4-trans-propenylveratrole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 06, 2014 to March 17, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 01, 2014/ signed on October 10, 2015)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-trans-propenylveratrole

EC Number:

228-958-7

EC Name:

4-trans-propenylveratrole

Cas Number:

6379-72-2

Molecular formula:

C11H14O2

IUPAC Name:

(E)-1,2-Dimethoxy-4-prop-1-en-1-ylbenzene

Test material form:

liquid

Details on test material:

- Physical state: Pale yellow, oily liquid
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Four healthy female mice were obtained from Harlan UK Ltd and a further 27 female mice were obtained from Charles River UK Ltd.
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 14.9-20.7 g
- Housing: Animals were housed two or three animals per cage, in solid bottomed polycarbonate cages with a stainless steel mesh lid.
- Diet: Standard rodent diet (Harlan Teklad 2014C Diet), ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From November 06, 2014 to March 17, 2015

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Main test: 10, 25 and 50 % v/v in acetone/olive oil 4:1

No. of animals per dose:

5

Details on study design:

PRELIMINARY STUDY:
- Compound solubility: A vehicle trial performed showed that the test item formed a clear solution in 4:1 v/v acetone: olive oil (AOO) at 50% v/v which was satisfactory for dose administration.
- Preliminary investigations were performed to ensure the highest concentration to be used on the main study did not result in systemic toxicity or excessive local irritation. Although the material could be dosed as supplied as a precautionary measure the preliminary investigation started at 50% v/v in AOO. As no toxicity was seen at 50% v/v in AOO the preliminary investigation continued with the test material as supplied. Preliminary investigations conducted in Harlan animals proved that 50% v/v in AOO was the suitable high dose level for the main study. However, it became necessary to perform the main study in mice provided by Charles River. As a precautionary measure when changing animal supplier the preliminary phase was repeated at 50% v/v using Charles River animals to ensure there was no difference in outcome before proceeding to the main study in Charles River mice.
- An assessment of local irritation was performed and an erythema score greater or equal to 3 and/or an increase in ear thickness of greater than 25% at Day 3 or 6 compared with predose would be regarded as irritation which precludes the use of that concentration on the main study.
- Two female mice (per concentration) received a daily application of 25 μL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3).
- Results of the preliminary investigations indicated that 50% v/v was a suitable high concentration for administration in the main phase of the study. The low and intermediate concentrations were selected from the concentration series given in regulatory guidelines and the concentrations administered on the main study were: 10, 25 and 50% v/v in AOO

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the SI is 3 or more, the test substance is regarded as a skin sensitizer with a consideration given to dose response and statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test substance was prepared for administration as a series of graded concentrations in the vehicle, by direct dilution. The test substance was used as supplied and all formulations were prepared on the day of dosing at the required concentration(s).
- The mice were treated by daily application of 25 μL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline containing 3H-methyl Thymidine (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse. Five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised for each experimental animal and placed in 1.0 mL of PBS. A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding 10 mL PBS, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash. After overnight incubation (minimum of 18 hours) with 5% TCA at 4 °C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. The 3HTdR incorporation was measured by β scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node).
- Stimulation Index: Results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group.

Positive control substance(s):

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

Statistics:

The individual dpm data was analysed statistically.

Results and discussion

Positive control results:

SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 5.5 which demonstrates the validity of this study.

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

9.5

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM / node for vehicle, 10, 25 and 50 % v/v were 466.3, 1876.3, 9956.7 and 13740.1, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 10, 25 and 50 % v/v were 4.0, 21.4 and 29.5, respectively

EC3 CALCULATION
As a SI of 3 or more was recorded for all of the concentrations tested, test item was considered to have the potential to cause skin sensitization. Based on the results of this study the EC3 value is calculated to be 9.5% v/v.

