Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 15 April 2014 and 08 May 2014

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:

2014

Report date:

2014

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:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

EC Number:

940-875-4

Molecular formula:

C3H8O3 - C99H182O20

IUPAC Name:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Test material form:

other: viscous amber liquid

Details on test material:

Description : amber coloured extremely viscous liquid
Purity : not supplied
Expiry date : 08 August 2014
Storage conditions: room temperature 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.

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:

dimethylformamide

Concentration:

25, 10%, or 5% w/w in dimethyl formamide

No. of animals per dose:

Five

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 |^ L of the test item at concentrations of 50% or 25% w/w in dimethyl formamide, 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 skin irritation was scored daily according to the scale as described in any other information. 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 on Day 6.
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 25%, 10%, or 5% w/w in dimethyl formamide. 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.
The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 15% v/v in dimethyl formamide was applied to the dorsal surface of each ear.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation) pre dose on Day 1, and on Days 2, 3, 4, 5 and on Day 6. Any changes in the ear thickness were noted. Group mean daily ear thickness changes were calculated. A group 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.

3H-Methyl Thymidine Administration
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 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  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 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 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

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in dimethyl formamide.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index are given in Table 4.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute per animal are given in Table 4.

Any other information on results incl. tables

Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in³HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in³HTdR incorporation will be classified as a "non‑sensitiser".

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. No signs of systemic toxicity were noted in the animal treated with the test item at a concentration of 50% w/w in dimethyl formamide but very slight erythema on both ears and a greater than 25% increase in mean ear thickness were noted. 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 animal treated with the test item at a concentration of 25% w/w in dimethyl formamide. Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in dimethyl formamide.

Estimation of the Proliferative Response of Lymph 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:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	5% w/w in dimethyl formamide	2.38	Negative
	10% v/v in dimethyl formamide	3.44	Positive
	25% v/v in dimethyl formamide	4.28	Positive
Positive Control Item	15% v/v in dimethyl formamide	5.35	Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 and local skin irritation is given in Table 6 (attachment 2). The ear thickness measurements and mean ear thickness changes are given in Table 7 (attachment 3).

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

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 8 (attachment 4).

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.

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

Concentration (% w/w) in butanone	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
50	S-1	19	20	0	0	0	0	0	0	0	0	0
25	S-2	19	18	0	0	0	0	0	0	0	0	0

0 =     No signs of systemic toxicity ;

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (% w/w) in butanone	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	0	0	1	1	1	1	0	0	0	0
25	S-2	0	0	0	0	0	0	0	0	0	0	0	0

na=     Not applicable

Table 4              Individual Disintegrations per Minute and Stimulation Indices

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle Dimethyl formamide	1-1	2713.03	2641.86 (±297.76)	N/A	N/A
	1-2	2124.93
	1-3	2861.30
	1-4	2689.44
	1-5	2820.62
Test Item 5% w/w in dimethyl formamide	2-1	8986.31	6294.31 (±1895.04)	2.38	Negative
	2-2	6770.96
	2-3	5608.18
	2-4	3761.06
	2-5	6345.06
Test Item 10% w/w in dimethyl formamide	3-1	7806.36	9081.76 (±3630.52)	3.44	Positive
	3-2	9451.21
	3-3	8722.49
	3-4	4712.87
	3-5	14715.87
Test Item 25% w/w in dimethyl formamide	4-1	14617.83	11301.99** (±2460.94)	4.38	Positive
	4-2	12410.24
	4-3	8333.72
	4-4	11606.45
	4-5	9541.69
Positive Control Item 15% v/v in dimethyl formamide	5-1	13961.96	14138.27* (±3801.42)	5.35	Positive
	5-2	9309.48
	5-3	19385.00
	5-4	12160.37
	5-5	15874.54

dpm = Disintegrations per minute

N/A =  Not applicable

a =     Total number of lymph nodes per animal is 2

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

** =     Significantly different from control group p<0.01

* =      Significantly different from control group p<0.05

Table 5              Individual Clinical Observations and Mortality Data

Treatment Group	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 Dimethyl formamide	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
Test Item 5% w/w in dimethyl formamide	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
Test Item 10% w/w in dimethyl formamide	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
Test Item 25% v/v in dimethyl formamide	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
Positive Control Item 15% v/v in dimethyl formamide	5-1	0	0	0	0	0	0	0	0	0
	5-2	0	0	0	0	0	0	0	0	0
	5-3	0	0	0	0	0	0	0	0	0
	5-4	0	0	0	0	0	0	0	0	0
	5-5	0	0	0	0	0	0	0	0	0

0 =     No signs of systemic toxicity

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 sensitizer under the conditions of the test.
The concentration of test item expected to cause a 3 fold increase in HTdR incorporation (EC 3value) was calculated to be 8%.
a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at aconcentration of 15% v/v in dimethyl formamide thus demonstrating the sensitivity and reliability of the test system.

Executive summary:

Introduction

A study in accordance with OECD Guideline No. 429 and EC Method B42 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 a concentration of 25% 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 dimethyl formamide at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with dimethyl formamide alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in dimethyl formamide.

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:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	5% w/w in dimethyl formamide	2.38	Negative
	10% w/w in diemthyl formamide	3.44	Positive
	25% w/w in dimethyl formamide	4.28	Positive
Positive Control Item	15% v/v in dimethyl formamide	5.35	Positive

The concentration of test item expected to cause a 3 fold increase in 3 HTdR incorporation (EC 3 value) was calculated to be 8%.

Conclusion

The test item was considered to be a sensitizer under the conditions of the test. The concentration of test item expected to cause a 3 fold increase in HTdR incorporation (EC 3 value) was calculated to be 8%. a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% w/w in dimethyl formamide thus demonstrating the sensitivity and reliability of the test system.