Registration Dossier

Administrative data

Description of key information

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

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
other justification
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September 2018 - 13 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: m-bis(2,3-epoxypropoxy)benzene
Batch:53715080
Purity:95-100%
Physical state / Appearance:Colourless slightly viscous liquid
Expiry Date: 05 August 2019
Storage Conditions:room temperature in the dark

For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given in the procedure section.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

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.
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands and Envigo RMS (UK) Limited, 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 5 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 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood flake bedding. 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. 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 at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test item at concentrations of 5%, 2.5% or 1% v/v in acetone/olive oil 4:1
No. of animals per dose:
Groups of four mice at each concentration.
Details on study design:
PRE-SCREEN TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using five mice. The mice were treated by daily application of 25 µL of the undiluted test item or test item formulation, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Day 4. Surviving mice were also observed once daily on Days 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 5. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6 or at death.
Where appropriate 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

Test Item Administration
Groups of four mice were treated with the test item at concentrations of 5%, 2.5% or 1% 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 0.25 mL (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.
Body Weights: 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. 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 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  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).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse – Pooled Method
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) 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)
Positive control results:
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Value:
8.96
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
18.49
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
20.81
Test group / Remarks:
5%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : Please see table in results section.

DETAILS ON STIMULATION INDEX CALCULATION :Please see table in results section.

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

BODY WEIGHTS: 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.

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

Concentration
(% v/v) in
acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

10360.32

1295.04

na

na

1

92848.80

11606.10

8.96

Positive

2.5

191580.80

23947.60

18.49

Positive

5

215572.10

26946.51

20.81

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

Concentration (%v/v) in
acetone/olive oil 4:1

Stimulation Index

Result

1

8.96

Positive

2.5

18.49

Positive

5

20.81

Positive

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
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

In an OECD Test Guideline 429 compliant study, following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of5%, 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) of the test item, as asolutioninacetone/olive oil 4:1,at concentrations of5%, 2,5% or1% v/v. A further group offour animals was treated withacetone/olive oil 4:1alone.

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

1

8.96

Positive

2.5

18.49

Positive

5

20.81

Positive

 

Conclusion

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

The threshold value for a significant sensitising effect is a stimulation index ≥ 3. The test substance had stimulation index value of 8.96 at the lowest concentration value 1%. Based on the results the EC3 value cannot be calculated as the SI values were higher then 3 even at the lowest dose.

The criteria for Skin sensitisation category 1A is EC3 value ≤ 2% according to the CLP regulation.

As the SI value is very high at the 1% dose group it is clear that the EC3 value will be ≤ 2% therefore the test item is classified as a skin sensitizer category 1A.