Diammonium 5-(hexylsulfonyl)-2-{[3-methyl-2,7-dioxo-1-(3-sulfonatobenzoyl)-2,7-dihydro-3H-naphtho[1,2,3-de]heterocycle-6-yl]amino}benzenesulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2009 to 19 March 2009

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 August 2008; Date of signature: 04 March 2009

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd) and CBA/Ca (CBA/CaBkl)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
- Water: Free access to mains tap water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

IN-LIFE DATES: From: 0 To: Termination

Study design: in vivo (LLNA)

Vehicle:

other: Prepared as a solution in ethanol/distilled water 7:3.

Concentration:

Each group was exposed to concentrations of 25%, 10% or 5% w/w in ethanol/distilled water 7:3.

No. of animals per dose:

Groups of five mice were treated.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Ethanol/distilled water 7:3
- Irritation: The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded.
- Lymph node proliferation response: Not performed for preliminary screening test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive .disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative tothat recorded for the control nodes (Stimulation Index).

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in ethanol/distilled water 7:3. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µI of the appropriate concentration of the test material to the dorsal surface of each ear for three
consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of five mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µI of phosphate buffered saline (PBS) containing 3H-methyl thymidine eHTdR:80µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:
P<0.001 = ***
P<0.01 = **
P<0.05 = *
P≥0.05 = (not significant)

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% v/v) in Stimulation Index Result
ethanol/distilled water 7:3
15 9.49 Positive

Conclusion. a-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A stimulation index of less than 3 was recorded for the three concentrations of the test material (25%, 10% and 5% w/w in ethanol/distilled water 7:3). The stimulation index are also given in Table 2 below.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute per lymph nodes for each individual animal are given in Table 2 on attachment 1 found in the attachments section.

Any other information on results incl. tables

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Red coloured staining of the ears and fur was noted post dose on Days 1 to 3 in all test animals.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBAlCa strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

• United States Environmental Protection Agency Health Effects Test Guidelines OPPTS 870.2600 Skin Sensitisation March 2003

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in ethanol/distilled water 7:3 at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with ethanol/distilled water 7:3 alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in ethanol/distilled water 7:3	Stimulation Index	Result
5	1.13	Negative
10	1.02	Negative
25	1.11	Negative

Conclusion. The test material was considered to be a non-sensitiser under the conditions of the test.