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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 429, EU Method B.42, and GLPs
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across: supporting information

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
-Test material (as cited in report): EKC 2004-0223
-Batch/Lot number: X-29363-079
-Physical state: Pale beige/white solid
-Date received: 2004-10-25
-Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
-Source: B & K Universal Ltd., Hull, UK
-Sex: female
-Age at study initiation: approximately 8-12 weeks old
-Acclimation period: 5 days
-Weight at study initiation: 15-23 g
-Housing: individually in polypropylene cages fitted with stainless steel mesh lids and furnished with softwood woodflakes
-Diet: Certified Rat and Mouse Diet (Code 5LF2; BCM IPS Limited, London, UK), ad libitum
-Environmental enrichment: items were provided that would not impact the outcome of the study
-Water: local municipality, ad libitum
-Method of animal identification: unique number that was written on the tail using a black indelible marker pen and a number written on a cage card
-Method of animal distribution: animals were selected at random

-Temperature (°C): 19-25
-Humidity (%): 30-70
-Photoperiod: 12 hours light/12 hours dark
-Air exchanges: 15 per hour

-Experimental Start Date: 2004-12-02
-Experimental Completion Date: 2004-12-22

Study design: in vivo (LLNA)

other: ethanol/distilled water 4:1
5, 10, and 25%
No. of animals per dose:
5 females/group
Details on study design:
Concentration, homogeneity and stability verifications of the test substance were not performed because they are not required by the test guidelines.

Preparation of Test Material:
The test material was freshly prepared in ethanol/distilled water 4:1 v/v. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Preliminary Screening Test:
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 25% w/w in ethanol/distilled water 4:1, to the dorsal surface of each ear for three consecutive days. Based on the results of this study, concentrations of 5%, 10% and 25% w/w were selected for the main study.

Test material administration:
Groups of 5 mice were treated with the test substance at concentrations of 5, 10, and 25% (w/w) in ethanol/distilled water 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days. The test substance was administered using a micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A group of 5 mice received the vehicle alone in the same manner and served as the study control group.

^3H-Methyl Thymidine administration:
Five days following the first topical application of the test substance 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, Amersham Biosciences UK Ltd.) giving a total of 20 µCi to each mouse.

Clinical observations:
All animals were observed twice daily on Days 1, 2, and 3 and on a daily basis on Days 4-6. Any signs of toxicity or signs of ill health were recorded during the study.

Body weights:
The body weight of each animal was recorded before dosing (Day 1) and prior to termination at Day 6.

Five hours following the administration of ^3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised from each animal and 1 mL PBS added.

Preparation of single cell suspension:
A single cell suspension of the lymph node cells from each 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, then the suspension was transferred to a 10-mL centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove any remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 X g) for ten 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 overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 X g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. The vials containing the samples and scintillation fluid were placed in the scintillator and left in the dark for approximately 20 minutes to reduce the risk of luminescence. After 20 minutes, the vials were shaken vigorously and ^3HTdR incorporation was measured by β-scintillation counting using a Beckman LS6500 scintillation system. The results were expressed as the Stimulation Index (SI) which was obtained by dividing the mean disintegrations per minute (dpm)/animal for each treatment group by the mean dpm/animal of the corresponding control group.

Interpretation of results:
The test substance was regarded as a sensitizer if at least one concentration of the test material resulted in a threefold or greater increase in ^3HTdR incorporation compared to control values. A test material that failed to produce a threefold or greater increase in ^3HTdR incorporation was classified as "not a sensitizer".
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Data were processed to provide group mean values for dpm and standard deviations, where appropriate. Individual and group mean dpm values were assessed 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:
A positive control was not performed concurrently with the study of EK 2004-0223. Positive control studies are routinely performed in the testing laboratory to confirm sensitivity of the method and the study report provided historical results for tests using a known sensitizer, α-hexylcinnamaldehyde. The most recent positive control test was performed from 28 April 2004 to 05 May 2004. In that study, mice responded as expected from exposure to α-hexylcinnamaldehyde. The Stimulation Index was 6.09 following exposure to 25% v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
Key result
Test group / Remarks:
Key result
Test group / Remarks:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 1151.48 ± 153.84 5%: 1145.61 ± 263.67 10%: 2142.94 ± 463.82 25%: 4245.45 ± 1129.30

Any other information on results incl. tables

Clinical observations:

No mortality or signs of toxicity were noted during the study. Fur loss on the neck and hardened residual test substance on the ears and head were noted in animals treated with the test substance at dose concentrations of 10 and 25%.


Body weights:

Body weight changes in test substance-treated animals were comparable to the controls. 

Applicant's summary and conclusion

Interpretation of results:
other: Sensitising
Criteria used for interpretation of results: EU
When EKC 2004-0223 (Resin acids and Rosin acids, hydrogenated, potassium salts) was tested in the local lymph node assay, it was determined to be a dermal sensitizer according to the interpretative criteria of the protocol.

Based on the positive effects for sensitisation obtained in the current study, EKC 2004-0223 (Resin acids and Rosin acids, hydrogenated, potassium salts) is classified for Skin Sensitisation (Category 1) according to the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Resin acids and Rosin acids, hydrogenated, potassium salts is not classified for Skin Sensitisation in Annex I of Directive 67/548/EEC.
Executive summary:

A study was conducted to assess the potential of Resin acids and Rosin acids, hydrogenated, potassium salts to produce delayed type hypersensitivity in the mouse. Using the Local Lymph Node Assay, five (CBA/CaCruBR) female mice per group were exposed to 50 µL (25 µL per ear) EKC 2004-0223 (Resin acids and Rosin acids, hydrogenated, potassium salts) at test concentrations of 5, 10, and 25% in a 4:1 ethanol:distilled water vehicle. Three days after the last test substance administration, the animals were injected via the tail vein with 250 µL of phosphate buffered saline containing ^3H-methyl thymidine and euthanized 5 hours later. Single cell suspensions of lymph node cells were made, incubated overnight, and the incorporation of ^3HTdR was measured by β-scintillation counting. A stimulation index of greater than 3 was recorded in the 25% dose concentration group. Since at least one concentration resulted in a Stimulation Index ≥ 3, EKC 2004-0223 (Resin acids and Rosin acids, hydrogenated, potassium salts) is a considered to be a dermal sensitizer according to the interpretative criteria of the protocol. However the reliability of the finding is unclear given the negative LLNA result obtained on a sample of Resin acids and rosin acids, potassium salts together with the large amount of supporting negative sensitisation data available on other members of this category.