Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 05 July 2016 and 04 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

1

Reference substance name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

EC Number:

944-405-9

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 13-0156
- Phystical state: clear, yellowish liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Specific details on test material used for the study:

Identification: IFF FRET 13-0156
Physical state/Appearance: pale amber liquid
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet and water (e.g. ad libitum): Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve limits of 19 to 25°C
- Humidity (%): Relative humidityas set to achieve limits of 30 to 70%,
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was 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 for erythema. Any clinical signs of toxicity, if present, were also recorded. The body weight 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 undiluted test item or the test item at concentrations of 50% or 25% 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 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 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

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: 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.

Bodyweights: 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, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness 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).

Data evaluation:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR 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 sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

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:

The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (8.64) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.51

Test group / Remarks:

Test item : 25% v/v in acetone/olive oil 4:1

Remarks on result:

other: negative

Results 2

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

Test item: 50% v/v in acetone/olive oil 4:1

Remarks on result:

other: negative

Results 3

Key result

Parameter:

SI

Value:

2.16

Test group / Remarks:

Test item: 100%

Remarks on result:

other: negative

Any other information on results incl. tables

Preliminary Screening Test:

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.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/vinacetone/olive oil 4:1were selected for the main test.

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:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	25%v/vin acetone/olive oil 4:1	1.51	Negative
	50%v/vin acetone/olive oil 4:1	0.97	Negative
	100%	2.16	Negative
Positive Control Item	25% v/v in acetone/olive oil 4:1	8.64	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.

Body Weight:

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

The radioactive disintegrations per minute per lymph node and the stimulation index are given below:

Individual Disintergrations per Minute and Stimulation Index

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle acetone/olive oil 4:1	1-1	576.23	981.53 (±325.31)	na	na
	1-2	917.18
	1-3	1465.81
	1-4	873.54
	1-5	1074.88
Test Item 25% v/vin acetone/olive oil 4:1	2-1	2239.12	1479.90 (±489.80)	1.51	Negative
	2-2	1207.35
	2-3	1617.79
	2-4	949.85
	2-5	1385.38
Test Item 50% v/vin acetone/olive oil 4:1	3-1	192.17	952.97 (±581.96)	0.97	Negative
	3-2	908.62
	3-3	612.32
	3-4	1552.88
	3-5	1498.86
Test Item 100%	4-1	3492.14	2121.22 (±1084.64)	2.16	Negative
	4-2	2807.12
	4-3	1458.18
	4-4	736.95
	4-5	2111.69
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	12575.92	8480.35** (±4605.71)	8.64	Positive
	5-2	4651.95
	5-3	5505.47
	5-4	5311.80
	5-5	14356.60

The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test item test groups 

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<0.01

Applicant's summary and conclusion

Interpretation of results:

other: The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: "Local Lymph Node Assay" method. At 25% (v/v in acetone/olive oil 4:1), 50% (v/v in acetone/olive oil 4:1) and 100% the substance showed SI values of 1.51, 0.97 and 2.16, respectively.

The test item was considered to be a non-sensitizerunder the conditions of the test.