Registration Dossier

Administrative data

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline; under GLP conditions.
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsD
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier.
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 20-24 g
- Housing: housed 5 animals per cage (IVC cages, type II L, polysulphone cages).
- Diet (e.g. ad libitum): Altromin 1324 maintainance diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): tap water (sulphur acidified to a pH of 2.8), ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): at least 10/h
- Photoperiod: 12 h light / 12 h dark (artificial light)
Vehicle:
other: 2% carboxymethylcellulose (CMC)
Concentration:
Preliminary screening test: 25% w/w
Main test: 6.25%, 12.5% and 25% w/w
No. of animals per dose:
Preliminary test: total of 3 animals were used (2 were treated with test material and the remainder served as the negative control).
Main test: 5 mice per dose group.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The maximum technically applicable concentration of the test material was found to be 25% in 2% CMC in aqua ad inject
- Irritation: no signs of irritation or systemic toxicity were observed in any animal in the preliminary test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were randomly selected for dose groups.
- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:

Preliminary test:
A solubility test and a preliminary screening test were performed to determine maximum concentration and test concentrations to be used in the main test. The maximum technically applicable concentration of the test item was found to be 25% in 2% CMC in aqua ad inject. Mice were treated by daily application of test substance at concentrations of 25% in the vehicle, to the dorsal surface of each ear for 3 consecutive days. An additional animal was treated with 100% of 2% CMC and served as the negative control. Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. Additionally clinical signs were recorded during this period.

Main test:
Topical aplication: Five groups of 5 mice were treated with the substance at concentrations of 0% (control), 6,25%, 12.5% or 25% w/v in the vehicle and 25% α-Hexylcinnamaldehyde (positive control) . The preliminary test suggested that the substance would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily topical application of 25µl of the appropriate concentration of the substance to the dorsal surface of each ear for 3 consecutive days.

3H-methyl thymidine administration: Five days after the first topical administration of the substance, all mice were injected via the tail vein with 250µl 3H-methyl thymidine (diluted to a working concentration of 80µCi/ml).

Preparation of single cell suspension: 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining “auricular lymph nodes” were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of administration3HTdR incorporation: The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Observations:

- Clinical observation: All animals were observed prior to application and daily thereafter. Any signs of toxicity or ill health were recorded.
- Body weights: Recorded prior to dosing and prior to treatment with 3HTdR.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The positive control group achieved the appropriate response with a stimulation index of 10.7. One animal from the positive-control group showed hair loss at the application site on day 6 of the study.
Parameter:
SI
Remarks on result:
other: - Negative control: 1.0 - Positive control: 10.7 (mean) - Test substance (6.25%): 1.3 (mean) - Test substance (12.5%): 1.1 (mean) - Test substance (25%): 1.2 (mean)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The following have been corrected for background DPM: - Negative control: 1212.6 (mean) - Positive control: 12957.4 (mean) - Test substance (6.25%): 1615.2 (mean) - Test substance (12.5%): 1374.1 (mean) - Test substance (25%): 1459.6 (mean) [background 3H-methyl thymidine levels were measured as 18.2)

All animals survived throughout the test period and no clinical signs were observed in any animal belonging to the test substance or negative control groups. Body weights were unaffected in all dose groups.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material is not considered to be a dermal sensitiser.
Executive summary:

The study was performed to assess the skin sensitisation potential of the test material in CBA/CaOlaHsD mice following topical application to the dorsal surface of the ear. The study was performed to GLP and the method was designed to meet the requirements of OECD 429 guideline, EPA OPPTS 870.2600 and EU Method B.42.

In a preliminary screening test in two mice, daily application of 25µL of substance at concentrations of 25% w/v in the vehicle to the dorsal surface of each ear was performed for 3 consecutive days. Prior to the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. Additionally clinical signs were recorded during this period. Body weights were recorded on Day 1 (prior to dosing) and, for the surviving mouse, on Day 6. Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal. Based on the preliminary test, the concentrations selected for the main test were 0%, 6.25%, 12.5% and 25% w/w.

 

Five groups of 5 mice were treated with the test substance at concentrations of 0% (control), 6.25%, 12.5% or 25% w/v and 25% α-Hexylcinnamaldehyde (positive control) in the vehicle. The mice were treated by daily application of 25µl of the appropriate concentration of the substance to the dorsal surface of each ear for 3 consecutive days. Five days after the first topical administration of the substance, all mice were injected via the tail vein with 3H-methyl thymidine. All animals were observed prior to application and daily thereafter and any signs of toxicity or ill health were recorded. Recorded prior to dosing and prior to treatment with 3HTdR. At test termination, five hours after administration of 3HTdR, the test animals were sacrificed. The draining auricular lymph nodes from each mouse were excised and individually pooled for each animal (2 lymph nodes per animal). Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured.

 

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensititising if at least one concentration of the substance resulted in a 3-fold or greater increase in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. A stimulation index (SI) of less than 3 was noted at each of the three test material concentrations evaluated. Accordingly, the test material was considered to be non-sensitising under the conditions of the test.

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

The study was performed to assess the skin sensitisation potential of the test material in CBA/CaOlaHsD mice following topical application to the dorsal surface of the ear. The study was performed to GLP and the method was designed to meet the requirements of OECD 429 guideline, EPA OPPTS 870.2600 and EU Method B.42.

In a preliminary screening test in two mice, daily application of 25µL of substance at concentrations of 25% w/v in the vehicle to the dorsal surface of each ear was performed for 3 consecutive days. Prior to the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. Additionally clinical signs were recorded during this period. Body weights were recorded on Day 1 (prior to dosing) and, for the surviving mouse, on Day 6. Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal. Based on the preliminary test, the concentrations selected for the main test were 0%, 6.25%, 12.5% and 25% w/w.

 

Five groups of 5 mice were treated with the test substance at concentrations of 0% (control), 6.25%, 12.5% or 25% w/v and 25% α-Hexylcinnamaldehyde (positive control) in the vehicle. The mice were treated by daily application of 25µl of the appropriate concentration of the substance to the dorsal surface of each ear for 3 consecutive days. Five days after the first topical administration of the substance, all mice were injected via the tail vein with 3H-methyl thymidine. All animals were observed prior to application and daily thereafter and any signs of toxicity or ill health were recorded. Recorded prior to dosing and prior to treatment with 3HTdR. At test termination, five hours after administration of 3HTdR, the test animals were sacrificed. The draining auricular lymph nodes from each mouse were excised and individually pooled for each animal (2 lymph nodes per animal). Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured.

 

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensititising if at least one concentration of the substance resulted in a 3-fold or greater increase in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. A stimulation index (SI) of less than 3 was noted at each of the three test material concentrations evaluated. Accordingly, the test material was considered to be non-sensitising under the conditions of the test.


Migrated from Short description of key information:
Skin sensitisation: non-sensitising, female mice, OECD 429, Schmid 2012

Justification for selection of skin sensitisation endpoint:
One study available; GLP compliant and has a Klimisch score 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for skin sensitisation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation.