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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 2011 - 22 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
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: the animals of the preliminary test were approximately 12-13 weeks old and the animals of the main test were approximately 9 weeks old
- Mean body weight at study initiation: in the main test, they had a mean body weight of 21.3 g (range: 19.5 g to 24.6 g)
- Fasting period before study: no
- Housing: the animals were housed by group of two (first preliminary test) or four (main test) or individually housed (second preliminary test) in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions 13 days (first preliminary test), 2 days (second preliminary test) or 11 days (main test) before the beginning of the treatment period.

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 (7:00 - 19:00).

IN-LIFE DATES: 31 August 2011 to 19 September 2011.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Range finding test: 1%, 2.5%, 5% and 10%.
Main test: 0%, 0.05%, 0.1%, 0.25%, 0.5% and 1%.
No. of animals per dose:
4 per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: as unsatisfactory solubility of the test item was obtained in AOO (i.e. a heterogeneous suspension to the naked eye was
obtained at the concentration of 50%), Propylene Glycol (PG) was used.
A homogenous suspension was obtained at the concentration of 50% in PG. However, as concentrations of 50% and 25% did not pass through a
plastic tip, it was not possible to treat animals using these concentrations.
Irritation: none at 1%; all animals treated at 2.5%, 5% and 10% were found dead before day 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
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.
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no
Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).
Parameter:
SI
Remarks on result:
other: 0.05% : SI = 0.71 0.1% : SI = 1.40 0.25% : SI = 0.83 0.5%: SI = 1.05 1%: SI = 1.46
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per group: Group 5: Vehicle: 980 Group 6: 0.05%: 700 Group 7: 0.1%: 1371 Group 8: 0.25%: 809 Group 9: 0.5%: 1025 Group 10: 1%: 1071

Results

A homogenous suspension of the test item was obtained at the concentration of 50% in PG. However, as concentrations of 50% and 25% did not pass through plastic tip, it was not possible to treat animals using these concentrations. Consequently, the concentrations selected for the preliminary test were 10%, 5%, 2.5% and 1%.

Since the concentrations higher or equal to 2.5% of the test item were lethal in the preliminary test, the highest concentration retained for the main test was 1%.

 

Clinical signs and mortality

In the main test, one female treated at the concentration of 1% (group 10) was found dead on day 2. Taking into account deaths observed in the preliminary assay, this death was attributed to dermal toxicity of the test item prepared in the vehicle.

No clinical signs were observed during the observation period in any other animal.

 

Local irritation

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


Proliferation assay

The acceptance criterion was met; this experiment was therefore considered valid.

The results of main test are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

0.05

non-irritant

0.71

Test item

0.1

non-irritant

1.40

Test item

0.25

non-irritant

0.83

Test item

0.5

non-irritant

1.05

Test item

1

non-irritant

1.46

HCA

25

-

12.48

 

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The objective of this study was to evaluate the potential of the test item 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 OECD Guideline for Testing of Chemicals No. 429 and the principles of Good Laboratory Practice.

 

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, 28 female CBA/J mice were allocated to 7 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 Propylene Glycol (PG)(vehicle),

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

. one positive control group of four animals receiving the positive control item, alpha‑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 positive control 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

A homogenous suspension of the test item was obtained at the concentration of 50% in PG. However, as concentrations of 50% and 25% did not pass through plastic tip, it was not possible to treat animals using these concentrations. Consequently, the concentrations selected for the preliminary test were 10%, 5%, 2.5% and 1%.

Since the concentrations higher or equal to 2.5% of the test item were lethal in the preliminary test, the highest concentration retained for the main test was 1%.

 

Clinical signs and mortality

In the main test, one female treated at the concentration of 1% (group 10) was found dead on day 2. Taking into account deaths observed in the preliminary assay, this death was attributed to dermal toxicity of the test item prepared in the vehicle.

No clinical signs were observed during the observation period in any other animal.

 

Local irritation

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


Proliferation assay

The acceptance criterion was met; this experiment was therefore considered valid.

The results of main test are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

0.05

non-irritant

0.71

Test item

0.1

non-irritant

1.40

Test item

0.25

non-irritant

0.83

Test item

0.5

non-irritant

1.05

Test item

1

non-irritant

1.46

HCA

25

-

12.48

 

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

Conclusion

The test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

LLNA on MTSC (Rokh 2011):

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA, OECD 429). Evaluation of local irritation was also carried out in parallel.

In the main test, 28 female CBA/J mice were allocated to 7 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 Propylene Glycol (PG)(vehicle), one negative control group of four animals receiving the vehicle, and one positive control group of four animals receiving the positive control item, alpha‑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 positive control 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.

A homogenous suspension of the test item was obtained at the concentration of 50% in PG. However, as concentrations of 50% and 25% did not pass through plastic tip, it was not possible to treat animals using these concentrations. Consequently, the concentrations selected for the preliminary test were 10%, 5%, 2.5% and 1%.

Since the concentrations higher or equal to 2.5% of the test item were lethal in the preliminary test, the highest concentration retained for the main test was 1%.

In the main test, one female treated at the concentration of 1% (group 10) was found dead on day 2. Taking into account deaths observed in the preliminary assay, this death was attributed to dermal toxicity of the test item prepared in the vehicle.

No clinical signs were observed during the observation period in any other animal. No cutaneous reactions and no increase in ear thickness were observed at any of the tested concentrations. No notable lymphoproliferation was noted with the test item at any tested concentrations.

The test item, MTSC, did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.


Migrated from Short description of key information:
A LLNA is available on MTSC. No notable lymphoproliferation was noted with the test item at any tested concentrations, therefore MTSC is not considered to be a skin sensitizer.

Justification for selection of skin sensitisation endpoint:
Only one reliable study is available for this endpoint.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Proposed self-classification

- Regulation (EC) No 1272/2008 : Not classified

- Directive 67/548/EEC : Not classified