Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-05-13 to 2008-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004/73/EC

Deviations:

no

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:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

641-048-8

Cas Number:

110839-13-9

Molecular formula:

C37H48N4O4

IUPAC Name:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Hungarian Supplier: WOBE Kereskedelmi Kft. H-1164, Budapest, Garmada u. 10.
- Age at study initiation: Young adult, 9 - 10 weeks old, age-matched within one week
- Weight at study initiation: The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Individual caging Type II. polypropylene/ polycarbonate
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes: 8 - 12 air exchange/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

25, 10, 5, 2.5 % (w/v)

No. of animals per dose:

4 female/treatment group

Details on study design:

In the main assay, twenty female CBA/Ca mice were allocated to five groups of four animals each:
- Four groups received the appropriate formulation of the test item at concentrations of 25, 10, 5 and 2.5 % (w/v)
- the negative control group received the solvent propylene glycol (PG)
Each substance was applied on the external surface of each ear (25 µL/ear) for three consecutive days (Day 0, 1 and 2) at the appropriate concentrations. On Day 5, the cell proliferation in the local lymph nodes was measured by in corporation of tritiated methyl-thymidine (3HTdR) and the values obtained were used to calculate the stimulation indices (SI)

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control substance alpha-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI >= 3) was noted for HCA with stimulation index value of 7.5, in accordance with previous data.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

26

Test group / Remarks:

25 % test group

Results 2

Key result

Parameter:

SI

Value:

13

Test group / Remarks:

10 % test group

Results 3

Key result

Parameter:

SI

Value:

12.3

Test group / Remarks:

5 % test group

Results 4

Key result

Parameter:

SI

Value:

7.5

Test group / Remarks:

2.5 % test group

Any other information on results incl. tables

A significant lymphoproliferative response (SI >= 3) was noted for the test item at all of the applied concentrations. The stimulation index values were 26.0, 13.0, 12.3 and 7.5 at concentrations of 25 %, 10 %, 5 % and 2.5 %, respectively. The stimulation index values were compatible with the conventional biological dose-response.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item, dissolved in an appropriate solvent up to the maximum feasible concentration (25 %), was shown to have sensitisation potential in the Local Lymph Node Assay.

Executive summary:

Local Lymph Node Assay according to OECD 429 and Commission Directive 2004/73/EC, B.42 was performed with CBA/Ca Ola Hsd mice. The aim of this study was to determine the skin sensitisation potential of the test item following dermal exposure in the Local Lymph Node Assay. The maximum concentration of test item in an acceptable solvent was established according to the principles of the relevant guidelines. The maximum attainable concentration based on solubility and applicability on the ears was 25 % in Propylene glycol (PG). A preliminary irritation/toxicity test was performed showing that the maximum concentration of test item of 25 % in PG was acceptable.

In the main assay twenty female CBA/Ca mice were allocated to five groups of four animals each:

- four groups received the appropriate formulation of the test item at concentrations of 25 %, 10 %, 5 % or 2.5 %,

- the negative control group received the solvent (PG).

Each substance was applied on the external surface of each ear (25 µL/ear) for three consecutive days (Day 1, 2 and 3) at the appropriate concentrations. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl-thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effect on the body weights was observed. No sign of a classical irritation was observed in any of the treated groups.

Significant loss of hair was observed in the 25 % dose group (4/4 animals) from Day 2 after the second treatment: the affected area enlarged after the subsequent treatment and the skin was dry and exfoliating on the hairless area (3/4 animals) from Day 4 or 5 to the study termination. No redness or damage of the skin integrity was observed. No cutaneous effect was observed in the other dose groups.

A lymphoproliferative response (SI >= 3) was noted for the test item at all of the applied concentrations. The stimulation index values (SI) were 26.0, 13.0, 12.3 and 7.5 at concentrations of 25 %, 10 %, 5 % and 2.5 %, respectively. The stimulation index values corresponded to the conventional biological dose-response. The EC3 value (potency) was below the lowest tested concentration of 2.5 %.

The result of the latest reliability check (performed within an interval of no longer than six months, Study code: 08/661-037E) was used to demonstrate the appropriate performance of the assay in accordance with the OECD guideline 429 (Ref. 1). The positive control substance alpha-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI >= 3) was noted for HCA with stimulation index value of 7.5 in accordance with the historical control data. The result confirms the validity of the LLNA in this laboratory.

Under the conditions of the present assay the test item, dissolved in an appropriate solvent up to the maximum feasible concentration (25 %), was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.