1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 September 2012- 25 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

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'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

EC Number:

228-846-8

EC Name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Cas Number:

6362-80-7

Molecular formula:

C18H20

IUPAC Name:

(4-methyl-4-phenylpent-1-en-2-yl)benzene

Details on test material:

- Name of test material (as cited in study report): 1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene
- Physical state: clear colourless liquid
- Analytical purity: 95 %
- Lot/batch No.: 2554
- Expiration date of the lot/batch: 28 February 2013
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing:The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100 % and 50 % v/v
Main test: 50 %, 25 % and 10 % v/v

No. of animals per dose:

Preliminary screening test: 1/ dose
Main test: 4/ dose

Details on study design:

RANGE FINDING TESTS:
One mouse was humanely killed that was treated with the undiluted test item. Animals in which any adverse effects were noted that were considered to approach the moderate severity limit set forth in the UK Home Office Project Licence, were humanely killed.

Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using mice, 1 mouse per test item concentration. The mice were treated by daily application of 25 μl of the undiluted test item or at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for 3 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 skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight of the mouse treated with the test item at a concentration of 50% in acetone/olive oil 4:1 was recorded on Day 1 (prior to dosing) and on Day 6. The bodyweight of the mouse that was humanely killed was recorded on Day 1 (prior to dosing) and immediately prior to termination (Day 4).

Where necessary 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 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 sensitisation.

MAIN STUDY
Test Item Administration: Groups of 4 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 produce systemic toxicity and slight skin irritation with the undiluted test item. However the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 suggested that the test item would not produce systemic toxicity or excessive local skin irritation at this 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

At a concentration of 25 % v/v in acetone/olive oil 4:1, the Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the control group was 5.76. Therefore, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

4.37

Test group / Remarks:

10 % v/v in acetone/olive oil 4:1.

Remarks on result:

other: Positive result

Results 2

Key result

Parameter:

SI

Value:

11.71

Test group / Remarks:

25 % v/v in acetone/olive oil 4:1.

Remarks on result:

other: Positive result

Results 3

Key result

Parameter:

SI

Value:

20.57

Test group / Remarks:

50 % v/v in acetone/olive oil 4:1.

Remarks on result:

other: Positive result

Any other information on results incl. tables

Preliminary Screening Test

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	21	*	0	0	0	0	0	0	HLPtK	*	*
50	S-2	21	22	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

K = Animal humanely killed due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Animal weighed 19g before death.

H = Hunched posture

L = Lethargic

Pt= Ptosis

*= No data animal dead

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
		Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
50	S-1	0	0	1	1	1	1	1	1	*	*	*	*
100	S-2	0	0	0	0	0	0	0	0	0	0	0	0

*= No data animal dead

Table 3 Measurement of Ear Thickness and 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	0.235	0.245	0.230	0.236	*	*
Overall mean (mm)		0.240		0.233		*
Overall mean ear thickness change (%)		na		-3.125		*

 

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	0.240	0.235	0.250	0.245	0.255	0.235
Overall mean (mm)		0.238		0.248		0.245
Overall mean ear thickness change (%)		na		4.211		3.158

na = Not applicable

*= No data animal dead

 

The animal treated with the undiluted test item was humanely killed, on Day 4, due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence.

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 in the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1.

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

Table 4 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/ olive oil 4:1	Dpm	Dpm/Node^a	Stimulation Index^b	Result
Vehicle	13828.85	1728.61	na	na
10	60472.80	7559.10	4.37	Positive
25	161890.50	20236.31	11.71	Positive
50	284486.40	35560.80	20.57	Positive

 

Clinical Observations and Mortality Data

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

Bodyweight

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.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The skin sensitisation potential of the test item was assessed according to OECD Guideline 429. The test item was considered to be a sensitiser under the conditions of the test.