Registration Dossier

Administrative data

Description of key information

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:
Between 17 January 2012 and 21 February 2012
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.
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)
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.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 10% or 5% v/v in acetone/olive oil 4:1
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 three mice, one mouse per test item concentration. The mice were treated by daily application of 25 µl of the test item at concentrations of 50%, 25% or 10% v/v in acetone/olive oil 4:1, 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 irritation was scored daily according to the scale included as Appendix 3. Any clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), 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 Days 1 to 3 and Days 1 to 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 sensitisation.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 25%, 10% or 5% 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 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.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA) pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Group mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. 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)
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 25% v/v in acetone/olive oil 4:1.
Parameter:
SI
Remarks on result:
other: The stimulation index are given in Table 4.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per animal are given in Table 4.

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".

For chemicals, which the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3value was extrapolated from the two lowest doses utilized. The extrapolated EC3value was calculated by log‑linear interpolation between the two points on a plane where thec-axis represents the dose level and theg‑axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3value was as follows:

aEC3= 2^ {log2(c) + (3-d)/(b-d)] x [log2(a)-log2(c)]}


a= mid concentration giving a stimulation index >3

b = actual stimulation index caused by ‘a’

c = lowest concentration giving a stimulation index of >3

d = actual stimulation index caused by ‘c’

Preliminary Screening Test

Clinical observations, bodyweight 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.

Very slight erythema was noted on both ears of all mice. No signs of systemic toxicity or excessive irritation indicated by anequal to or greater than 25% increase in mean ear thicknesswere noted in animals treated with the test item at a concentration of 25% or10% v/v in acetone/olive oil 4:1. No signs of systemic toxicity were noted in the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1 but excessive irritation, indicated by a greater than 25% increase in mean ear thickness, was noted.

Based on this information the dose levels selected for the main test were 25%,10% and 5% v/v in acetone/olive oil 4:1.

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% v/v in
acetone/olive oil4:1

4.45

Positive

10% v/v in
acetone/olive oil4:1

9.96

Positive

25% v/v in
acetone/olive oil4:1

13.74

Positive

Positive
Control Item

25% v/v in
acetone/olive oil4:1

6.62

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.

Very slight erythema was noted on both ears of mice treated with the test item at concentrations of 25% or 10% v/v in acetone/olive oil 4:1and mice treated with the positive control item.

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.

CALCULATION OF EC3VALUE

aEC3= 2^ {log2(c) + (3-d)/(b-d)] x [log2(a)-log2(c)]}

a

=

10

b

=

9.96

c

=

5

d

=

4.45

EC3= 2^ {log2(5) + (3-4.45)/(9.96-4.45)] x [log2(10)-log2(5)]} = 4.2

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (extrapolated EC3value) was calculated to be 4.2%.


a= mid concentration giving a stimulation index >3

b = actual stimulation index caused by ‘a’

c = lowest concentration giving a stimulation index of >3

d = actual stimulation index caused by ‘c’

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

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

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

10

S-1

20

21

0

0

0

0

0

0

0

0

0

25

S-2

19

19

0

0

0

0

0

0

0

0

0

50

S-3

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
(% 
v/v) in
acetone/olive oil 4:1

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

10

S-1

0

0

0

0

1

1

1

1

0

0

0

0

25

S-2

0

0

0

0

1

1

1

1

1

1

1

1

50

S-3

0

0

0

0

1

1

1

1

1

1

1

1

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

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

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

10

S-1

0.250

0.240

0.265

0.255

0.260

0.250

overall mean (mm)

0.245

0.260

0.255

overall mean
ear thickness change (%)

na

6.122

4.082

 

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

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

25

S-2

0.235

0.235

0.260

0.250

0.220

0.220

overall mean (mm)

0.235

0.255

0.220

overall mean
ear thickness change (%)

na

8.511

-6.383

 

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

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

50

S-3

0.230

0.240

0.235

0.240

0.340

0.375

overall mean (mm)

0.235

0.238

0.358

overall mean
ear thickness change (%)

na

1.064

52.128


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 Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

842.71

1292.88
(±664.18)

N/A

N/A

1-2

684.77

1-3

1389.12

1-4

1173.88

1-5

2373.92

Test Item
5% v/v in
acetone/olive oil 4:1

2-1

4566.60

5750.74**
(±1297.86)

4.45

Positive

2-2

4524.82

2-3

7373.05

2-4

5477.36

2-5

6811.88

Test Item
10% v/v in
acetone/olive oil 4:1

3-1

9372.04

12875.10**
(±3440.25)

9.96

Positive

3-2

11489.32

3-3

18401.53

3-4

11459.78

3-5

13652.81

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

4-1

8426.12

17758.98*
(±7588.48)

13.74

Positive

4-2

16743.45

4-3

20557.59

4-4

14258.08

4-5

28809.68

Positive Control Item

25% v/v in
acetone/olive oil 4:1

5-1

5786.90

8562.97
(±3571.37)

6.62

Positive

5-2

8061.03

5-3

5688.11

5-4

14456.01

5-5

8822.82


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
acetone/olive oil 4:1

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% v/v in
acetone/olive oil 4:1

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% v/v in
acetone/olive oil 4:1

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
acetone/olive oil 4:1

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

25% v/v in
acetone/olive oil 4:1

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

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test.
The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.
Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% v/v, 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 acetone/olive oil 4:1at concentrations of 25%,10% or 5% v/v. A further group of five animals was treated withacetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser,α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

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% v/v in
acetone/olive oil4:1

4.45

Positive

10% v/v in
acetone/olive oil4:1

9.96

Positive

25% v/v in
acetone/olive oil4:1

13.74

Positive

Positive
Control Item

25% v/v in
acetone/olive oil4:1

6.62

Positive

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (extrapolated EC3value) was calculated to be 4.2%.

Conclusion. 

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

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A key study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% v/v, 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 acetone/olive oil 4:1at concentrations of 25%,10% or 5% v/v. A further group of five animals was treated withacetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser,α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was found to be 4.45 using 5% v/v in acetone/olive oil, the lowest concentration tested. 

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (extrapolated EC3value) was calculated to be 4.2%.

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

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.


Migrated from Short description of key information:
In a LLNA the Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was found to be 4.45 using 5% v/v in acetone/olive oil, the lowest concentration tested. 
The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (extrapolated EC3value) was calculated to be 4.2%.
The test item was considered to be a sensitiser under the conditions of the test.

Justification for selection of skin sensitisation endpoint:
GLP Guideline study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance tested positive in a LLNA with a stimulation index of > 3 at the lowest concentration tested.