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Description of key information

A 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 (Skin sensitization: Local Lymph Node Asay)

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

The test item was classified as a contact sensitizer (Category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

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:
The study was conducted between 07 July 2016 and 26 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Version / remarks:
EC 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
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.
Vehicle:
dimethylformamide
Concentration:
10%, 25% and 50% w/w
No. of animals per dose:
Preliminary screening test = 1
Main Test = 4
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 50% w/w in dimethyl formamide, 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
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. 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.

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.

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).

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  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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None
Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in
dimethyl formamide Stimulation Index Result
25 6.08 Positive

Conclusion
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test
Key result
Parameter:
EC3
Value:
>= 0.15
Remarks on result:
other: Sensitizer
Key result
Parameter:
SI
Value:
6.34
Test group / Remarks:
10%
Remarks on result:
other: Positive
Remarks:
Sensitizer
Key result
Parameter:
SI
Value:
7.07
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
7.98
Test group / Remarks:
50%
Cellular proliferation data / Observations:
Preliminary screening test:
Red colored staining on the ears and fur was noted post dose on Days 1 to 3. 
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in dimethyl formamide.

DETAILS ON STIMULATION INDEX CALCULATION
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
dimethyl formamide Stimulation Index Result
10 6.34 Positive
25 7.07 Positive
50 7.98 Positive

EC3 CALCULATION
The EC3 value was calculated. The following extrapolation equation was used for the calculation.
EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a)  log2(c)]}
a = 25
b = 7.07
c = 10
d = 6.34

EC3 = 2^ {log2(10) + (3-6.34)/(7.07-6.34) x [log2(25)  log2(10)]}= 0.15

CLINICAL OBSERVATIONS:
Red colored staining on the ears and fur was noted in all test animals.
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test

BODY WEIGHTS
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:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
The test item was classified as a contact sensitizer (Category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals
Executive summary:

A 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.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of50% 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 asolutionindimethyl formamide at concentrations of 50%,25% or10% w/w. A further group offour animals was treated withdimethyl formamidealone.

Results

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
dimethyl formamide

Stimulation Index

Result

10

6.34

Positive

25

7.07

Positive

50

7.98

Positive

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolatedEC3value) was calculated to be 0.15%.

Conclusion

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

The test item was classified as a contact sensitizer (Category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

The test item was classified as a contact sensitizer (Category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals