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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:
2014-02-19 until 2014-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks
- Weight at study initiation: 18.8-20.8 g
- Housing: Group caging in Type II. polypropylene / polycarbonate cages
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.4-25.1
- Humidity (%): 22-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 19 February 2014 To: 25 February 2014 (preliminary experiment 11 December 2013 to 16 December 2013)
Vehicle:
other: acetone:olive oil (4:1 v/v)
Concentration:
0 (controls), 50%, 25%, 10% w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test using Acetone: Olive oil 4:1 (v/v) mixture (AOO). A formulation at 50% (w/v) was suitable for the test.
- Irritation/toxicity/ear thickness: A preliminary irritation/toxicity tests was carried out on 2 mice/dose level at concentrations of 50% and 25% in AOO. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were scored for erythema. Ear thickness was measured using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness by ear punch weight determination after euthanasia

Based on these results, 50, 25 and 10% (w/v) doses were considered to be acceptable for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation assay
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than 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:
- TOPICAL APPLICATION: Each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the appropriate test item applied using a pipette. Each animal was dosed once a day for 3 consecutive days (Days 1, 2 and 3).

- PROLIFERATION ASSAY: On Day 6, each mouse was iv injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR. Five hours (± 30 minutes) after iv injection, the mice were killed by asphyxiation with CO2 and the draining auricular lymph nodes removed. A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregating of the lymph nodes through a cell strainer. The cell strainer was washed with PBS and the lymph node cells for each mouse were pelleted in a centrifuge. After centrifugation supernatants were discarded and pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

After the final washing step, supernatants were removed. Pellets were gently agitated, re-suspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight incubation at 2-8 °C, precipitates were centrifuged and supernatants removed. Pellets were re-suspended in 1 mL of 5% (w/v) TCA solution and dispersed using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

All animals were observed at least once daily for any clinical signs, including local irritation and systemic toxicity. Individual body weights were recorded on Day 1 (beginning of the assay) and Day 6 (prior to 3HTdR injection).

DPM was measured for each animal. The measured DPM values were corrected with the background DPM value (“DPM”). The measured DPM value of 5 % (w/v) TCA solution was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable
Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed with 25% (w/v) α-Hexylcinnamaldehyde within 6 months of the current study (27 November 2013).

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lympho-proliferative response (stimulation index value of 19.5) was noted for α-Hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.
Parameter:
SI
Remarks on result:
other: 1.0, 3.4, 3.1 and 3.7 at concentrations of 0 (control), 50, 25 and 10 % (w/v), respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 1140, 3748, 3431, 4169 at concentrations of 0 (control), 50%, 25% and 10%, respectively.

No mortality or signs of systemic toxicity were observed for the test item treated animals during the study. Test item precipitate / minimal amount of test item precipitate was detected on the ears of the animals in the 50 and 25 % (w/v) dose groups on Days 1-3. There were no indications of irritancy at the site of application. No treatment related effects were observed on body weight in the test item treated groups.

The appearance of the lymph nodes was normal in the negative (vehicle) control group, in the 10 % (w/v) dose group and for three animals in the 25 % (w/v) group. The appearance of one of the lymph nodes of one animal in the 25 % (w/v) dose group was normal and the other was larger than normal. Slightly enlarged lymph nodes were observed in the 50 % (w/v) dose group.

Table 1: DPM, DPN and stimulation Index

 Group

Measured DPM per group

DPM

Number lymph nodes

DPN

Stimulation index

Background (5% w/v TCA)

31

-

-

-

-

Negative control

1140

1109

8

138.6

1.0

50% test item

3748

3717

8

464.6

3.4

25% test item

3431

3400

8

425.0

3.1

10% test item

4169

4138

8

517.3

3.7

 

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was shown to have skin sensitisation potential (sensitizer) in the Local Lymph Node Assay.
Executive summary:

The test item solutions were applied on the dorsal surface of ears of female CBA/J Rj mice (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI). In the main assay sixteen female CBA/J Rj mice were allocated to four groups of four animals each: - three groups received 50, 25 and 10 % (w/v) of test item(formulated in AOO) - the negative (vehicle) control group received the vehicle (AOO).

No mortality or signs of systemic toxicity were observed during the study. Test item precipitate / minimal amount of test item precipitate was detected on the ears of the animals in the 50 and 25 % (w/v) dose groups on Days 1-3. There were no indications of any irritancy at the site of application. The appearance of the lymph nodes was normal in the negative (vehicle) control group, in the 10 % (w/v) dose group and for three animals in the 25 % (w/v) group. The appearance of one of the lymph nodes of one animal in the 25 % (w/v) dose group was normal and the other was larger than normal. Slightly enlarged lymph nodes were observed in the 50 % (w/v) dose group. The observed stimulation index values were 3.4, 3.1 and 3.7 at concentrations of 50, 25 and 10 % (w/v), respectively.

In conclusion, under the conditions of the present assay, the test item was shown to have skin sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In 1970, Jones published a report on an in vivo study involving guinea pigs. The original document was not available and no description on the method or the results assessment was given. A moderate skin sensitization was observed during the study. However, the lack of details does not allow to draw a reliable conclusion on sensitization.

Another in vivo study was conducted without following any relevent guideline by Sapegin (1985) on 36 guinea pigs. The method details and results description were not provided. Under the testing conditions the substance did not show signs of allergenic properties. However, the present data are insufficient to draw a reliable conclusion, and was thus disregarded.

Váliczkó (2014) carried out an in vivo study under GLP and following OECD guideline 429 and EU method B.42: Local Lymph Node Assay. The substance was applied as a solution on the dorsal surface of ears of female mice (25 μL/ear: 50, 25 and 10 %w/w) for 3 consecutive days. On Day 6, animals were intravenously injected via the tail vein with tritiated methyl thymidine. Cell proliferation in the local lymph nodes was measured and the values obtained were used to calculate stimulation indices. No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. The observed stimulation index values were 3.4, 3.1 and 3.7 at concentrations of 50, 25 and 10 % (w/v), respectively. Under the conditions of the assay, the test item was shown to have skin sensitisation potential (sensitiser).

Based on the available data from Váliczkó (2014) which is considered to be adequate, reliable and conclusive for skin sensitisation, the substance is considered to be a sensitiser.


Migrated from Short description of key information:
Skin: Sensitising, in vivo, mouse LLNA, Váliczkó 2014
Skin: Moderate sensitizer, in vivo, guinea pig, Jones 1970

Justification for selection of skin sensitisation endpoint:
Study conducted to current guidelines under GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

Results from validated in vivo studies for skin sensitisation demonstrated the substance to be a skin sensitizer. The LLNA study was conducted following OECD guideline 429 and in compliance with GLP Directive 2004/10/EC. The result is valid, reliable and adequate for the purpose of risk assessment, classification and labelling. As a result the substance is classified as a skin sensitizer Category 1 according to Regulation (EC) No. 1272/2008, Part 3, 3.4.2.2.

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