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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
Study period:
The study was performed between 28 August 2012 and 11 September 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

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)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
other: solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd.
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was controlled to remain within target range of 19 to 25°C
- Humidity (%): The relative humidity was controlled to remain within target range of 30 to 70%,
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary Screening Test: 25%
Main Test: 25%, 10% and 5%
No. of animals per dose:
Preliminary Screening Test: 1 mouse
Main Test: Five mice per dose
Details on study design:
PREPARATION OF TEST ITEM:
For the purpose of the study, the test item was freshly prepared as a suspension in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

PRELIMINARY SCREENING TEST:
Using available information 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 test item at a maximum attainable concentration of 25% 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. Local irritation was scored daily according to the scale for erythema below. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

Scale for Erythema :
Observation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4

The thickness of each ear was measured using an Oditest micrometer, 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% w/w in dimethyl formamide. 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 15% v/v in dimethyl formamide was applied to the dorsal surface of each ear.

The thickness of each ear was measured using an Oditest micrometer, pre dose on Day 1 and on Days 2, 3, 4, 5 and 6. Any changes in the ear thickness were noted. Daily mean ear thickness changes were calculated. 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.

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,) 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.

Local Skin Irritation Observations: All animals were observed daily. Any signs of local skin irritation noted 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 beta-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.








Positive control substance(s):
other: α-Hexylcinnamaldehyde, tech., 85%. The positive control item was freshly prepared as a 15% v/v dilution in dimethyl formamide.
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)

Results and discussion

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 15% w/w in dimethyl formamide.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.81
Test group / Remarks:
Test item 5% w/w in dimethyl formamide
Parameter:
SI
Value:
1.51
Test group / Remarks:
Test item 10% w/w in dimethyl formamide
Parameter:
SI
Value:
1.24
Test group / Remarks:
Test item 25% w/win dimethyl formamide

Any other information on results incl. tables

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 (attached background material).

Off white residual test item on the ears was noted on Days 1 to 5. No signs of systemic toxicity,visual local skin irritation or excessive irritation indicated by anequal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 25%,10% and 5% w/w in dimethyl formamide.

Main Test

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 (attached background material).

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% w/w in
dimethyl formamide

0.81

Negative

10% w/w in
dimethyl formamide

1.51

Negative

25% w/w in
dimethyl formamide

1.24

Negative

Positive
Control Item

15% v/v in
dimethyl formamide

5.19

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. The ear thickness measurements and mean ear thickness changes are given in Table 7 (see attached background material).

Off white residual test item on the ears was noted in animals treated with the test item. 

There were no deaths. No signs of systemic toxicity or excessive irritation indicated by anequal to or greater than 25% increase in mean ear thickness were noted in the test or control group animals during the test. No visual local skin irritation was noted in animals treated with the test item or vehicle control item during the test. Very slight erythema was noted on Days 2 and 3 on both ears of the animals treated with the positive control item. 

Bodyweight:

Individual bodyweights and bodyweight changes for test and control animals are given in Table 8 (attached background material).

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.

The test item did not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals.

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 15% w/w in dimethyl formamide
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 tobe 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 maximum attainable 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 five animals, were treated with 50 µl (25 µl per ear) of the test item as a suspension in dimethyl formamide at concentrations of 25%,10% or 5% w/w. A further group of five animals was treated with dimethyl formamide alone. 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 15% v/v in dimethyl formamide.

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% w/w in
dimethyl formamide

0.81

Negative

10% w/w in
dimethyl formamide

1.51

Negative

25% w/w in
dimethyl formamide

1.24

Negative

Positive
Control Item

15% v/v in
dimethyl formamide

5.19

Positive

Conclusion

The test item was considered to be a non-sensitiserunder the conditions of the test.

The test item did not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals.

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 15% w/wi n dimethyl formamide.