2-hydroxynaphthalene-1-carbaldehyde [(2-hydroxy-1-naphthyl)methylene]hydrazone

Skin sensitisation

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: Data is from BASF study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The Murine Local Lymph Node Assay (LLNA) is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans.The study was performed based on the method of Kimber I. et al. (1994)1. The study procedure was extended according to the publications described in section “Test Guidelines”, which evaluate the skin sensitizing potential of test items by determination of lymph node response using lymph node weight and lymph node cell count as parameters which are set into perspective to ear weight as an indicator of skin irritation.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxynaphthalene-1-carbaldehyde [(2-hydroxy-1-naphthyl)methylene]hydrazone

EC Number:

219-210-0

EC Name:

2-hydroxynaphthalene-1-carbaldehyde [(2-hydroxy-1-naphthyl)methylene]hydrazone

Cas Number:

2387-03-3

Molecular formula:

C22H16N2O2

IUPAC Name:

1,1'-(hydrazine-1,2-diylidenedimethylylidene)di(2-naphthol)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report):Pigment Yellow 101 (Chemical Name: 2-hydroxynaphthalene-1-carbaldehyde [(2-hydroxy-1-naphthyl)methylene]hydrazone)- Molecular formula (if other than submission substance):C22H16N202- Molecular weight (if other than submission substance):340,385g/mol- Smiles notation (if other than submission substance):Oc1c(c2c(cccc2)cc1)\C=N\N=C\c1c2c(cccc2)ccc1O- InChl (if other than submission substance):1S/C22H16N2O2/c25-21-11-9-15-5-1-3-7-17(15)19(21)13-23-24-14-20-18-8-4-2-6-16(18)10-12-22(20)26/h1-14,25-26H/b23-13+,24-14+- Substance type:Organic- Physical state:Solid-Purity: 100%- Lot/batch No.:29010809T0

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source:Charles River Laboratories, Research Models and Services,Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld- Age at study initiation:6 – 12 weeks- Weight at study initiation:17.3 g – 21.2 g- Housing:Makrolon cage, type II;Nest-building material: wood wool (Typ NBF E-011);Abedd ® Lab. and Vet. Service GmbH Vienna, Austria.PLEXX mouse tunnel (red, transparent) EMSICON Jung GmbH, Forstinning, Germany- Diet (e.g. ad libitum):Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum- Water (e.g. ad libitum):Tap water ad libitum- Acclimation period:7 days before the first test-item applicationENVIRONMENTAL CONDITIONS- Temperature (°C):20 – 24°C- Humidity (%):30 – 70% for relative humidity- Air changes (per hr):No details- Photoperiod (hrs dark / hrs light):12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, open

Vehicle:

propylene glycol

Induction 2

Concentration / amount:

30% in propylene glycol

Challenge

Challenge 1

Route:

other: intravenously

Vehicle:

propylene glycol

Challenge 2

Concentration / amount:

30% in propylene glycol

No. of animals per dose:

Total no of animals-10 Teastitem-5 animalsControl-5 animals

Details on study design:

RANGE FINDING TESTS:MAIN STUDYA. INDUCTION EXPOSURE- No. of exposures: once- Exposure period: day 0 – day 2- Test groups:5- Control group:5- Site: Dorsal part of both ears- Frequency of applications:3 consecutive applications (day 0 – day 2) to the same application site- Duration: - Concentrations:30% in vehicleB. CHALLENGE EXPOSURE- No. of exposures:once- Day(s) of challenge: 5 days- Exposure period: 66 to 72 hours after the last application of test item to the ears- Test groups:5- Control group:5- Site: tail vein.- Concentrations:30% in vehicle- Evaluation (hr after challenge): Immediately after death OTHER:

Positive control substance(s):

no

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Control group 1: Vehicle: propylene glycolTest group 2: Test item 30% in propylene glycol

No. of animals per dose:

Group 1 -5 animalsGroup 2- 5 animals

Details on study design:

Conduct of the study:The study comprised one treatment group and a vehicle control group. Each group consisted of 5 mice.Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York,San Francisco, London, 1978, pp. 62 – 64“.Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/orlocal inflammation at the application sites were noted in the raw data.Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical useconditions.Application volume: 25 μL per earSite of application: Dorsal part of both earsFrequency of application: 3 consecutive applications (day 0 – day 2) to the same application siteMortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.Terminal procedures:The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.Removal and weightdetermination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.Preparation of cell suspension and determination of cell count:After weight determination, the pooled lymph nodes of each test group were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, analiquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®- Counter.Measurement of 3Hthymidine incorporation of the lymph node cells:The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde

Statistics:

Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value.

