Registration Dossier

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Positive control results:
Appendix 1 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 Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test Material: α-Hexylcinnamaldehyde, tech., 85%
Project number: 0039/1116
Study dates: 11 November 2009 to 17 November 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide alone.

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

Concentration (% v/v) in dimethyl formamide Stimulation Index Result

15 5.16 Positive

Conclusion. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
1.47
Test group / Remarks:
5% w/w
Key result
Parameter:
SI
Value:
1.44
Test group / Remarks:
10% w/w
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
25% w/w
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Table 1               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

25%

S-1

16

18

0

0

0

0

0

0

0

0

0


0= No signs of systemic toxicity

Table 2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

3106.38

388.30

na

na

5

4551.35

568.92

1.47

Negative

10

4488.50

561.06

1.44

Negative

25

4531.39

566.42

1.46

Negative


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


Table 3              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
dimethyl formamide

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

5

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

10

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

25

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


0=      No signs of systemic toxicity

Table 4              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
dimethyl formamide

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

22

20

-2

1-2

21

21

0

1-3

20

19

-1

1-4

21

19

-2

5

2-1

18

18

0

2-2

19

18

-1

2-3

19

18

-1

2-4

21

22

1

10

3-1

19

20

1

3-2

19

19

0

3-3

19

19

0

3-4

19

20

1

25

4-1

20

21

1

4-2

20

19

-1

4-3

20

20

0

4-4

19

19

0


Appendix 2      Summary of Positive Control Data for the Local Lymph Node Assay

Project Number

Start Date

Finish Date

Test Material

Concentration

Vehicle

Stimulation Indexa

Classificationb

0039/1091

25/06/09

01/07/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

ethanol/distilled water 7:3

10.68

Positive

0039/1092

25/06/09

01/07/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone

10.72

Positive

0039/1099

08/07/09

14/07/09

α-Hexylcinnamaldehyde, tech., 85%

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, tech., 85%

15% v/v

acetone/olive oil 4:1

3.70

Positive

0039/1108

01/10/09

07/10/09

α-Hexylcinnamaldehyde, tech., 85%

25% v/v

1% pluronic L92
in distilled water

5.11

Positive

0039/1115

05/11/09

11/11/09

Phenylacetaldehyde (90%)

2.5% v/v

propylene glycol

4.20

Positive

0039/1116

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl formamide

5.16

Positive

0039/1117

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

50% v/v

cottonseed oil

6.40

Positive

0039/1118

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

butanone

7.45

Positive

0039/1119

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl sulphoxide

6.07

Positive

0039/1120

05/11/09

11/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone/olive oil 4:1

3.12

Positive

0039/1138

12/02/10

18/02/10

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

ethanol/distilled water 7:3

9.61

Positive

0039/1139

17/02/10

23/02/10

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone

5.55

Positive


a=      Ratio of test to control lymphocyte proliferation

b=      Stimulation index greater than 3.0 indicates a positive result

Interpretation of results:
other: Not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test
Executive summary:

This data is being read across from the source study that tested Resin acids and rosin acids, calcium salts based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

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" (adopted24 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 25% w/w, 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 material as a suspension in dimethyl formamide at concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with dimethyl formamide 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 (%w/w) in
dimethyl formamide

Stimulation Index

Result

5

1.47

Negative

10

1.44

Negative

25

1.46

Negative

Conclusion. 

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification: CAS No 9007-13-0 Resin and rosin acids, calcium salts
Supplier : FLINT Group Frankfurt GmbH
Description : amber coloured solid
Batch number : not supplied
Date received : 04 March 2010
Expiry date : 04 March 2011
Storage conditions: room temperature 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:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, 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 (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, 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.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 25% , 10% or 5% v/v (in dimethylformamide)
No. of animals per dose:
Groups of four mice were treated
Details on study design:
RANGE FINDING TESTS:
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%, 10 and 5% v/v in dimethylformamide.

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:
For the purpose of the study, the test material was used freshly prepared in dimethylformamide. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.

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 and 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.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided.

Results and discussion

Positive control results:
Appendix 1 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 Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test Material: α-Hexylcinnamaldehyde, tech., 85%
Project number: 0039/1116
Study dates: 11 November 2009 to 17 November 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide alone.

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

Concentration (% v/v) in dimethyl formamide Stimulation Index Result

15 5.16 Positive

Conclusion. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.47
Test group / Remarks:
5% w/w
Key result
Parameter:
SI
Value:
1.44
Test group / Remarks:
10% w/w
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
25% w/w
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Any other information on results incl. tables

Table 1               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

25%

S-1

16

18

0

0

0

0

0

0

0

0

0


0= No signs of systemic toxicity

Table 2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

3106.38

388.30

na

na

5

4551.35

568.92

1.47

Negative

10

4488.50

561.06

1.44

Negative

25

4531.39

566.42

1.46

Negative


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


Table 3              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
dimethyl formamide

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

5

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

10

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

25

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


0=      No signs of systemic toxicity

Table 4              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
dimethyl formamide

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

22

20

-2

1-2

21

21

0

1-3

20

19

-1

1-4

21

19

-2

5

2-1

18

18

0

2-2

19

18

-1

2-3

19

18

-1

2-4

21

22

1

10

3-1

19

20

1

3-2

19

19

0

3-3

19

19

0

3-4

19

20

1

25

4-1

20

21

1

4-2

20

19

-1

4-3

20

20

0

4-4

19

19

0


Appendix 2      Summary of Positive Control Data for the Local Lymph Node Assay

Project Number

Start Date

Finish Date

Test Material

Concentration

Vehicle

Stimulation Indexa

Classificationb

0039/1091

25/06/09

01/07/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

ethanol/distilled water 7:3

10.68

Positive

0039/1092

25/06/09

01/07/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone

10.72

Positive

0039/1099

08/07/09

14/07/09

α-Hexylcinnamaldehyde, tech., 85%

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, tech., 85%

15% v/v

acetone/olive oil 4:1

3.70

Positive

0039/1108

01/10/09

07/10/09

α-Hexylcinnamaldehyde, tech., 85%

25% v/v

1% pluronic L92
in distilled water

5.11

Positive

0039/1115

05/11/09

11/11/09

Phenylacetaldehyde (90%)

2.5% v/v

propylene glycol

4.20

Positive

0039/1116

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl formamide

5.16

Positive

0039/1117

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

50% v/v

cottonseed oil

6.40

Positive

0039/1118

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

butanone

7.45

Positive

0039/1119

11/11/09

17/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl sulphoxide

6.07

Positive

0039/1120

05/11/09

11/11/09

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone/olive oil 4:1

3.12

Positive

0039/1138

12/02/10

18/02/10

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

ethanol/distilled water 7:3

9.61

Positive

0039/1139

17/02/10

23/02/10

α-Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone

5.55

Positive


a=      Ratio of test to control lymphocyte proliferation

b=      Stimulation index greater than 3.0 indicates a positive result

Applicant's summary and conclusion

Interpretation of results:
other: Not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test
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" (adopted24 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 25% w/w, 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 material as a suspension in dimethyl formamide at concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with dimethyl formamide 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 (%w/w) in
dimethyl formamide

Stimulation Index

Result

5

1.47

Negative

10

1.44

Negative

25

1.46

Negative

Conclusion. 

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