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. the cell viability was higher than 80 % in each group.

A dose-related increase in the SI was recorded at the concentration ≥ 0.05 %, and the threshold positive value of 3 was exceeded at the concentration of 0.25%.

With the test item at the concentration of 0.025%, the incorporation of 3H-TdR was slightly higher than in the vehicle control group, but did not follow the dose-response relationship observed at the other tested concentrations.

The irritation level was I.

In the absence of local irritation, the positive lymphoproliferative responses observed at the concentration ≥ 0.25 % were attributed to delayed contact hypersensitivity.

The EC3 value for the test item Produit Y is equal to 0.2%.

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, the test item Produit induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained in this experiment, the test item Produit Y should be considered as a strong sensitizer.
According to EC-Regulation 1272/2008, and subsequent regulations, the test item is classified as sub-category 1A.