[1,2,4]Triazolo[1,5-a]pyrimidin-2-amine, 5,7-dimethoxy-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5,7-dimethoxy-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine

EC Number:

603-562-0

Cas Number:

13223-43-3

Molecular formula:

C7H9N5O2

IUPAC Name:

5,7-dimethoxy-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine

Details on test material:

- Name of test material (as cited in study report): ADTP
- Molecular formula (if other than submission substance): C7H9N5O2
- Molecular weight (if other than submission substance): 195.2
- Physical state: Solid, white to off-white
- Analytical purity: Non-GLP characterization lists the purity of the test material as 98.8% wt. by liquid chromatography (Albers, 2006).
- Lot/batch No. GuN 06/05
- Radiochemical purity (if radiolabelling): 99.3% (HPLC - GE Healthcare Amersham, 2005)
- Locations of the label (if radiolabelling): 3H-Thymidine

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc. (Raleigh, North Carolina)
- Age at study initiation: Approximately 9-12 weeks
- Weight at study initiation: 17.5 - 22.9 g
- Housing: Animals were housed one per cage in stainless steel cages after assignment to the study. Cages had wire-mesh floors that were suspended above absorbent paper and contained a feed container and a pressure activated lixit valve-type watering system. On the day the animals were euthanized, and following the injection of 3H-thymidine, each treatment group of mice was housed in shoebox cages containing corncob bedding, food pellets, and a crock filled with water. The mice were euthanized five hours later.
- Diet (e.g. ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed was provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants.
- Water (e.g. ad libitum): Municipal water was provided ad libitum. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.
- Acclimation period: Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5%, 20%, and 60%.

No. of animals per dose:

Six female mice/group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Concentrations tested for the irritancy screen were selected based upon maximum solubility in an appropriate LLNA vehicle while maintaining a solution suitable for application. Propylene glycol was selected as the vehicle based upon maximum solubility of ADTP.
- Irritation: Prior to the LLNA study, several concentrations of the test material were evaluated for irritation potential as measured by erythema of the ears. Mice (one female/concentration) received one application of ADTP (1%, 5%, 15%, 30%, or 60%), on the dorsal surface of each ear (25 µl) on three consecutive days. Using an adjustable pipette with a disposable tip, the test solutions (25 µl/ear) were spread over the dorsal surface of each ear in a manner to prevent material loss. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 2, 3, and 6. All mice were weighed on days 1 and 6. Erythema scores and body weight data following test material applications were compared to the response of the animals treated with vehicle alone. Irritation to the mouse ears is only relevant in the context of the LLNA and should not be interpreted as an indication of irritation potential in humans.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Any test material that produces a SI of > 3 in the LLNA should be considered "positive" for contact sensitization

TREATMENT PREPARATION AND ADMINISTRATION:
The application of the test material (25 µl/ear) was made on the dorsal surface of both ears as described above. Six female mice/group received one of three concentrations of ADTP (5%, 20%, or 60%) or vehicle (propylene glycol (PG)) once daily for three consecutive days. HCA at 30% (v/v) in vehicle was run concurrently as a positive dermal sensitization control. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 2, 3, and 6. All mice were weighed on days 1 and 6. On day 6, all mice received a 250 µl intravenous injection (i.v.) via the lateral tail vein containing 20 µCi of 3H-thymidine (specific activity 2Ci/mmol; Amersham code TRA310, Buckinghamshire, United Kingdom) diluted in phosphate-buffered saline (PBS).
Approximately five hours post administration, the mice were euthanized via CO2 asphyxiation and both auricular lymph nodes located at the bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of the auricular lymph nodes from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer (Stomacher 80 Lab System, Seward Ltd., London, United Kingdom). The cells were washed two times and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of Aquasol-2 scintillation cocktail (Packard Instrument Company, Meridan, Connecticut).
Two additional 2 ml aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vials containing the 1 ml of pellet in TCA and cocktail. The radioactivity in each precipitate was measured using a β-scintillation counter and reported as disintegrations per minute (dpm) per mouse.
A mean dpm value + SD (standard deviation) was calculated for each experimental group. The SI was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator; the mean SI ± SD was calculated for each experimental group. Any test material that produces a SI of > 3 in the LLNA should be considered "positive" for contact sensitization (.Kimber et at, 1994). While the criterion for a positive response (SI > 3) was originally developed empirically, a recent robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999a). Furthermore, by determining EC3 values (estimated concentration resulting in a 3-fold SI), one can compare relative sensitization potency of chemicals and/or formulations (Basketter et at., 1999b). Based on the EC3 values derived from the LLNA, it has been proposed that contact allergens can be categorized as weak (≥ 10% - 100%), moderate (≥ 1% - < 10%), strong (≥ 0.1% - < 1%), or extreme (< 0.1%); (ECETOC, 2003). The EC3 value is determined by interpolating between two values one above and one below the SI value of 3.