CLINICAL OBSERVATIONS:
There were no deaths and no signs of ill health or toxicity were observed during this study.
Wet/greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.
In addition partially closed eye lids were seen pre dose on Day 3 for all positive control animals and from approximately 5.5 hours post dose on Day 3 for two animals receiving 50%v/v (animal numbers A25 and A26). Recovery for all animals was seen by the end of Day on Day 3.
No signs of dermal irritation were seen on the ear during the study. There was no evidence of an effect of treatment on ear thickness.

BODY WEIGHTS
There was no indication of an effect of treatment on body weight gain.
A minor loss in body weight was noted for several mice (Nos. A7, A9, A12, A14, A17, A26 and A27) over the study period, however, there was no evidence of a dose relationship and as a small loss in body weight is not uncommon in young laboratory mice this is considered not to be an effect of treatment.

Any other information on results incl. tables

Preliminary investigation

Mortality and clinical signs: There were no deaths and no signs of ill health or toxicity were observed during this study for any of the mice receiving 50% v/v. Group 2 animals dosed with the test substance as supplied (Nos. A3 and A4) were killed for welfare reasons on Day 2 due to poor condition.

Group 2 animals both exhibited partially closed eyelids (both), hunched posture, underactive behaviour and irregular breathing on Day 2 after dosing. The female animal (No. A3) was also noted to have closed eyelids (both), lachrymation in both eyes and tremors before being killed for welfare reasons.

Wet/greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

Dermal reactions: No erythema was observed on the ears for any of the mice receiving 50% v/v on Days 1 to 6.

Measurement of ear thickness: There was no evidence of an effect of treatment on ear thickness.

Body weight:There was no indication of an overt effect of treatment on body weight gain.

A loss in body weight was noted for both mice from Charles River receiving 50% v/v (animal Nos. A5 and A6) over the study period, however, a small loss in body weight is not uncommon in young laboratory mice and this was considered not to be an effect of treatment.

On the basis of the results from the preliminary investigation, 50% v/v was considered a suitable high concentration for administration in the main phase of the study.

Table 7.4.1/1: Group dpm/node and Stimulation Index

Test article	dpm	Number of lymph nodes per animal	dpm/node	Stimulation Index†	Result + = positive - = negative
Vehicle (AOO)	932.5	2.0	466.3	n/a	n/a
10% v/v	3752.5**	2.0	1876.3	4.0	+
25% v/v	19913.3***	2.0	9956.7	21.4	+
50% v/v	27480.2***	2.0	13740.1	29.5	+
HCA 25% v/v	5107.7*	2.0	2553.9	5.5	+

 

†         Stimulation Index of 3 or more indicates a positive result

n/a       Not applicable

dpm    Disintegrations per minute (less background count of 35.8 dpm)

AOO    Acetone:olive oil (4:1 v/v) (vehicle control)

HCA    Hexyl cinnamic aldehyde (positive control)

 

Individual approach only:

*p<0.05 for comparisons with Group 4 using t-test.

**p<0.01 p-values for comparisons with Group 4 using Williams’ test

***p<0.001 p-values for comparisons with Group 4 using Williams’ test

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the test conditions, test material is classified as a skin sensitizer 1B according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

The study comprised three treated groups, each comprising five female mice receiving test item at concentrations of 10, 25 or 50% v/v. Similarly constituted groups received the vehicle 4:1 v/v acetone: olive oil (AOO) or positive control substance (25% v/v hexyl cinnamic aldehyde). The mice were treated by daily application of 25mL of the appropriate concentration or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of ³H-methyl Thymidine (³HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of ³HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

The SI obtained for 10, 25 and 50% v/v were 4.0, 21.4 and 29.5 respectively which indicates that test item showed the potential to induce skin sensitization. The EC3 value was calculated to be 9.5% v/v. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 10, 25 and 50 % v/v.

The SI for the positive control substance hexyl cinnamic aldehyde was 5.5, which demonstrates the validity of this study.

 

Under the test conditions, test material is classified as a skin sensitizer 1B in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.