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

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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.)

Route:

epicutaneous, open

Vehicle:

propylene glycol

Concentration / amount:

30% in propylene glycol

Route:

other: intravenously

Vehicle:

propylene glycol

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

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.

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.

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.

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

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Target CAS no. 2387-03-3 -C.I. Pigment Yellow 101: Summary

Skin sensitisation

BASF study report (Report no. 58H0031/012312), 2010:

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.

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.

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.

In a supporting study (Prediction report, Sustainability Support Services - 2016): The skin sensitisation potential for C.I. Pigment Yellow 101 is estimated using OECD QSAR toolbox version 3.3. No sensitising effects were observed in the test animal guinea pig (male/female).The test material C.I. Pigment Yellow 101 was found to be a Not-sensitizing to guinea pig (male/female).

In a supporting studies on similar substance (CAS no.1948-33-0);

 

Mary Ann Liebert, Inc., Publishers, 1986:

1) Skin sensitization study was carried out on humans using Modified Draize-Shelanski-Jordan patch procedure.Test chemical conc. of 0.11% by weight used for the study.Total 55 humans were used for the study. Out of 55 test humans, 49 are females and 6 are males.

The test material was applied under occlusive patches for 24 h every other day for a total of 10 induction patches. After a 13-day non treatment period, a 48-h challenge patch was applied to the back of each subject. A second 48-h challenge patch was applied 7 days later.The challenge sites were evaluated 48 and 72 h after application.

No skin reactions were observed in any other subject during either induction or challenge phases. Thus the test chemical was evaluated to benon-sensitizer to test humans.

2) Skin sensitization study was carried out on humans using Modified Draize-Shelanski-Jordan patch procedure.Test chemical conc. of 0.15% by weight used for the study.Total 52 humans were used for the study. Out of 52 test humans, 41 are females and 11 are males.

The test material was applied under occlusive patches for 24 h every other day for a total of 10 induction patches. After a 13-day non-treatment period, a 48-h challenge patch was applied to the back of each subject. A second 48-h challenge patch was applied 7 days later.The challenge sites were evaluated 48 and 72 h after application.

No skin reactions were observed in any other subject during either induction or challenge phases. Thus the test chemical was evaluated to benon-sensitizer to test humans.

3) Skin sensitization study was carried out on humans using Modified Draize-Shelanski procedure.Test chemical conc. of0.054% by weight used for the study.Total 110 humans were used for the study. Out of 110 test humans, 92 are females and 18 are males.

The test material was applied under occlusive patches to the same skin site for a total of eight 24-h inductions. After a 11-day non-treatment period, a 48-h challenge patch was applied to the induction site. A second 48-h challenge patch was applied following removal of the initial challenge patch. Challenge sites were evaluated immediately and 24 h after patch removal.

No skin reactions were observed in any other subject during either induction or challenge phases. Thus the test chemical was evaluated to be non-sensitizer to test humans.

On the basis of available information for the target as well as read across substance , the test substance can be considered as not sensitising to the skin, in accordance with the CLP criteria. 

 

Migrated from Short description of key information:
In the skin sensitization study conducted using OECD guideline 429, 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.
Thus it was concluded that Pigment Yellow 101 does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Justification for selection of skin sensitisation endpoint:
The data is considered to be a highly reliable data since it is an exprimental data conducted using OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).Data purcahased from BASF

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data for Pigment Yellow 101, the test chemical is classified as being “Non-sensitizing”as per the CLP criteria.