5-fluoro-2-methoxypyrimidin-4(3H)-one

Endpoint summary

Description of key information

Under the conditions of the test, the test material does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and the material is not considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2006 to 11 December 2006

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)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 9 weeks old
- Weight at study initiation: Mean body weight ± standard deviation of 20.4 ± 1.1 g
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22 x 8.5 x 8 cm). Each cage contained autoclaved sawdust.
- Diet: ad libitum
- Water: ad libitum access to tap water filtered using a 0.22 micron filter
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 to 70 % (relative)
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hours of light / 12 hours of darkness

IN-LIFE DATES: Not reported

Vehicle:

other: The vehicle used in the first experiment was DMSO; the vehicle used in the second experiment was propylene glycol

Concentration:

- Experiment 1: 0.05, 0.1, 0.25, 0.5 and 1 %
- Experiment 2: 5, 10 and 25 %

No. of animals per dose:

4 females per dose

Details on study design:

PRE-SCREEN TESTS:
To assess the irritant potential of the test material (through ear thickness measurement), a preliminary test was performed on a small number of animals. The test material was prepared at the concentrations of 1, 2.5, 5 and 10 %. For 3 consecutive days, the animals received applications of 25 μL of the dosage form preparations to the external surface of both ears (one concentration per ear); measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application. The test material was irritating in the preliminary test at concentrations ≥2.5 %. The highest concentration retained for the first experiment was therefore 1 %. However, following a request from the British Authorities (HSE) a second experiment was performed with a maximum concentration of 25 %.

MAIN STUDY
- Administration of the test material
On days 1, 2 and 3 of each experiment, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimised application of the test materials, the animals were placed under light isoflurane anaesthesia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

- Proliferation Assay:
On day 6 of each experiment, all animals of all groups received a single intravenous injection of 250 μL of 0.9 % NaCl containing 20 μCi of 3H-methyl-thymidine (3H-TdR; specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe.
Cell suspensions were washed with 15 mL of 0.9 % NaCl and pellets obtained were re-suspended in 0.9 % NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5 % (w/v) trichloroacetic acid (TCA) in purified water at +4 °C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5 % TCA. Three mL of Ultima Gold® scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The results were expressed as disintegrations/mn (dpm) per group and per node.
Stimulation Indices (SI) were calculated according to the following formula:
SI = dpm of treated group / dpm of control group (group 1)

- Clinical Examinations
The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality. In each experiment, the animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test material, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before application), as well as on day 6 (after sacrifice) of each experiment, the thickness of the left ear of each animal of groups 1 to 6 was measured using a micrometer. No measurement of ear thickness was performed for the animals of the positive control group.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test material, etc.) was noted.
The irritation level of the test material was determined as follows: % increase in ear thickness between day 1 and day 3 or 6 of <10 %, 10 to 30 % and >30 % were interpreted as irritation levels I, II and III, respectively. These correspond to non-irritant, slightly irritant and irritant.

INTERPRETATION OF RESULTS
The test material was considered as a skin sensitiser when the SI for a dose group is ≥3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Positive control substance(s):

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

Positive control results:

In the positive control groups given HCA at the concentration of 25 % in both experiments 1 and 2, a stimulation index exceeding the threshold value of 3 (SI = 5.32 and 24.65, respectively) were noted. The cellularity index also increased in the positive control groups. The study was therefore considered valid.

Key result

Parameter:

SI

Value:

0.63

Test group / Remarks:

0.05 %

Remarks on result:

other: Irritation level I

Key result

Parameter:

SI

Value:

0.78

Test group / Remarks:

0.1 %

Remarks on result:

other: Irritation level II

Key result

Parameter:

SI

Value:

0.82

Test group / Remarks:

0.25 %

Remarks on result:

other: Irritation level II

Key result

Parameter:

SI

Value:

0.86

Test group / Remarks:

0.5 %

Remarks on result:

other: Irritation level II

Key result

Parameter:

SI

Value:

0.86

Test group / Remarks:

1 %

Remarks on result:

other: Irritation level II

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

5 %

Remarks on result:

other: Irritation level I

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

10 %

Remarks on result:

other: Irritation level I

Key result

Parameter:

SI

Value:

1.29

Test group / Remarks:

