Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.02.2011-04.10.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

EC Number:

700-714-9

Cas Number:

1254469-57-2

Molecular formula:

C14H20ClN3O3

IUPAC Name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

Details on test material:

- Name of test material (as cited in study report): Tinocat ES 96000
- Physical state: Solid, white
- Analytical purity: see analytical report, study code 11L00011
- Lot/batch No.: RD173323
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed
- Storage condition of test material: Room temperature (protected from light, protected against humidity)

- Other: pH-value: Ca. 4 (undiluted test substance, moistened with water); CAS name: Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl] -2-oxoethyl]-4-methyl-, choride (1:1)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.6 g – 21.6 g
- Housing: single housed animals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
- Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands)

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic® L 92 Surfactant in highly de-ionized water

Concentration:

5%, 10% and 25% of Tinocat ES 96000 were formulated in 1% aqueous Pluronic®

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: pre-test revealed that the 50% concentration is unsuitable for appropriate application on the ears and therefore a 25% preparation was selected as the high concentration
- Irritation: no relevant signs of local irritation
- Lymph node: increase in lymph node weights was noted

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: The increase in 3H-thymidine incorporation by a factor of ≥ 3 (SI ≥ 3) and/or the increase in cell count by a factor of ≥ 1.5 (SI ≥ 1.5) as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.

Positive control substance(s):

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

Statistics:

The EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: vehicle: 1.00 5%: 1.69 10%: 2.30 25%: 3.17

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: vehicle: 168.8 5%: 285.8 10%: 388.9 25%: 535.9

Any other information on results incl. tables

Table of Lymph Node Weights

Test Item Concentration % (w/w)	Mean Lymph Node Weight (mg per group)	SD	Index
0	4.4	0.4	1.00
5	5.1	0.6	1.14
10	5.9	0.8	1.32
25	6.7	0.9	1.51

Table of Lymph Node Cell Counts

Test Item Concentration % (w/w)	Mean Lymph Node Cell Count (per Node Pair)	SD	Index
0	6,024,750	557,378	1.00
5	7,532,400	886,241	1.25
10	7,785,000	1,604,462	1.29
25	8,652,000	1,541,965	1.44

Table of Ear Weights

Test Item Concentration % (w/w)	Mean Ear Weight (mg per group)	SD	Index
0	30.4	1.3	1.00
5	33.2	1.0	1.09
10	30.9	1.3	1.02
25	30.7	1.1	1.01

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU