Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 2014 to 02 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
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)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
EC Number:
943-540-0
Molecular formula:
Too complex
IUPAC Name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
Details on test material:
Purity: 100%
Physical state / Appearance: pale amber liquid
Expiry date: 09 May 2016
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were nulliparous and non-pregnant.
Acclimatization period of at least five days.
Animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
Free access to mains tap water and food.
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 approximately fifteen changes per hour.
The lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
other: Neat and in Butanone
Concentration:
100% neat and 10% and 1% v/v in butanone
No. of animals per dose:
5
Details on study design:
Groups of five mice were treated with the test item at concentrations of 100%, 10% or 1.0% v/v in butanone. 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 ofthe ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, alpha-Hexylcinnamaldehyde, tech., 85%, at a concentration of 15% v/v in butanone as applied to the dorsal surface of each ear.
The thickness of each ear was measured and recorded as described in the preliminary screening test.

Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation. 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 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 centrifiige tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 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).
3HTdR incorporation was measured by B-scintillation counting. 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 meandisintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of ariances, then parametric one way analysis of variance (ANOVA) and Durmett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

Positive control results:
15% v/v of the positive control in butanone gives positive results with a stimulation Index score of 8.23

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
1% v/v of the test item in butanone gave negative results with a stimulation Index score of 1.55 10% v/v of the test item in butanone gave negative results with a stimulation Index score of 2.79 100% of the neat test item gave positive results with a stimulation Index score of 10.01
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
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 pL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/rnL, specific activity 20 Ci/mmoL) giving a total of 20µCi to each mouse.

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No observations of erythema were noted on the ears of all animals treated with the test item or positive control item.

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.

Calculation of EC3 value

EC3 = 10 + [[(3 -2.79) / (10.01 - 2.79)] x (100 - 10)] = 13

The concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation (EC3 value) was calculated to be 13 %.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test and the concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation (EC3 value) was calculated to be 13 %. Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

Introduction

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. The method was designed to be compatible with the OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in butanone at concentrations of 100% or 10% or 1 % v/v in butanone. A further group of five animals was treated with butanone alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, alpha-Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in butanone.

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:

1% v/v of the test item in butanone gave negative results with a stimulation Index score of 1.55

10% v/v of the test item in butanone gave negative results with a stimulation Index score of 2.79

100% v/v of the test item gave positive results with a stimulation Index score of 10.01

25% v/v of the positive control in butanone gave positive results with a stimulation Index score of 8.23

Conclusion

The test item was considered to be a sensitiser under the conditions of the test and the concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation (EC3 value) was calculated to be 13 %. Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone thus demonstrating the sensitivity and reliability of the test system.