Butanamide, 2-[2-(2-methoxy-5-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxo-

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version April 24, 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Version March 2003

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:

2-[(1E)-2-(2-methoxy-5-nitrophenyl)diazen-1-yl]-N-(2-methoxyphenyl)-3-oxobutanamide

EC Number:

617-143-5

Cas Number:

80675-49-6

Molecular formula:

C18H18N4O6

IUPAC Name:

2-[(1E)-2-(2-methoxy-5-nitrophenyl)diazen-1-yl]-N-(2-methoxyphenyl)-3-oxobutanamide

Test material form:

solid: particulate/powder

Details on test material:

- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Specific details on test material used for the study:

- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Analytical purity: 95%
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/J mouse from Charles River Laboratories, Research Models and Services, Germany GmbH, 97633 Sulzfeld, Germany
- Weight at study initiation: 18.2 g – 21.3 g
- Housing: single housed in Makrolon cages, type II
- Diet: ad libitum, Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland
- Water: ad libitum, tap water
- Acclimation period: 7 days before the first application of the test substance.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. – 6.00 a.m. / 6.00 a.m. – 6.00 p.m.)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

Used as vehicle because a good homogeneity of the preparation could be achieved.

Concentration:

30 % preparation (maximum technically applicable concentration); 25 μL test substance suspension in vehicle per ear

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS: not conducted
- Compound solubility: the homogeneity of the test substance preparation during application was provided by stirring with a high speed homogenizer and/or a magnetic stirrer.
- The homogeneity of the test substance preparation was investigated once by analysis during the study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
The parameters used to characterize the response are:
(1) Lymph node cell count,
(2) [3]H-thymidine incorporation into the lymph node cells,
(3) And to a certain extent lymph node weight.
(4) Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
(5) The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
(6) If applicable, the estimated concentration (EC) 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.
(7) In addition the evaluation uses the following considerations:
(a) If biologically relevant increases in ear weights are running in parallel to the increase in cell count, [3]H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Depending on the magnitude of lymph node response the evaluation of the sensitizing potential may be modified or additional studies might be necessary by expert judgment.
(b) If a test substance does not elicit a biological relevant increase in cell count, 3H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
The animals of negative control (group 1) and test (group 2) groups were treated with vehicle or test-substance
1) Treatment
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Mortality: twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.

2) [3]H-thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μL of sterile saline into a tail vein.

3) Terminal procedures
The animals were sacrificed on study day 5 about 5 hours after [3]H-thymidine injection by cervical dislocation.
- Determination of ear weight: to detect a potential inflammatory ear swelling a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals immediately after the death of each animal. The weight of the pooled punches was determined for each test group.
- Removal and weight determination 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 (PBS) in ice water 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 PBS. For determination of cell counts, an aliquot 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 [3]H-thymidine incorporation of the lymph node cells: the remaining cell suspensions were washed twice with PBS and precipitated with 5 % trichloroacetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of [3]H-thymidine into the cells was measured in a ß-scintillation counter.

Positive control substance(s):

other: studies using Alpha-Hexylcinnamaldehyde are performed twice a year in the laboratory

Statistics:

The stimulation indices of cell count, [3]H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

Results and discussion

Positive control results:

The result of the historical positive control tests show that the test system is able to detect sensitizing compounds under the test conditions chosen.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

1.31

Test group / Remarks:

cut off value: 1.5

Results 2

Key result

Parameter:

SI

Remarks:

[3]H-thymidine incorporation

Value:

2.33

Test group / Remarks:

cut off stimulation index: 3

Any other information on results incl. tables

Ear weight:

The test substance (TS) preparation caused some increase in ear weights indicating an ear skin irritation. The ears of all animals showed a yellow discoloration on study days 1 and 2 and on the day of lymph node removal.

Lymph node weight:

no relevant increase was observed.

Body weights:

The expected body weight gain was generally observed in the course of the study.

Other findings:

No abnormalities were observed during general observation. No signs of systemical toxicity were observed.

 

Table 1: Cell count, [3]H-thymidine incorporation and lymph node weight: test group mean values and stimulation indices

Test group	Treatment	Cell counts
		[Counts/Lymph Node Pair]	Stimulation Index(a)
1	Vehicle propylene glycol	7385333	1.00
2	30 % TS in propylene glycol	9642667	1.31
		[3]H-thymidine incorporation
		[DPM/Lymph Node Pair]	Stimulation Index(a)
1	Vehicle propylene glycol	418.3	1.00
2	30 % TS in propylene glycol	973.2	1.12
		Lymph Node Weight
		[mg/Lymph Node Pair]	Stimulation Index(a)
1	Vehicle propylene glycol	4.4	1.00
2	30 % TS in propylene glycol	4.9	1.12
TS: test substance; (a): test group x / test group 1 (vehicle control)

 

Table 2: Ear weight: test group mean values and stimulation indices

Test group	Treatment	Ear weight
		[Counts/Lymph Node Pair]	Stimulation Index(a)
1	Vehicle propylene glycol	31.9	1.00
2	30 % TS in propylene glycol	34.5	1.08

 

Table 3: Mean body weight (bw) and body weight changes (bwc)

Test group	Treatment	Mean values ± standard deviations
		bw at day 0	bw at day 5	bwc day5-day0
1	Vehicle propylene glycol	19.7 ± 1.2	20.3 ± 0.9	0.6 ± 0.5
2	30 % TS in propylene glycol	19.8 ± 0.6	20.9 ± 0.7	1.1 ± 0.7

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met