[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 2009 - May 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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

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:

2-Amino-5-cyano-N,3-dimethylbenzamide

Cas Number:

890707-29-6

Molecular formula:

C10H11N3O

IUPAC Name:

2-Amino-5-cyano-N,3-dimethylbenzamide

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 21.6-22.8 g
- Housing: During dosing and resting phases of the study, animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. After final weighing (test Day 5) until sacrifice, animals were housed 1 group per plastic shoebox cage with bedding.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: For a minimum of 6 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): Not reported
- Photoperiod: Alternating 12-hour light and dark cycles
- IN-LIFE DATES: 15 April 2009 - 21 April 2009

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0% (vehicle control), 1%, 10%, 20%, or 40% test substance

No. of animals per dose:

5 female

Details on study design:

Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle control), 1%, 10%, 20%, or 40% test substance. Dimethylsulfoxide (DMSO) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMSO as a positive control. Animals were assigned to dose groups by computerised randomisation so that individual body weights did not vary more than 20% from the group mean.
On each day of dosing (test Days 0-2), 25 μL of test or control substance were administered topically to the dorsum of each mouse ear. Test Days 3-4 were days of rest followed by intravenous (tail vein) injection of 20 μCi of ³H-thymidine per mouse on test Day 5.
Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 6, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm).
A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. Statistically significant increases in cell proliferation in the test concentration groups compared to the vehicle control group and/or SIs of greater than or equal to 3.0 indicated a positive response.

Positive control substance(s):

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

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice, SI = 6.79. Therefore, the LLNA test system was valid for this study.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION:
Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 10%, 20%, and 40% test concentrations. However, stimulation indexes of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., stimulation index = 3) for the test substance under the conditions of this study was not calculable.

CLINICAL OBSERVATIONS:
No clinical signs of toxicity were observed in the study. Stained skin/fur was observed on the ear of one mouse dosed with 40% test substance concentration.

BODY WEIGHTS:
No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test substance did not produce a dermal sensitisation response in mice.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitisation response in mice using the local lymph node assay (LLNA) according to OECD Guideline 429.

Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle control), 1%, 10%, 20%, or 40% test substance on both ears. Dimethylsulfoxide (DMSO) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMSO as a positive control. On test Day 5 of the assay, mice received ³H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears of each mouse from the test substance groups was then evaluated and compared to the vehicle control group.
No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs of toxicity were observed in the study. Stained skin/fur was observed on the ear of one mouse dosed with 40% test substance concentration.
Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 10%, 20%, and 40% test concentrations. However, stimulation indexes (SI) of less than 3.0 were observed at all test concentrations. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., stimulation index = 3) for the test substance under the conditions of this study was not calculable. Based on these data, the test substance is not a dermal sensitiser.
A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice, SI = 6.79. Therefore, the LLNA test system was valid for this study. Under the conditions of this study, the test substance did not produce a dermal sensitisation response in mice.