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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 29 December 2009 and 13 January 2010.
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.

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
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 inspection: 15-09-2009 Date of Signature: 26-11-2009
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
1,3-Cyclohexanedione
IUPAC Name:
1,3-Cyclohexanedione
Details on test material:
Sponsor's identification: 1,3-Cyclohexanedione
Description : pale beige solid
Batch number : 1091224
Date received : 24 November 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:
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
Concentration:
50%, 25% or 10% w/w in dimethyl formamide.
No. of animals per dose:
Groups of four mice were treated
Details on study design:
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 Commission Regulation (EC) No. 440/2008

The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.
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.

Preparation of Test Material
For the purpose of the study, the test material was freshly prepared as a solution in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section. The vehicle determination record is included as Appendix 3.
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 Guideline.


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 test material at a concentration of 50% w/w in dimethyl formamide, 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 of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
No signs of systemic toxicity were noted.

Main Test
Test Material Administration
Groups of four mice were treated with the test material at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. 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 four mice received the vehicle alone in the same manner.

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

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.

Project number: 0039/1116

Test material: α Hexylcinnamaldehyde, tech., 85%

Study dates: 11 November 2009 to 17 November 2009

Methods.
A test 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 incorporation of 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
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
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 10 1.55 Negative 25 1.82 Negative 50 1.45 Negative
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2.

Any other information on results incl. tables

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 dose levels selected for the main test were50%,25% and10% w/win dimethyl formamide.

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.

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

Concentration (%w/w) in
dimethyl formamide

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

50

S-1

20

19

0

0

0

0

0

0

0

0

0

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

11398.73

1424.84

na

na

10

17653.87

2206.73

1.55

Negative

25

20701.06

2587.63

1.82

Negative

50

16555.58

2069.45

1.45

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

10

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

25

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

50

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

18

19

1

1-2

18

19

1

1-3

19

19

0

1-4

18

19

1

10

2-1

20

20

0

2-2

19

18

-1

2-3

17

19

2

2-4

20

20

0

25

3-1

21

22

1

3-2

21

22

1

3-3

20

20

0

3-4

19

19

0

50

4-1

19

19

0

4-2

17

18

1

4-3

19

20

1

4-4

21

21

0

Appendix 3      Vehicle Determination Record

Vehicle

Concentration

Method of Preparation

Description of Formulation

Suitability*

acetone/olive oil (4:1)

50%
0.5 g test material + 0.5 g vehicle

Vortex mixer

N/A

X

dimethyl formamide

50%
0.5 g test material + 0.5 g vehicle

Vortex mixer

Solution


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

N/A =   Not applicable

Please also see attached Appendix 2    Summary of Positive Control Data for the Local Lymph Node Assay & Appendix 4  Certificate of Analysis

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC.
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are 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 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 50% 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 asolutionindimethyl formamideat concentrations of 50%,25% or10% w/w. A further group of four animals was treated withdimethyl formamidealone.

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

10

1.55

Negative

25

1.82

Negative

50

1.45

Negative

Conclusion. 

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

The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.

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