1,1,3,3-tetramethylbutyl hydroperoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 28 February 2012 and 20 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted 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:

2012

Report date:

2012

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)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3,3-tetramethylbutyl hydroperoxide

EC Number:

227-369-2

EC Name:

1,1,3,3-tetramethylbutyl hydroperoxide

Cas Number:

5809-08-5

Molecular formula:

C8H18O2

IUPAC Name:

2,4,4-trimethylpentane-2-peroxol

Test material form:

other: clear colorless liquid

Details on test material:

Sponsor's identification: 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5)
Description : clear colourless liquid
Batch number : 1010519063
Purity : 91.1%
Date received : 15 November 2011
Expiry date : 01 November 2012
Storage conditions: approximately 4°C in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.
A Certificate of Analysis supplied by the Sponsor is given in Appendix 5 (attachment 1)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five 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 eight to twelve weeks old.
The animals were individually 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 controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve 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:

The test item was used at concentrations of 50%, 25% or 10% v/v in acetone/olive oil 4:1.

No. of animals per dose:

Groups of five mice were treated

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 two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µl of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were 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 scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse 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 Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% v/v 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 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 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:80µCi/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.
Bodyweights: The bodyweight 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 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 4°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 QTM" 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 removed from the sample changer, shaken vigorously and then returned to the sample changer for counting. 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 were 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.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks on result:

other: See Table 4

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 4

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

No signs of systemic toxicity were noted. No visual local skin irritation was noted in the animal treated with the undiluted test item but a greater than 25% increase in mean ear thicknesswas observed. Very slight erythema was noted on Day 3 on both ears of the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1but no excessive irritation, indicated by an equal to or greater than 25% increase in mean ear thickness,was noted.

Based on this information, the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

Main Test

Estimation of the Proliferative Response of Lymh Node Cells

The radioactive disintegrations per minute per animal and the stimulation index are given in Table 4.

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 (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
10	2.21	Negative
25	3.91	Positive
50	6.51	Positive

Clinmical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

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

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

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.

Calculation of EC₃ Value

EC₃= c + [[(3-d)/(b-d)] x (a-c)]a

a	=	25
b	=	3.91
c	=	10
d	=	2.21
EC3= 10 + [[(3-2.21)/(3.91-2.21)] x (25-10)] =17

The concentration of test item expected to cause a 3 fold increase in ³HTdR incorporation (EC₃ value) was calculated to be 17%.

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’

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
100	S-1	18	19	0	0	0	0	0	0	0	0	0
50	S-2	19	19	0	0	0	0	0	0	0	0	0

0=     No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Local Skin Irritation
		Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		pre‑dose		post dose		post dose
		left	right	left	right	left	right	left	right	left	right	left	right
100	S-1	0	0	0	0	0	0	0	0	0	0	0	0
50	S-2	0	0	0	0	1	1	0	0	0	0	0	0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
100%	S-1	0.250	0.240	0.330	0.310	0.290	0.260
overall mean (mm)		0.245		0.320		0.275
overall mean ear thickness change (%)		na		30.612		12.245
Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-2	0.235	0.240	0.260	0.265	0.265	0.270
overall mean (mm)		0.238		0.263		0.268
overall mean ear thickness change (%)		na		10.526		12.632

na=     Not applicable

Table 4              Individual Disintegrations per Minute and Stimulation Indices

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	1020.89	2647.62 (±997.56)	na	na
	1-2	3626.62
	1-3	2993.81
	1-4	2473.94
	1-5	3122.83
10	2-1	6262.78	5860.51 (±1449.69)	2.21	Negative
	2-2	4820.07
	2-3	3926.77
	2-4	7386.70
	2-5	6906.22
25	3-1	9868.68	10341.85*** (±2479.25)	3.91	Positive
	3-2	6392.37
	3-3	11601.61
	3-4	12966.26
	3-5	10880.32
50	4-1	20440.41	17241.49*** (±2498.04)	6.51	Positive
	4-2	13700.83
	4-3	16831.89
	4-4	18550.25
	4-5	16684.05

dpm=Disintegrations per minute

a=     Total number of lymph nodes per animal is 2

b=     Stimulation Index of 3.0 or greater indicates a positive result

na=     Not applicable

***=   Significantly different from control group p<0.001

Table 5              Individual Clinical Observations and Mortality Data

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
	1-5	0	0	0	0	0	0	0	0	0
10	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
	2-5	0	0	0	0	0	0	0	0	0
25	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
	3-5	0	0	0	0	0	0	0	0	0
50	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0
	4-5	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	20	19	-1
	1-2	21	20	-1
	1-3	19	20	1
	1-4	18	19	1
	1-5	17	17	0
10	2-1	21	20	-1
	2-2	19	20	1
	2-3	20	21	1
	2-4	20	21	1
	2-5	17	18	1
25	3-1	19	20	1
	3-2	20	21	1
	3-3	21	22	1
	3-4	20	20	0
	3-5	20	19	-1
50	4-1	20	19	-1
	4-2	18	18	0
	4-3	21	20	-1
	4-4	19	20	1
	4-5	17	18	1

Appendix 1      Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of CommissionRegulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non‑Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26^thmeeting held on 26 ‑ 27 April 2007 at ECVAM, Ispra, Italy.

Test Item                   :                                 α‑Hexylcinnamaldehyde, tech., 85%

Project number       :                                  41201832

Study dates              :                                  06 April 2012 to 12 April 2012

Methods. A group of five animals was treated with 50 µl (25 µl per ear) ofα‑Hexylcinnamaldehyde, tech., 85%as a solution inacetone/olive oil 4:1at a concentration of 25% v/v. A further control group of five animals was treated withacetone/olive oil 4:1alone.

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	5.76	Positive

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

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.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation 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 following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% v/v, 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 asolutioninacetone/olive oil 4:1at concentrations of 50%, 25% or 10% v/v. 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 (% v/v) in acetone/olive oil 4:1	Stimulation Index	Result
10	2.21	Negative
25	3.91	Positive
50	6.51	Positive

The concentration of test item expected to cause a 3 fold increase in ³HTdR incorporation (EC₃ value) was calculated to be 17%.

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