2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-06-11 to 2007-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

29th Adaption of Guideline 67/548/EEC, B.42

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modification refer to the measurement of cell proliferation by cell
counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes.

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:

Copolymer of hexahydro-2H-azepin-2-one and 1,6-diisocyanatohexane

EC Number:

607-997-7

Cas Number:

26776-30-7

Molecular formula:

Exact identification is not feasible

IUPAC Name:

Copolymer of hexahydro-2H-azepin-2-one and 1,6-diisocyanatohexane

Test material form:

other: liquid

Details on test material:

- Appearance: clear
- Physical state: liquid
- Content 72 % in solvent

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win: NMRI
- Age at study initiation: 9 weeks
- Weight at study initiation: 26 - 32 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark/ hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

The stability of the test item in the vehicle was analytically verified for up to 2 hours.

Concentration:

50 % test item in MEK for induction and challenge phase
0.5 % DNCB (positive control) for induction and challenge phase

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated immediately before each administration in methyl ethyl ketone.
- Induction: The test item in the formulation was applied epicutaneously onto the pre-shaven flank of the animals. This treatment was repeated on
three consecutive days (d1, d2 and d3). The volume administered was 50µl/ear.
- Challange: The test item in the formulation was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was
repeated on three consecutive days (d15, d16 and d17). The volume administered was 25µl/ear.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d18). The appropriate organs were
then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight

Positive control substance(s):

other: 1-Chloro-2,4-dinitrobenzene (DNCB) CAS-No: 97-00-7

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group(s; vehicle) by a one-way
analysis of variance (ANOVA) when the variances are considered homogeneous according to an homogeneity testing like Cochrans test. Outlying
values in the LN weights were eliminated at a probability level of 99% by Nalimov's method (Statistik für naturwissenschaftliche Berufe, 1982, 88-89).
In addition, for the LLNA/IMDS the smallest significant differentes in the means were calculated by Scheffe's method (Biometrica 40, 1953, 87-104),
which according to Sachs (Angewandte Statistik 6th and 10th edition, Springer Verlag, Berlin, 1978/2002 ) can be used for both equal and unequal
sample sizes.

Results and discussion

Positive control results:

Performed in 2002. The "positive level" which is 1.3 for cell counts has been statistically significant exceeded in the highest dose group (30%).
(Cell index/concentration: 0.88 / 3%; 1.13/ 10%; 1.77/ 30%)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The "positive level" which is 1.4 for the cell index was statistically significant exceeded after application of 0.5 % DCNB

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: modified LLNA; measurement of cell counts instead of radioactive labeling

Any other information on results incl. tables

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Conclusions:

Sensitizing property can be excluded after application of 50 % concentration of the test item for induction and challenge.

Executive summary:

The aim of these investigations was to verify any specific (sensitizing) stimulating potential of the test item as assumed after primary response investigations (Bayer AG, 2003).

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of the test item for 3 consecutive days, each onto the flank (induction) and both ears (challenge) of the animals. Application with the positive control compound DNCB led to the effects expected after specific challenge, i.e. significant increase in cell counts and ear swelling compared to primary response. Compared to vehicle treated animals there was a clear increase in cell counts in group 2 (vehicle/test item). This change is of statistical significance and the "positive level" of index 1.4 has been exceeded. In contrast, cell count indices of challenge (group 3, 2x test item) did not reach or exceeded this cut-off. The "positive level" of ear swelling has been exceeded in group 2 and 3. These increases are of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was detected in these groups, too.

Taken together, no activation of antigen specific cells via dermal route was determined after challenge application of

the test item by the used method.

In conclusion, these results show that the test item has an irritant potential in mice after dermal application of a 50% concentration, while a specific memory development has not been observed. Therefore, a sensitizing property can be excluded after application of 50 % concentration of the test item for induction and challenge. These results are verified by the comparision with the results of the positive control group.