2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Endpoint summary

Description of key information

The sensitization potential of SP02 (trade name: 5OHMIC) was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline 429, the test item was suspended in Dimethyl sulfoxide (DMSO). The positive control (a-Hexylcinnamic aldehyde) (25%) was dissolved in Acetone:Olive Oil 4:1. The Pre-screen test was performed using a dose of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test. Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI). After application of the test item at three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity. In this study the mean Stimulation Indices (SI) of 1.10, 1.90 and 1.47 were determined with the test item at concentrations of 25%, 50%, and 100% in DMSO, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item SP02 (trade name: 5OHMIC) is not considered a skin sensitizer under the test conditions of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 103901
- Expiration date of the lot/batch: 1 February 2019

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5oC), handling according to SPPA-00147-BIO, Test and Reference Items
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Velaz, Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 17.89 - 18.75 g
- Housing: The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. Bedding material was Lignocel S3/4, Lufa - ITL GmbH, Germany. Sanitation was performed according to standard operation procedures.
- Diet: A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time. The
certificate of analysis is included in the raw data.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodically monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
- Acclimation period: The animals were acclimated in identical conditions as during the experiment for 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
- Indication of any skin lesions: The health condition of animals was examined by a veterinarian before initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 - 60 %
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle

Vehicle:

dimethyl sulphoxide

Remarks:

The vehicle Dimethyl sulfoxide (DMSO) was selected from the recommended vehicles according to OECD Guideline No. 429. The test item was not soluble in the vehicle; therefore, a homogeneous suspension was obtained.

Concentration:

Pre-test: 100% (w/v)
Main test: 25%, 50% and 100% (w/v)

No. of animals per dose:

5 females – negative control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test plus spare animals

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle Dimethyl sulfoxide (DMSO) was selected from the recommended vehicles according to OECD Guideline No. 429. The test item was not soluble in the vehicle; therefore, a homogeneous suspension was obtained
- Irritation: None observed
- Systemic toxicity: None observed
- Ear thickness measurements: Mean difference: -3.63 (S.D. 2.42). The increase in ear thickness did not meet the criteria which are considered
as signs for excessive local skin irritation
- Erythema scores: No erythema was observed in both mice after test item administration

MAIN STUDY
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The increase of mean body weight was observed at all used concentrations. The increase was not statistically significant.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation Index (SI) ≥3

TREATMENT PREPARATION AND ADMINISTRATION:
Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal was recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Positive control substance(s):

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

Statistics:

For calculation of mean, SD. and difference (mm, %) values of body weights and ear thickness MS Excel was used.

Positive control results:

Disintegrations per minute (DPM): 3763
Stimulatin Index (SI): 6.15

Key result

Parameter:

SI

Value:

ca. 1.1

Test group / Remarks:

25% (w/v)

Key result

Parameter:

SI

Value:

ca. 1.9

Test group / Remarks:

50% (w/v)

Key result

Parameter:

SI

Value:

ca. 1.47

Test group / Remarks:

100% (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In comparison with the control group, a minor increase of the pooled lymph node weights at all concentrations was observed. The increase was not dose dependent. The pooled lymph node weights of treated groups were 0.0342g for 25% concentration, 0.0406g for 50% concentration and 0.0315g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0298g and 0.0677g, respectively. The DPM values for the three treated groups were 853 (25%), 1470 (50%) and 1141 (100%), respectively. The SI values for the three treated groups were 1.10 (25%), 1.90 (50%) and 1.47 (100%), respectively.

EC3 CALCULATION
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The increase of mean body weight was observed at all used concentrations. The increase was not statistically significant.

The Lymph node weight, DPM, SI, EC3 values.

	Lymph node weight (g)	Number of lymph nodes	DPM	SI	EC3 (%)
Control	0.0298	10	775	-	-
Positive Control	0.0677	10	3763	6.15	-
Test item 25%	0.0342	10	853	1.1	-
Test item 50%	0.0406	10	1470	1.9	-
Test item 100%	0.0315	10	1141	1.4	-

Interpretation of results:

GHS criteria not met

Conclusions:

The results demonstrate that the test item SP02 (trade name: 5OHMIC) was not a skin sensitizer under the test conditions of this study.

Executive summary:

The sensitization potential of SP02 (trade name: 5OHMIC) was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline 429, the test item was suspended in Dimethyl sulfoxide (DMSO). The positive control (-Hexylcinnamic aldehyde) (25%) was dissolved in Acetone:Olive Oil 4:1. The Pre-screen test was performed using a dose of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test. Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI). After application of the test item at three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity. In this study the mean Stimulation Indices (SI) of 1.10, 1.90 and 1.47 were determined with the test item at concentrations of 25%, 50%, and 100% in DMSO, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item SP02 (trade name: 5OHMIC) is not considered a skin sensitizer under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The GHS criteria for classification were not met.