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Description of key information

Skin senstising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 03 to 31, 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: the animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK ad libitum
- Water: ad libitum
- Acclimation period: at least 5 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness
Vehicle:
other: ethanol/distilled water 7:3
Concentration:
5 %, 10 %, 25 % w/w in ethanol/distilled water 7:3
No. of animals per dose:
four
Details on study design:
PRELIMINARY SCREENING TEST
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a concentration of 25 % w/w in ethanol/distilled water 7:3, 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 body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 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 sensitization.

MAIN TEST
1.Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25 %, 10 % or 5 % w/w in ethanol/distilled water 7:3. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin 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 four mice received the vehicle alone in the same manner.
2. 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: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.
3. 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.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
4. Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labeled with the study number and dose concentration. The lymph node cell 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: the proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitizer 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-sensitizer". The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]

TREATMENT PREPARATION AND ADMINISTRATION: The test item was freshly prepared as a solution/suspension in ethanol/distilled water 7:3. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. The test item was applied to the dorsal surface of each ear.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
α-Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitizer under the conditions of the test.
Parameter:
EC3
Value:
ca. 6
Parameter:
SI
Value:
2.52
Test group / Remarks:
5 % w/w of test item
Parameter:
SI
Value:
4.86
Test group / Remarks:
10 % w/w of test item
Parameter:
SI
Value:
5.67
Test group / Remarks:
25 % w/w of test item

Estimation of the Proliferative Response of Lymph Node Cells

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in ethanol/distilled water 7:3

Stimulation Index

Result

5

2.52

Negative

10

4.86

Positive

25

5.67

Positive

Clinical Observations and Mortality Data

White residual test item on the ears was noted, post dose on Days 1 to 3, in animals treated with the test item at a concentration of 25 % w/w in ethanol/distilled water 7:3. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:
other: Category 1B according to the CLP Criteria
Conclusions:
Skin sensintising
Executive summary:

Method

The study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD guideline 429.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution/suspension in ethanol/distilled water 7:3 at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with ethanol/distilled water 7:3 alone.

Results

The stimulation index results to be greater than 3 for the concentration of 10 and 25 %. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 6 %.

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.

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

The study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD guideline 429.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution/suspension in ethanol/distilled water 7:3 at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with ethanol/distilled water 7:3 alone.

SI > 3; EC3 value = 6 %

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

SKIN SENSITIZATION

Category 1

Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

For a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive.

Furthermore, stimulation index of three or more is considered a positive response in the local lymph node assay.

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Based on animal test (Local lymph node assay) results performed, according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is classified in Sub Category 1B, as the EC3 value is > 2 % (EC3 = 6% ).