Dioctanoyl peroxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 10th February 2015 Experimental completion date: 9th April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Dioctanoyl peroxide

EC Number:

212-094-2

EC Name:

Dioctanoyl peroxide

Cas Number:

762-16-3

Molecular formula:

C16H30O4

IUPAC Name:

octanoyl octaneperoxoate

Test material form:

solid: flakes

Details on test material:

Identification: Dioctanoyl peroxide (CAS# 762-16-3)
Chemical Name: Dioctanoyl peroxide
Trade Name: Perkadox SE-8
CAS Number: 762-16-3
Batch: 1204350050
Purity: 99.2 %
Physical state / Appearance: White flakes
Expiry Date: 06 March 2016
Storage Conditions: approximately 20 °C in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaCrl)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaCrl) strain mice were supplied by Charles River (UK), Kent, UK. 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.

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 Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 degrees C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light (06.00 to 18.00) 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.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary screening test: 50%, 25%, 10%, 5%, 2.5% and 1% in acetone/olive oil 4:1

Main test: 2.5%, 1% and 0.5% in acetone/olive oil 4:1

No. of animals per dose:

Preliminary screening test: 1 mouse per test item concentration

Main test: Five mice

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 six mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50%, 25%, 10%, 5%, 2.5% or 1% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The surviving mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. The mouse treated at a concentration of 50% w/w in acetone/olive oil 4:1 was observed on Days 1, 2 and 3. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mice on Day 6. The body weight of the mouse that was humanely killed was recorded immediately prior to termination.

Where necessary 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 five mice were treated with the test item at concentrations of 2.5%, 1% or 0.5% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at a concentration of 2.5% w/w in acetone/olive oil 4:1. 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 five 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. 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 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 centrifuge tube. The 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, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 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 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:

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. 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 variances, then parametric one way analysis of variance (ANOVA) and Dunnett’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.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:

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

Concentration (% v/v) in
acetone/olive oil 4:1 Stimulation Index Result
25 13.93 Positive


Conclusion
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

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 acetone/olive oil 4:1 Stimulation Index Result 0.5 1.89 Negative 1 2.83 Negative 2.5 8.15 Positive The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 1.05%.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Please refer to the atatched Table 4

Any other information on results incl. tables

  Preliminary Screening Test

The animal treated with the test item at a concentration of 50% w/winacetone/olive oil 4:1was humanely killed, post dose on Day 3, due to the occurrence of clinical signs of toxicity that approachedthe moderate severity limit set forth in the UK Home Office Project Licence. Hunched posture, lethargy and body weight loss were noted in this animal.

 

Very slight erythema was noted on both ears of the animals treated with the test item at concentrations of 50%, 25%, 10%, 5% or 1% w/winacetone/olive oil 4:1. Novisual local skin irritation was notedon the ears of the animal treated with the test item at a concentration of2.5% w/winacetone/olive oil 4:1. 

 

A greater than 25% increase in mean ear thickness was notedthe animals treated with the test item at concentrations of 50%, 25%, 10% or 5% w/winacetone/olive oil 4:1. Noirritation indicated by an equal to or greater than 25% increase in mean ear thickness was notedthe animals treated with the test item at concentrations of2.5% or 1% w/winacetone/olive oil 4:1.

 

Based on this information the dose levels selected for the main test were2.5%,1% and0.5% w/winacetone/olive oil 4:1.

 

Main Test

  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 acetone/olive oil 4:1	Stimulation Index	Result
0.5	1.89	Negative
1	2.83	Negative
2.5	8.15	Positive

 

 

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animalsduring the test.

 

 Body Weight

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

 

Calculation of EC₃Value

aEC₃= c + [[(3-d)/(b-d)] x (a-c)]

 

a	=	2.5
b	=	8.15
c	=	1
d	=	2.83
EC3=1+ [[(3-2.83)/(8.15-2.83)] x (2.5-1)] =1.05

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be1.05%.

a=   lowest concentration giving stimulation index >3

b =  actual stimulation index caused by ‘a’

c =  highest concentration failing to produce a stimulation index of 3

d =  actual stimulation index caused by ‘c’

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

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

The test item was also classified as a contact sensitizer (Category 1A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

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:

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.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of 2.5% 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 five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1at concentrations of2.5%,1% or 0.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1alone.

 

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 acetone/olive oil 4:1	Stimulation Index	Result
0.5	1.89	Negative
1	2.83	Negative
2.5	8.15	Positive

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be1.05%.

 

Conclusion

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

 

The test item was also classified as a contact sensitizer (Category 1A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

 

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