Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2009 to 20 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 429

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
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)
Remarks:
Date of GLP inspection: 2009-11-26 Date of signature: 26-11-2009
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of divinylbenzene and ethylstyrene
EC Number:
910-757-7
Cas Number:
N/A
Molecular formula:
Divinylbenzene: C10H10 Ethylstyrene: C10H12
IUPAC Name:
Reaction mass of divinylbenzene and ethylstyrene
Details on test material:
Sponsor's identification : Divinylbenzene 55
Description : clear colourless liquid with black inclusions
Batch number : XF30012V44
Date received : 07 August 2009
Storage conditions : approximately 4°C in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
EXAMPLE:
TEST ANIMALS
- Source:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited, Bicester, Oxon, UK.

The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitisers and non-sensitisers during the in-house validation. The results of routine positive control studies are shown in Appendix 1 and Appendix 2. The results of the study are believed to be of value in predicting the sensitisation potential of the test material to man.

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
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.

- Diet (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES:
From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Groups of five mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1.
No. of animals per dose:
Groups of five mice were treated
Details on study design:
RANGE FINDING TESTS:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test material 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.


- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in the results section (table 1).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 25% and 50% v/v in acetone/olive oil 4:1 and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- 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(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
Test Material Administration
Groups of five mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material 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 material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material 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.




Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
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)

Results and discussion

Positive control results:
Current Positive Control Study for the Local Lymph Node Assay
Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Method (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test Material: α Hexylcinnamaldehyde
Project number: 0039/1107
Study dates: 11 September 2009 to 17 September 2009
Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. The control group was shared with Project Number 0673/0012.
Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
acetone/olive oil 4:1 Stimulation Index Result
15 3.70 Positive
Conclusion. α Hexylcinnamaldehyde was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index greater than 3 was recorded for the undiluted test material and the test material at concentrations of 50% and 25% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean radioactive disintegrations per minute (dpm) per animal and the stimulation index (SI) are given in Table 2.

Any other information on results incl. tables

RESULTS

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1.

No signs of systemic toxicity were noted.

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

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given in Table2.

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

4.09

Positive

50

5.00

Positive

100

6.78

Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

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

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 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 THE EXTRAPOLATED EC3VALUE

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

a=      50
b =      5
c =      25
d =      4.09

EC3= 2^ {log2(25) + (3-4.09)/(5-4.09)] x [log2(50)-log2(25)]}

The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (extrapolated EC3value) was calculated to be 10.9%.

a= middle 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

Table1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration

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

100%

S-1

18

18

0

0

0

0

0

0

0

0

0

 


Table2              Individual Disintegrations per Minute and Stimulation Indices

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

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle

1-1

2457.06

1998.59
(±708.09)

N/A

N/A

1-2

2001.58

1-3

1656.20

1-4

2853.11

1-5

1024.98

25

2-1

12453.04

8181.37
(±4506.61)

4.09

Positive

2-2

2308.30

2-3

11160.93

2-4

4436.91

2-5

10547.65

50

3-1

11442.35

9989.32**
(±2492.73)

5.00

Positive

3-2

11810.86

3-3

8533.27

3-4

11877.56

3-5

6282.57

100

4-1

18148.28

13558.00**
(±3172.07)

6.78

Positive

4-2

12422.15

4-3

9516.08

4-4

13021.79

4-5

14681.72


Table3              Individual Clinical Observations and Mortality Data

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

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

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

25

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

50

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

100

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


Table4              Individual Bodyweights and Bodyweight Changes

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

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

16

18

2

1-2

19

20

1

1-3

21

22

1

1-4

19

19

0

1-5

18

19

1

25

2-1

18

18

0

2-2

19

19

0

2-3

21

22

1

2-4

19

19

0

2-5

18

19

1

50

3-1

20

19

-1

3-2

19

19

0

3-3

19

20

1

3-4

18

18

0

3-5

18

19

1

100

4-1

18

19

1

4-2

19

20

1

4-3

19

19

0

4-4

19

19

0

4-5

18

19

1

 


Appendix1      Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of CommissionRegulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non‑Commission members of the European Centre for the Validation of Alternative Method (ECVAM) Scientific Advisory Committee (ESAC) at its 26thmeeting held on 26 – 27 April 2007 at ECVAM,,.

