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Description of key information

Skin sensitisation: sensitising, EC3 23.0% female mice, OECD TG 429, 2014

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:
2012-11-23 to 2013-01-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected July 2012; signature: November 2012
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: 8-12 weeks
- Weight at study initiation: 15 - 23 grams
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: certified rodent diet; ad libitum
- Water: mains tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark


IN-LIFE DATES: From: 23 November 2012 To: 22 January 2013
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: undiluted (100%)
- Main test: undiluted (100%), 50%, 25% and 0% (vehicle control)
The test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
No. of animals per dose:
Preliminary test: 1 mouse
Main test: 5 mice per dose group
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test substance, a preliminary screening test was performed using two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item (100% v/v), to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale (included in the full study report). Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a micrometer, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the undiluted test item or the test item at concentrations of undiluted (100% v/v), 50% v/v or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80μCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 μCi to each mouse.

Observations:
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness measurements: The thickness of each ear was measured using a micrometer, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Termination:
Five hours following the administration of 3HTdR all mice were humanly terminated. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid . 3HTdR incorporation was measured by β-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Positive control results:
In a 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 25% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 6.29 and met the criteria for a 'positive' result. The positive control study was within the range of the laboratory historic PC studies (provided in the full study report).
Parameter:
SI
Value:
13.56
Test group / Remarks:
100%v/v test item in Acetone:Olive Oil (4:1)
Parameter:
SI
Value:
6.69
Test group / Remarks:
50%v/v test item in Acetone:Olive Oil (4:1)
Parameter:
SI
Value:
3.4
Test group / Remarks:
25%v/v test item in Acetone:Olive Oil (4:1)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : see table 1.

DETAILS ON STIMULATION INDEX CALCULATION: see ‘any other information on results incl. tables’

EC3 CALCULATION: see ‘any other information on results incl. tables’

CLINICAL OBSERVATIONS: No signs were noted.

BODY WEIGHTS: Body weights were comparable to controls.

In the preliminary screening test: No local irritation responses were noted for both ears. There were no signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the undiluted test item (100%), 50% and 25% v/v in acetone/olive oil 4:1 were selected for doses in the main test.

In the main test: There were no deaths or signs of systemic toxicity, local skin irritation or marked increases in the ear thicknesses were noted during the test. Body weights were comparable to that observed in the corresponding control group over the same period.

Table 1.0 – Main test results including mean DP and SI

Concentration (% v/v)

Mean dpm

SI #

Result

0 (vehicle control)

2074.19 ±887.21

N/A

-

25

7061.81 ±6263.82)

3.40

Positive

50

13877.86 ±7826.82

6.69

Positive

100

28122.56*** ±10913.61

13.56

Positive

 

 

 

 

dpm = Disintegrations per minute

# = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

*** = Significantly different from control group p<0.001

 

Calculation of EC3 Value

Where the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value is extrapolated from the two lowest doses utilised. The extrapolated EC3 value was calculated by log linear interpolation between the two points on a plane where the α axis represents the dose level and the γ axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d).

EC3 = 2^ {log2(c) + (3 - d)/(b - d) x [log2(a) - log2(c)]}

a = 50

b = 6.69

c = 25

d = 3.40

EC3 = 23.0%

The concentration of test substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 24.8%.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of this study, the item is considered to sensitising to skin. The concentration of test substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 23.0%.
Executive summary:

The study was performed according OECD TG 429 and EU Method B.42 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the test item undiluted (100%) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest concentration. Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% (acetone/olive oil 4:1) and 100% v/v (undiluted). Groups of five mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes. 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 animal 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 sensitising 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 comparable to controls. A stimulation index (SI) of more than 3 was noted at the 25, 50and 100% test concentration groups. The 25% v/v generated a SI = 3.40. Accordingly, the test material was considered to be sensitising under the conditions of the test. The EC3 value was calculated by log-linear extrapolation to be 23.0%.

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

OECD TG 429, 2014 – The study was performed according OECD TG 429 and EU Method B.42 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. Based on the preliminary test, the concentrations selected for the main test were 0%, 25%, 50% (in acetone/olive oil 4:1) and 100% v/v (undiluted). Groups of five mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes. 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 animal 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 sensitising 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 comparable to controls. A stimulation index (SI) of more than 3 was noted at the 25, 50and 100% test concentration groups. The 25% v/v generated a SI = 3.40. Accordingly, the test material was considered to be sensitising under the conditions of the test. The EC3 value was calculated by log-linear extrapolation to be 23.0%. Under the conditions of this study, the test substance would be considered to be classified as skin sensitizer (Category 1B) under Regulation (EC) No 1272/2008.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B.

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.

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