4,4'-isopropylidenebis[2-allylphenol]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 21st May 2014 and 12th June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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)

Remarks:

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

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 acclimatization 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 group 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 °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 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.

Justification:
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.

Vehicle:

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

Remarks:

For the purpose of the study, the test item was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen because it was the first vehicle tried in order of preference that was suitable.

Concentration:

Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% v/v in acetone/olive oil 4:1.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
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 test item at concentrations of 50% and 25% 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 (Day 6 observations were not performed for the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1). Local skin irritation was scored daily according to the scale for Erythema. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse treated at a concentration of 25% v/v in acetone/olive oil 4:1 was recorded on Day 1 (prior to dosing) and on Day 6. The body weight of the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 was recorded on Day 1 (prior to dosing) and on Day 5 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 STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% 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 skin 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 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.



TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

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).

TERMINATION PROCEDURES:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 cell 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 pooled 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 re suspended 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, re suspended 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 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 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)

Positive control 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 12.76 Positive

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

Parameter:

SI

Remarks on result:

other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4. * Table 4 can be found in the any other information on results section

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4*. * Table 4 can be found in the any other information on results section

Parameter:

SI

Value:

2.02

Test group / Remarks:

5%

Parameter:

SI

Value:

4.42

Test group / Remarks:

10%

Parameter:

SI

Value:

11.55

Test group / Remarks:

25%

 Preliminary Screening Test:

Clinical observations, body weight 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*.

 * For all tables, please see below.

The animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1 was humanely killed, on Day 5, 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, or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted with the mouse treated at a concentration of 25% v/v in acetone/olive oil 4:1. Very slight erythema was noted on Day 3.

 

Based on this information the dose levels selected for the main test were 25%, 10% and 5% v/v in acetone/olive oil 4:1.

 Main Test:

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4 (please see below).

 

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:

 

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

Concentration (%v/v) in acetone/olive oil 4:1	Animal Number	Body Weight (g)		Clinical Observations
				Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	22	*	0	0	0	0	0	0	0	H L Et Ri K	*
25	S-2	20	20	0	0	0	0	0	0	0	0	0

0=          No signs of systemic toxicity

H =        Hunched posture

L=        Lethargy

Et =       Elevated tail

*=          No data, animal dead

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 19 g prior to termination

 

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	1	1	[1]	*
25	S-2	0	0	0	0	1	1	0	0	0	0	0	0

 

[1]=          No data, animal dead

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

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-1	0.21	0.21	0.22	0.22	*	*
overall mean (mm)		0.21		0.22		*
overall mean ear thickness change (%)		na		4.76		*

 

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
25	S-2	0.21	0.22	0.22	0.23	0.21	0.21
overall mean (mm)		0.22		0.23		0.21
overall mean ear thickness change (%)		na		4.65		-2.33

 

na=        Not applicable

*=          No data, animal dead

 

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	25825.87	3228.23	na	na
5	52156.77	6519.60	2.02	Negative
10	114040.30	14255.04	4.42	Positive
25	298330.50	37291.31	11.55	Positive

dpm = Disintegrations per minute

^a=          Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

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

na =        Not applicable

 

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

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 25% 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 four animals, were treated with 50 µL (25 µL per ear) ofthe test item as asolutioninacetone/olive oil 4:1at concentrations of 25%, 10% or 5% v/v. A further group offour animals was treated withacetone/olive oil 4:1 alone.

 

 

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
5	2.02	Negative
10	4.42	Positive
25	11.55	Positive

 

The concentration of test item expected to cause a 3 fold increase in ³HTdR incorporation (EC₃value) was calculated to be 7.04%.

 

 

Conclusion:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

This 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.

  

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of 25% 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 four animals, were treated with 50 µL (25 µL per ear) ofthe test item as asolutioninacetone/olive oil 4:1at concentrations of 25%, 10% or 5% v/v. A further group offour animals was treated withacetone/olive oil 4:1 alone.

 

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
5	2.02	Negative
10	4.42	Positive
25	11.55	Positive

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be 7.04%.

 

 

Conclusion:

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

Migrated from Short description of key information:
The 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 according to the OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

Justification for selection of skin sensitisation endpoint:
Only this study is available

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

- skin sensitisation:

Based on the above stated assessment of the skin sensitisation potential, the substance needs to be classified as R43 (May cause sensitisation by skin contact)according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and accordingCLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.

- respiratory sensitisation:

As no data on respiratory sensitization is available for the substance a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and accordingCLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.