Purine-2(3H),6(1H)-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test item solubility was tested in acetonitrile, water, 1: 1 (v:v) acetonitrile:water, isopropanol, methanol, ethanol, 1,4-butandiol, N,N-dimethylformamide (DMF) and tertbutanol at 100 mM. The test item was not soluble in any of these solvents. Therefore, in vitro studies could not be performed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Purine-2(3H),6(1H)-dione

EC Number:

200-718-6

EC Name:

Purine-2(3H),6(1H)-dione

Cas Number:

69-89-6

Molecular formula:

C5H4N4O2

IUPAC Name:

xanthine

Specific details on test material used for the study:

Purity: > 99%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Inc.
- Age at study initiation: Preliminary Animals: Young adult (11 weeks); Test and Control Animals: nulliparous and non-pregnant; young adult (11 or 12 weeks)
- Weight at study initiation: 19.9- 24.8 grams
- Housing: Individually housed in plastic solid bottom cages during dosing and resting phase of study; after final weighing until sacrifice, animals housed in respective dose groups in plastic cages with bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22°C
- Humidity (%): 67-79% (Humidity was above the targeted upper limit for 4 days during the study. A portable dehumidifier was used to lower the humidity levels during this time. This excursion was considered minor and had no impact on this study.)
- Air changes (per hr): 12
- Photoperiod (hrs dark/ hrs light): 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic L92

Concentration:

10, 25, 50%

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
- Dosing: 10%, 25%, and 50% w/w mixtures in 1% Pluronic® L92; 25 µL concentration of test substance or vehicle alone applied to dorsum of both ears of each mouse for three consecutive days; dose spread evenly as possible over dorsal surface of ear using tip of pipette; no treatment made on Days 4 and 5; on Day 6, sites for each mouse evaluated for local reactions (erythema & edema); animals were observed daily for signs of toxicity. Data from pre-screen used to select the three concentrations to be tested in main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: If test substance produces an SI ≥ 3, it is considered "positive" for dermal sensitization potential.

TREATMENT PREPARATION AND ADMINISTRATION: Beginning on Day 1, a quantity of 25 µL of test substance concentration, positive control substance, or vehicle alone applied to dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, material was spread evenly as possible over dorsal surface of ear using tip of pipette. On Day 6 of study (three days after the final topical application), 250 µL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine was injected intravenously via tail vein of each mouse. Approximately five hours after injections, all test and control mice were euthanized and draining auricular lymph nodes were excised.

TEST SYSTEM/ASSESSMENT: Lymph nodes evaluated for each individual mouse. A single cell suspension of lymph node cells (LNC) prepared in PBS by gently massaging lymph nodes between frosted ends of microscope slides over collection vessel. The slides were then rinsed briefly with PBS into vessel. Contents of vessel transferred to centrifuge tube and washed with excess of PBS and centrifuged- process carried out twice. In both cases, supernatant decanted and discarded following each centrifugation. After second wash, 5 mL of 5% trichloroacetic acid (TCA) in distilled water added to sediment and tube vortexed. DNA was precipitated in 5% TCA in distilled water at 4°C overnight (approximately 18 hours). Following the overnight precipitation of DNA, tubes centrifuged again and supernatant discarded. Resulting precipitate re-suspended using 1 mL of 5% TCA in distilled water and transferred to 10 mL of scintillation fluid. Incorporation of 3 H-methyl thymidine was measured by beta-scintillation counting and expressed as disintegrations per minute, minus background dpm.

All mice were observed for signs of mortality, gross toxicity, and/or behavorial changes daily. Individual body weight were recorded on Day 1 (initial) shortly before test substance application and prior to IV injections on test Day 6.

Positive control substance(s):

other:

Statistics:

Statistical analysis was performed on DPM minus background values. Significance was judged at p < 0.05. Treated groups and negative vehicle control group compared using a One­Way Analysis of Variance (ANOVA), followed by comparison of treated groups to control by Dunnett's t-test for multiple comparisons. Where variances were considered significantly different by Bartlett's test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn's test).

Results and discussion

Positive control results:

The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI = 7.96).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10%

Remarks on result:

other: test substance was not considered positive for a dermal sensitization potential

Results 2

Key result

Parameter:

SI

Value:

1.43

Test group / Remarks:

25%

Remarks on result:

other: test substance was not considered positive for a dermal sensitization potential

Results 3

Key result

Parameter:

SI

Value:

1.56

Test group / Remarks:

50%

Remarks on result:

other: test substance was not considered positive for a dermal sensitization potential

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: None

DETAILS ON STIMULATION INDEX CALCULATION: Stimulation Index = Average dpm of Test Substance/Average dpm of Vehicle.

EC3 VALUE: Not calculated since all dose levels induced a stimulation index (SI) of less than 3.0.

CLINICAL OBSERVATIONS: All animals appeared active and healthy throughout study.

BODY WEIGHTS: Six mice from test and two from positive control groups lost body weight during study. All other mice gained body weight during study.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness). None

Any other information on results incl. tables

As a stimulation index (SI) of less than 3.0 was observed in all the treatment groups, the test substance was not considered positive for a dermal sensitization potential. The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI = 7.96). Therefore, the LLNA test system was valid for this study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not considered to be a contact dermal sensitizer at concentrations less than or equal to 50% in the LLNA.

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for the test substance to produce sensitization after repeated topical applications. Three concentrations of the test substance (10%, 25% and 50%) w/w in distilled water (1% Pluronic® L92) were topically applied to fifteen healthy test mice (five mice/group) for three consecutive days. Three days after the last application, the mice were given an IV injection containing 20 µCi of 3H-methyl thymidine. Approximately five hours later, all animals were euthanized via an overdose of inhaled lsoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. During the study, each animal's ears were evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection. A vehicle control group (five animals) and a positive control group (five animals) were maintained under the same environmental conditions and treated in the same manner as the test animals. The vehicle control animals were treated with 1% Pluronic® L92 and the positive control group animals were treated with a 25% w/w mixture of HCA in 1% Pluronic® L92. In an effort to reduce the total number of animals used, this study was run concurrently with another study to utilize a common positive control and common vehicle control group. All animals appeared active and healthy throughout the study. Six mice from the test and two from the positive control groups lost body weight during the study. All other mice gained body weight during the study. Mean DPM (minus bsckground) scores were 1196.43, 9520.82, 1200.49, 1710.02, and 1867.91 for the vehicle control, positive control, 10%, 25% and 50% test substance groups, respectively. Simulation index was not calculated, 7.96, 1.00, 1.43, 1.56, respectively. Based on the results of the study, the test substance is not considered to be a contact dermal sensitizer at concentrations less than or equal to 50% in the LLNA. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.