Test Material:                                               α‑Hexylcinnamaldehyde

Project number:                                          0039/1107

Study dates:                                                 11 September 2009 to 17 September 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) ofα‑Hexylcinnamaldehydeas a solution in acetone/olive oil 4:1at a concentration of 15% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. The control group was shared with Project Number 0673/0012.

Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

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

Stimulation Index

Result

15

3.70

Positive

Conclusion. α‑Hexylcinnamaldehydewas considered to be a sensitiser under the conditions of the test.

 


Appendix2      Summary of Positive Control Data for the Local Lymph Node Assay

Project Number

Start Date

Finish Date

Test Material

Concentration

Vehicle

Stimulation Indexa

Classificationb

0039/1061

05/11/08

11/11/08

α‑Hexylcinnamaldehyde

15% v/v

acetone

4.21

Positive

0039/1062

05/11/08

11/11/08

α‑Hexylcinnamaldehyde

15% v/v

ethanol/distilled water 7:3

9.49

Positive

0039/1078

18/03/09

24/03/09

2,4-Dinitrobenzenesulfonic acid, sodium salt

10% w/w

1% pluronic L92
in distilled water

13.71

Positive

0039/1079

20/03/09

26/03/09

Phenylacetaldehyde (90%)

2.5% v/v

propylene glycol

8.00

Positive

0039/1080

24/04/09

30/04/09

α‑Hexylcinnamaldehyde

15% v/v

acetone/olive oil 4:1

8.34

Positive

0039/1081

30/04/09

06/05/09

α‑Hexylcinnamaldehyde

15% v/v

dimethyl formamide

4.24

Positive

0039/1082

17/04/09

23/04/09

α‑Hexylcinnamaldehyde

50% v/v

cotton seed oil

6.54

Positive

0039/1083

16/04/09

22/04/09

α‑Hexylcinnamaldehyde

15% v/v

butanone

4.08

Positive

0039/1084

17/04/09

23/04/09

α‑Hexylcinnamaldehyde

15% v/v

dimethyl sulphoxide

4.60

Positive

0039/1091

25/06/09

01/07/09

α‑Hexylcinnamaldehyde

15% v/v

ethanol/distilled water 7:3

10.68

Positive

0039/1092

25/06/09

01/07/09

α‑Hexylcinnamaldehyde

15% v/v

acetone

10.72

Positive

0039/1099

08/07/09

14/07/09

α‑Hexylcinnamaldehyde

5%, 10% 25% v/v

1% pluronic L92
in distilled water

1.30, 2.37, 8.14

Positive

0039/1107

11/09/09

17/09/09

α‑Hexylcinnamaldehyde

15% v/v

acetone/olive oil 4:1

3.70

Positive

0039/1108

01/107/09

07/10/09

α‑Hexylcinnamaldehyde

25% v/v

1% pluronic L92
in distilled water

5.11

Positive

 


Appendix3      Vehicle Determination Record

Vehicle

Concentration

Method of Preparation

Description of Formulation

Suitability*

acetone/olive oil (4:1)

50%
0.5 ml test material + 0.5 ml vehicle

Vortex mixer

solution

suitable for dosing


0=     No signs of systemic toxicity

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

N/A=  Not applicable

**=     Significantly different from control group p<0.01

0=     No signs of systemic toxicity

a=      Ratio of test to control lymphocyte proliferation

b=      Stimulation index greater than 3.0 indicates a positive result

*=      Suitable for dosing if formulation is a solution or fine homogenous suspension which can be     administered via a micropipette

Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual 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 material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in3HTdR incorporation will be classified as a "non-sensitiser".

The results were evaluated according to Commission Directive 2001/59/EC for classification and labelling of dangerous substances.

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 SI (Stimulation Index). The point with the higher SI was denoted (a, b) and the point with the lower SI 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= middle 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

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a weak sensitiser under the conditions of the test. The test material should be classified as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, indication of danger ‘irritant’ and the risk phrase R 43 “May Cause Sensitisation by Skin Contact” are therefore required.
Executive summary:

Introduction. 

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

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

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

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 undiluted test material or the test material as a solution in acetone/olive oil 4:1at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

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

4.09

Positive

50

5.00

Positive

100

6.78

Positive

The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 10.9%.

Conclusion. 

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

The test material was classified as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, indication of danger ‘irritant’ and the risk phrase R 43 “May Cause Sensitisation by Skin Contact” are therefore required.