Registration Dossier

Administrative data

Description of key information

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

25

4.99

Positive

50

4.29

Positive

100

No data as animals terminated

 

Conclusion

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
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2018 - 08 August 2018
Reliability:
1 (reliable without restriction)
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)
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 Envigo 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:
100%, 50% or 25% v/v in acetone/olive oil 4:1
No. of animals per dose:
4 mice per dose.
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. 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.
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 four mice were treated with the test item at concentrations of 100%, 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 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.

Key result
Parameter:
SI
Value:
4.99
Test group / Remarks:
25% v/v dose group
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
4.29
Test group / Remarks:
50%(v/v) dose group
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
0
Test group / Remarks:
100% (v/v) dose group
Remarks on result:
not measured/tested
Cellular proliferation data / 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).


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/v in acetone/olive oil 4:1 were selected for the main test.

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

8531.97

1066.50

na

na

25

42612.26

5326.53

4.99

Positive

50

36572.03

4571.50

4.29

Positive

100

No data due to termination of group


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:
Category 1 (skin sensitising) 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

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of100%, 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 undiluted or the test item, as asolutioninacetone/olive oil 4:1,at concentrations of50% or25% 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

25

4.99

Positive

50

4.29

Positive

100

No data as animals terminated

 

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

Stimulation indices of 4.99 and 4.29 were produced by test concentrations of 25% and 50% and there was no evidence of systemic toxicity or skin irritation being produced by these test concentrations, so these results could indicate skin sensitisation potential.  However, as no data were obtained for a third concentration, and due to the shallow dose response, it is not possible to determine the EC3 value.

As an EC3 value could not be determined therefore according to the CLP criteria substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub-categorisation.

The test item is classified as Skin sensitisation category 1 based on the CLP criteria.