Positive control substance(s):

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

Statistics:

1. The Stimulation Index (SI) was calculated for each mouse using the following equation:

SI = Disintegration per minute (dpm.) of individual mouse / Average dpm of the VH control mice

2. EC3 Calculation:

EC3 = XL + [(3-YL) / (Yh-YL)] (Xh-XL)

Where,
YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

Means and standard deviation (SD) were generated for body weight data (absolute and gain) and the LLNA response (dpm & SI values).

These body weight and dpm data were analyzed by a one-way analysis of variance (Steele and Tonle, 1960). When differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett's t-test (Steele and Torrie, 1960). The alpha level at which all tests were conducted was 0.05. The final interpretation of the biological significance of the responses was based on both statistical outcome and scientific judgment.

Results and discussion

Positive control results:

Proper conduct of the LLNA was demonstrated via the positive response from the positive control, 30% HCA which elicited a stimulation index (SI) that was 12.1 in comparison to vehicle-treated mice

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Dose Mean SI Vehicle 1.0 5% ADTP 1.2 20% ADTP 1.3 See Table 3 attached for individual scores

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Dose Mean DPM Vehicle 577.36 5% ADTP 669.97 20% ADTP 737.06 See Table 3 attached for individual scores

Any other information on results incl. tables

During the screening study, the mice were treated with three daily applications 1%, 5%, 15%, 30%, or 60% ADTP. Erythema was absent in the mice dosed with 1%, 5%, or 15%, while the mice dosed 30% and 60% demonstrated slight erythema on days 2 and 3. Body weights were unaffected in all dose groups.

Based on the results of the screening study, 60% ADTP was tested in the LLNA along with 20% and 5% to characterize the dose response. Erythema was absent in the mice treated with 5% and 20% ADTP, while the mice dosed with 60% demonstrated slight erythema on days 2 and 3, however, erythema resolved by day 6. Body weights were unaffected in the mice dosed with 5% and 20% ADTP, while the mice dosed with 60% lost an average of 1.4 grams of body weight. This average was primarily due to the fact that one mouse lost 6.3 grams of body weight. This mouse was moribund on day 6, and was immediately euthanized.

Proper conduct of the LLNA was demonstrated via the positive response from the positive control, 30% HCA which elicited a stimulation index (SI) that was 12.1 in comparison to vehicle-treated mice. ADTP did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold (Table 3).

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

ADTP did not elicit a stimulation index (SI) that met the 3X threshold, indicating a lack of dermal sensitization potential in the mouse LLNA.

Executive summary:

The Local Lymph Node Assay (LLNA) was conducted to assess the potential of ADTP to cause contact sensitization by measuring the lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear.

Screening Study: Three daily topical applications of 1%, 5%, 15%, 30%, or 60% ADTP were given to one animal at each dose level. Erythema was absent in the mice treated with 1%, 5%, and 15% ADTP, while the mice dosed with 30% and 60% ADTP demonstrated slight erythema on days 2 and 3, that resolved by day 6. Body weights were unaffected in all dose groups. Results from this study were used to determine the dosing concentrations for ADTP in the LLNA.

Six female mice/group received 5%, 20% or 60% ADTP, or vehicle (propylene glycol (PG)), on days 1-3. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 30% a-hexylcinnamaldehyde (HCA), a moderate contact sensitizer, which elicited proliferation that was 12.1% in comparison to vehicle-treated mice.

Erythema was absent in the mice treated with 5% and 20% ADTP, while the mice dosed with 60% demonstrated slight erythema on days 2 and 3 that resolved by day 6. Body weights were unaffected in all dose groups.

ADTP elicited proliferative responses with stimulation indices (SI) that were respectively 1.2, 1.3 and 1.2 in comparison to the vehicle-treated mice. ADTP did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold proliferation when compared to vehicle-treated mice.