2-Thiazolamine, 4-(2-naphthalenyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 07 August 2012 and 23 August 2012

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:

2012

Report date:

2012

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:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(naphthalen-2-yl)-1,3-thiazol-2-amine

EC Number:

606-740-6

Cas Number:

21331-43-1

Molecular formula:

C13H10N2S

IUPAC Name:

4-(naphthalen-2-yl)-1,3-thiazol-2-amine

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification : 4-(2-naphthalenyl)-2-thiazolamine
Description : Off white powder
Purity : 98.8%
Batch number : 107 (ex Pentagon)
Date received : 08 May 2012
Expiry date : Not supplied
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 Ltd., 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 (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:

dimethylformamide

Concentration:

The test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide

No. of animals per dose:

Groups of four mice were treated

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 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. 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 bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), 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 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 sensitisation.
Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. 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.
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. 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 labelled 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, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß 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 (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

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 Item: α-Hexylcinnamaldehyde, tech., 85%
Project number:41202697
Study dates: 16 May 2012 to 22 May 2012
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 incorporation of the vehicle control group is as follows:
Concentration (% v/v) in
dimethyl formamide Stimulation Index Result
15 5.74 Positive
Conclusion. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: See Table 4

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 4

Any other information on results incl. tables

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

The results wereinterpretedaccording to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous substances andRegulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

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.

White residual test item on the ears was noted from post-dose on Day 1 to Day 6. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than25% increase in mean ear thicknesswere noted.

Based on this information the dose levels selected for the main test were50%,25% and10% w/windimethyl formamide.

Main Test

Estimation of the Proliferative Response of Lymph Node cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

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	3.89	Positive
25	4.61	Positive
50	3.03	Positive

Clinical Observations and Mortality Data

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

White residual test item on the ears was noted in all test animals. 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 6.

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.

Table 1              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

21

0

0Rt

0Rt

0Rt

0Rt

0Rt

0Rt

0Rt

0Rt

0=      No signs of systemic toxicity

Rt =     White residual test item on the ears

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.240	0.230	0.255	0.260	0.225	0.220
overall mean (mm)		0.235		0.258		0.223
overall mean ear thickness change (%)		na		9.574		-5.319

na=     Not applicable

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%w/w) in dimethyl formamide	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	13291.87	1661.48	na	na
10	51676.80	6459.60	3.89	Positive
25	61319.28	7664.91	4.61	Positive
50	40230.22	5028.78	3.03	Positive

dpm= Disintegrations per minut

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 5              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	0Rt	0	0Rt	0	0Rt	0	0	0
	2-2	0	0Rt	0	0Rt	0	0Rt	0	0	0
	2-3	0	0Rt	0	0Rt	0	0Rt	0	0	0
	2-4	0	0Rt	0	0Rt	0	0Rt	0	0	0
25	3-1	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0	0
	3-2	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0	0
	3-3	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0	0
	3-4	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0
50	4-1	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt
	4-2	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt
	4-3	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt
	4-4	0	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt	0Rt

0=      No signs of systemic toxicity

Rt =     White residual test item on the ears

Table 6              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	17	18	1
	1-2	18	18	0
	1-3	21	20	-1
	1-4	18	18	0
10	2-1	20	18	-2
	2-2	19	19	0
	2-3	19	20	1
	2-4	17	19	2
25	3-1	18	21	3
	3-2	18	19	1
	3-3	20	18	-2
	3-4	18	19	1
50	4-1	19	20	1
	4-2	19	18	-1
	4-3	19	18	-1
	4-4	20	20	0

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item was considered to be a sensitiser under the conditions of the test.
The test item 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.
The test item was classified as a contact sensitiser (Category 1) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances. The Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

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 withthe 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 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 item as a solution in dimethyl formamide at concentrations of 50%, 25% or 10% 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
10	3.89	Positive
25	4.61	Positive
50	3.03	Positive

Conclusion. The test item was considered to be a sensitiser under the conditions of the test.

The test item 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.

The test item was classified as a contact sensitiser (Category 1) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances. The Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.