N-alkylester N-aryl substituted aryl diazo aryl amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 to 12 May 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant with OECD 429 and GLP guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

Name: DYNS 2246

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species and strain: CBA/CaOlaHsd mice
- Source: Harlan Hungarian Supplier: WOBE Kereskedelmi Kft. H-1164, Budapest, Garmada u. 10.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 19.1 – 20.8 grams
- Housing: single
- Diet: ssniff SM R/M-Z+H ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 06 May 2009 To: 12 May 2009

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5%, 10%, 25%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: maximum 25% in DMF
- Irritation: no
- Lymph node proliferation response: NA


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node proliferation assay with incorporated tritiated thymidine
- Criteria used to consider a positive response:
-- exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
AND
-- data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically dosed with 25 µl of the appropriate formulation (5%, 10% or 25%) of the test item using a pipette, on the dorsal surface of each ear.

Positive control substance(s):

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

Statistics:

NA

Results and discussion

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed. A significant lymphoproliferative response (SI >= 3) was noted for HCA with a stimulation index value of 12.7 in the relevant vehicle. The results of the positive control demonstrated the appropriate performance of the assay in accordance with the OECD guideline 429

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 5%: 1.1 10%: 1.3 25%: 1.1

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 5%: 2388.5 10%: 2765.5 25%: 2723.5

Any other information on results incl. tables

The test item is regarded as a sensitiser if both of the following criteria are fulfilled:

• exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR >= 3-fold than recorded in control mice, as indicated by the stimulation index,
• data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM/group	DPM	No. of LNN	DPN	Stimulation Index Values
Background 5 (w/v) % TCA	36,5	-	-	-	-
Negative control DMF	2115	2078.5	8	259.8	1.0
DYNS 2246 25 (w/v) %	2760	2723.5	8	340.4	1.3
DYNS 2246 10 (w/v) %	2802	2765.5	8	345.7	1.3
DYNS 2246 5 (w/v) %	2425	2388.5	8	298.6	1.1
Positive control 25 (w/v) % HCA	26529	26492.5	8	3311.6	12.7

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of the present assay, DYNS 2246 (Batch No.: DYNS 3677 (D06-00693-008)), tested in a suitable vehicle, proved to have no sensitizing potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of DYNS 2246 following dermal exposure.

Based on results of the Preliminary Compatibility Test and on recommendations of the OECD Test Guideline 429, the test item was dissolved in N,N-Dimethyl-formamide (DMF). The maximum attainable concentration based on solubility of the test item in DMF was 25%.

A Preliminary Irritation/Toxicity Test was performed with the test item at concentrations of 25% and 10% in the selected vehicle. According to this test, the applicability and biocompatibility of the test item to the animals’ ears (2 female CBA/CaOlaHsd mice/dose) at the maximum concentration of 25% was acceptable.

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

-      three groups received the appropriate formulation of DYNS 2246 at concentrations of 25%, 10% and 5%,

-      the negative control group received the vehicle (DMF),

-      the positive control group received 25% a-Hexylcinnamaldehyde (HCA) in DMF.

The test item solutions were applied to the dorsal surface of both ears of the mice (25 µl/ear) for three consecutive days (Days 1, 2, and 3). There was no treatment on Days 4, 5, and 6. 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 effects were observed on animal body weights in any of the treated groups. No cutaneous reactions were observed at the site of the treatment in any of the treated groups.

No significant lymphoproliferative response (SI >= 3) of DYNS 2246 was observed at the concentrations applied compared to the negative control. The stimulation index values of the test item were 1.3, 1.3, and 1.1 at treatment concentrations of 25%, 10%, and 5%, respectively. No conventional dose-response to treatment was observed.

A significant lympho­proliferative response (stimulation index value of 12.7) was noted for the positive control chemical HCA. This result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay DYNS 2246 (Batch Number: DYNS 3677 (D06-00693-008)), tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.