Results and discussion

Positive control results:

The sensitivity of mice (CBA/CaOlaHsd, Harlan Winkelmann GmbH, Borchen, Germany or CBA/J, Charles River Laboratories, Research Models and Services, Germany GmbH,Sandhofer Weg 7, 97633 Sulzfeld) and the reliability of experimental techniques was assessed regularly using a known sensitizer as recommended by the test guidelines.Positive results were consistently obtained over the years using several variations of the methods and different vehicles.

In vivo (LLNA)

Results

Results 1

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: 1st reading. Group: test group. Dose level: 30%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: not sensitizing according to stimulation index reading.

Results 2

Parameter:

SI

Remarks on result:

other: no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5no increase above the cut off stimulation index of 3

Any other information on results incl. tables

CELL COUNT, 3H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT: TEST GROUP MEAN VALUES AND STIMULATION INDICES

		Cell Counts
Test Group	Treatment	[Counts/Lymph Node Pair]	Stimulation Index^1
1	vehicle propylene glycol	5,808,000	1.00
2	30% in propylene glycol	6,902,667	1.19

 

		³H-thymidine incorporation
Test Group	Treatment	[DPM/Lymph Node Pair]	Stimulation Index^1
1	vehicle propylene glycol	362.1	1.00
2	30% in propylene glycol	883.3	2.44

 

		Lymph Node Weight
Test Group	Treatment	[mg/Lymph Node Pair]	Stimulation Index^1
1	vehicle propylene glycol	4.3	1.00
2	30% in propylene glycol	4.8	1.13

 

EAR WEIGHT: TEST GROUP MEAN VALUES AND STIMULATION INDICES

		Ear Weight
Test Group	Treatment	[mg/animal]	Stimulation Index^1
1	vehicle propylene glycol	31.2	1.00
2	30% in propylene glycol	34.3	1.00

¹test group x / test group 1 (vehicle control)

BODY WEIGHT DATA

Treatment	Animal No.	Body Weight d0 [g]			Body Weight d5 [g]			Body Weight Change d5-d0 [g]
		Individual	Mean	S.D.	Individual	Mean	S.D.	Individual	Mean	S.D.
vehicle propylene glycol	1	21.2	19.4	1.6	23.1	20.3	1.9	1.9	0.8	0.8
	2	20.0			20.9			0.9
	3	17.8			19.0			1.2
	4	20.4			20.2			-0.2
	5	17.8			18.2			0.4
30% in propylene glycol	1	19.6	18.6	1.1	20.0	19.5	1.4	0.4	0.9	0.5
	2	19.5			21.1			1.6
	3	17.3			17.8			0.5
	4	17.4			18.3			0.9
	5	19.0			20.1			1.1

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

No signs of systemic toxicity were noticed.When applied as a 30% preparation in propylene glycol, the test item did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.There was no relevant increase in lymph node weights, as well.Concomitantly, the increase of 3H-thymidine incorporation into the cells was not biologicallyrelevant (no increase above the cut off stimulation index of 3) at this concentration.The test-item preparation caused some increase in ear weights as indication of ear skin irritation. The ears of the mice were slightly yellow discolored on study days 1 and 2 and on the day of lymph node removal.Thus it is concluded that Pigment Yellow 101 does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of Pigment Yellow 101 was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test item to the dorsal skin of the ears.

Groups of 5 female CBA/J mice each were treated with a 30% w/w preparation of the test item in propylene glycol or with the vehicle alone. The 30% preparation was the maximum technically applicable concentration.

The study used 1 test group and 1 control group. Each test animal was applied with 25 μL per ear of the test-item preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone.

Three days after the last application the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and 3H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed.

When applied as a 30% preparation in propylene glycol, the test item did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well.

Concomitantly, the increase of 3H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3) at this concentration.

The test-item preparation caused some increase in ear weights as indication of ear skin irritation. The ears of the mice were slightly yellow discolored on study days 1 and 2 and on the day of lymph node removal.

Thus it is concluded that Pigment Yellow 101 does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.