N-(2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date:13 December 2012 Experimental end date: 08 January 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

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:

N4-Benzoylcytosine

EC Number:

628-907-2

Cas Number:

26661-13-2

Molecular formula:

C11H9N3O2

IUPAC Name:

N4-Benzoylcytosine

Specific details on test material used for the study:

Sponsor's identification: N-Benzoyl Cytosine
Description: off white powder
Batch number: NBC-5-11001
Purity: not supplied
Date received: 09 July 2012
Expiry date: not supplied
Storage conditions: room temperature in the dark over silica gel

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card . At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old. The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary Screening Test: 25% w/w
Main Test: 25%, 10% and 5% w/w

No. of animals per dose:

Preliminary Screening Test: 1 mouse at 25% w/w
Main Test: 4 mice at 25%, 10% and 5% w/w

Details on study design:

Preparation of Test Item
For the purpose of the study, the test item was freshly prepared as a suspension in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a maximum attainable concentration of 25% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the following scale.

Scale for Erythema
Observation Score
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar
formation preventing grading of erythema: 4

Any clinical signs of toxicity, if present,were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes. Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA) .
Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q " vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Interpretation of Results
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

Positive control substance(s):

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

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control
group is as follows:
Concentration (% v/v) in acetone/olive oil 4:1: 25
Stimulation Index: 6.29
Result: Positive

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

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

5 % w/w

Value:

1.42

Remarks on result:

other: negative

Results 2

Key result

Parameter:

SI

Remarks:

10% w/w

Value:

1.04

Remarks on result:

other: negative

Results 3

Key result

Parameter:

SI

Remarks:

25% w/w

Value:

1.49

Remarks on result:

other: negative

Cellular proliferation data / Observations:

Preliminary Screening Test
White residual test item on the ears was noted post dose on Days 2 and 3. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in acetone/olive oil 4:1.

Main Test
Clinical Observations and Mortality Data
White residual test item on the ears was noted post-dose on Day 2 in animals treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1 and post-dose on Day 3 in animals treated with the test item at a concentration of 25% or 10% w/w in acetone/olive oil 4:1.
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

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.

Any other information on results incl. tables

Estimation of the Proliferative Response of Lymph Node Cells

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 acetone/olive oil 4:1	Stimulation Index	Result
5	1.42	negative
10	1.04	negative
25	1.49	negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 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 item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable 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 four animals, were treated with 50 pi (25 pi per ear) of the test item as a suspension in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with acetone/olive oil 4:1 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:

Conclusion.

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