[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2013 and 07 January 2014

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:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

See Attachment section

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 65088/A TE
Batch: CUF 176, 3011 SCE 346
Description: light yellow powder
Purity: 93.8%
Expiry date: 29 September 2017
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:

Animals and Animal Husbandry

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 acclimatization 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 group 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.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

EXAMPLE: Please see below for Vehicle Determination Record

Concentration:

Preliminary screening test
25% w/w in dimethyl sulphoxide
Main Test
Each group was exposed to concentrations of 25%, , 10% or 5% w/w in dimethyl sulphoxide

No. of animals per dose:

5

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 one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 25% w/w in dimethyl sulphoxide, 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 skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.

The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), 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 time periods Days 1 and 3 and Days 1 and 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 sensitization.


Main Test
Test Item Administration

Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in dimethyl sulphoxide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin 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 four mice received the vehicle alone in the same manner.


3H-Methyl Thymidine Administration

Five days following the first topical application of the test item or vehicle (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, 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.

Body Weights: The body weight 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. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labeled with the project number and dose concentration. The lymph node cell 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 pooled 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, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by  scintillation counting. The "Poly Q™" 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:

None

Results and discussion

Positive control results:

No positive control group was conducted in this test. Positive control data was used in the report and shown in the Attachment section.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The radioactive disintegrations per minute per animal and the stimulation index are shown in the other information section on results.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The individual DPM's are shown in the other information section on results.

Any other information on results incl. tables

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

Concentration (%w/w) in dimethyl sulphoxide	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	18994.55	2374.32	na	na
5	21895.33	2736.92	1.15	Negative
10	22234.91	2779.36	1.17	Negative
25	23250.62	2906.33	1.22	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 applicab

            

Preliminary Screening Test

The preliminary screening test results are shown in the appendix.

Clinical observations, body weight 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.

 

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal 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 sulphoxide.

Main Test

           

Individual clinical observations and mortality data for test and control animals are given in Table 5.

 

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

      

Individual body weights and body weight change for test and control animals are given in Table 6.

 

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Table5     Individual Clinical Observations and Mortality Data

Concentration (% w/w) in dimethyl sulphoxide		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

  

Table6     Individual Body Weights and Body Weight Change

Concentration (% w/w) in dimethyl sulphoxide	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	18	18	0
	1-2	17	17	0
	1-3	19	19	0
	1-4	18	18	0
5	2-1	19	17	-2
	2-2	19	20	1
	2-3	19	18	-1
	2-4	17	17	0
10	3-1	17	18	1
	3-2	18	18	0
	3-3	18	18	0
	3-4	18	17	-1
25	4-1	18	19	1
	4-2	18	18	0
	4-3	18	18	0
	4-4	19	19	0

 

         

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

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

Executive summary:

INTRODUCTION AND PURPOSE

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Guidelines / Regulations

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

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

2. Method B42 Skin Sensitization (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 items that are moderate to strong sensitizers.

 

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

SUMMARY

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration 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) ofthe test item as asolutionindimethyl sulphoxideat concentrations of 25%,10% or 5% w/w. A further group offour animals was treated withdimethyl sulphoxidealone.

 

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 sulphoxide	Stimulation Index	Result
5	1.15	Negative
10	1.17	Negative
25	1.22	Negative

 

Conclusion

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