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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 jan 2012 to 01 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
LLNA is used because the test has been performed before REACH regulation came into force requesting in vitro studies (October, 2016)

Data source

Reference
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
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:
Harlan Laboratories Ltd, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Chemical structure
Reference substance name:
3-(p-cumenyl)propionaldehyde
EC Number:
231-885-3
EC Name:
3-(p-cumenyl)propionaldehyde
Cas Number:
7775-00-0
Molecular formula:
C12H16O
IUPAC Name:
3-[4-(propan-2-yl)phenyl]propanal
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation:15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet: Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Water: Free access to mains tap water was allowed throughout the study
- Acclimation period: at least 5 days

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

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100%
No. of animals per dose:
5
Details on study design:
PRELIMINARY SCREENING TEST
Using available information regarding the 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 undiluted test item 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 irritation was scored daily according to the erythema scores. 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 thickness of each ear was measured using an Oditest micrometer (Dyer, PA), 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 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.

MAIN STUDY
- 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.
- Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3 H-methyl thymidine (3HTdR:80pCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 pCi to each mouse.
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. 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 project 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 40°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 (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß-scintillation counting. The "Poly Q" 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 using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

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).
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.

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde gave a Stimulation Index of 6.63 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
4.32
Test group / Remarks:
25%
Parameter:
SI
Value:
7.43
Test group / Remarks:
50%
Parameter:
SI
Value:
15.53
Test group / Remarks:
100%
Key result
Parameter:
EC3
Value:
18.6
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
Mild redness was noted on the ears of animals treated with the undiluted test item. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

EC3 CALCULATION
EC3 = 2^ {log2(c) +(3 -d)/(b-d)] x [log2(a) - log2(c)]}
a= 50
b= 7.43
c= 25
d= 4.32

EC3 = 2^ {log2(25) +(3 - 4.32)/ (7.43 - 4.32)] x [log2(50) - log2(25)]}= 18.6
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 18.6%.

Any other information on results incl. tables

PRELIMINARY SCREENING TEST

No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on both ears on Day 3. Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitizer category 1B
Remarks:
in accordance with CLP (EC 1272/2008 and its updates)
Conclusions:
The application of the test substance at concentration of 25% in acetone/olive oil (4:1 v/v), the lowest concentration tested, resulted in an increase in isotope incorporation which was greater than 3-fold. The extrapolated EC3 was 18.6%, indicating that the substance is a skin sensizer.
Executive summary:

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP principles. Using available information regarding the irritancy potential of the test item, a preliminary screening test was performed using one mouse which was treated by daily application of 25 µl of the undiluted test item to the dorsal surface of each ear for three consecutive days. Based on the results, the test substance was applied at 25, 50 and 100% in a vehicle of acetone/olive oil (4:1 v/v) for the main test. The application of the test substance at concentration of 25% in acetone/olive oil (4:1 v/v), the lowest concentration tested resulted in an increase in isotope incorporation, which was greater than 3-fold. The extrapolated EC3 value was 18.6% and therefore the substance is considered to be sensitising to the skin.