25 %

Remarks on result:

other: Irritation level I

Cellular proliferation data / Observations:

- Systemic clinical signs and mortality: No clinical signs and no mortality were observed during the study, in both experiments.
- Body weight: In both experiments, the body weight change of the treated animals was similar to that of the control animals.
- Local irritation: In the first experiment, 13/20 treated animals showed a dryness of the skin of the ears on day 6. A very slight increase in ear thickness was observed in the animals at the concentrations of 0.1, 0.25 and 0.5 %, without clear evidence of dose-response relationship.
In the second experiment, no cutaneous reactions and no noteworthy increase in the ear thickness were observed in the animals of the treated groups.
- Proliferation assay: In both experiments, the incorporation of 3H-TdR in the vehicle control group was as specified in acceptance criteria, the quantity of cells obtained in each group was satisfactory and the cell viability in each group was higher than 80 %. The study was therefore considered valid.
In both experiments, no noteworthy lymphoproliferation was noted at any of the tested concentrations.

Table 1: Study Results

Conc. (%)	Cell Count		Viability (%)	Amount of cells (x10^6 cells)	Cellularity Index	No. of nodes / group	dpm / group	dpm / node	SI	Irritation level
	Viable	Dead
Experiment 1
Vehicle	272	30	90.07	13.60	-	8	812.31	101.54	-	-
0.05	138	19	87.90	6.90	0.51	8	509.35	63.67	0.63	I
0.1	158	18	89.77	7.90	0.58	8	637.29	79.66	0.78	II
0.25	219	6	97.33	10.95	0.81	8	664.84	83.11	0.82	II
0.5	193	28	87.33	9.65	0.71	8	696.59	87.07	0.86	II
1	190	9	95.48	9.50	0.70	8	697.13	87.14	0.86	II
HCA	365	39	90.35	36.50	2.68	8	4321.06	540.13	5.32	-
Experiment 2
Vehicle	106	10	91.38	10.60	-	8	355.09	44.39	-	-
5	80	17	82.47	8.00	0.75	8	401.91	50.24	1.13	I
10	80	11	87.91	8.00	0.75	8	399.50	49.94	1.13	I
25	131	4	97.04	13.10	1.24	8	456.54	57.07	1.29	I
HCA	539	29	94.89	53.90	5.08	8	8753.25	1094.16	24.65	-

Viability = [viable cells / (viable cells + dead cells)] x 100

Cellularity index = amount of cells (x 10^6 cells) in the treated group / amount of cells (x 10^6 cells) in the vehicle group

Stimulation index = dpm of treated group / dpm of control group

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of the test, the test material does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and the material is not considered to be a skin sensitiser.

Executive summary:

The potential of the test material to act as a skin sensitiser was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

Following a preliminary test, the test material was tested in two independent experiments in female CBA/J mice.

In the first experiment, the test material was tested at the concentrations of 0.05, 0.1, 0.25, 0.5 and 1 % in DMSO on groups of 4 mice per dose level. At the request of the UK Competent Authority (HSE), the test material was tested in a second experiment at higher concentrations of 5, 10 and 25 % in propylene glycol. Concurrent positive (HCA) and negative (vehicle) controls were carried out.

In each experiment, the test material, vehicle or positive control was applied over the ears (25 μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test material was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

In the first experiment, no mortality and no clinical signs were observed during the study. Almost all the treated animals showed a dryness of the skin of the ears on day 6. A very slight increase in ear thickness was observed in the animals at the concentrations of 0.1, 0.25 and 0.5 %, without clear evidence of dose-response relationship. No lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with HCA at 25 %. The SI values were 0.63, 0.78, 0.82, 0.86 and 0.86 at concentrations of 0.05, 0.1, 0.25, 0.5 and 1 %, respectively.

In the second experiment, no mortality and no clinical signs were observed during the study. No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups. No noteworthy lymphoproliferation was noted at any of the tested concentrations, while significant lymphoproliferation was observed with HCA at 25 %. The SI values were 1.13, 1.13 and 1.29 at 5, 10 and 25 %, respectively.

Under the conditions of the test, the test material does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and the material is not considered to be a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test material to act as a skin sensitiser was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). 

Following a preliminary test, the test material was tested in two independent experiments in female CBA/J mice.

In the first experiment, the test material was tested at the concentrations of 0.05, 0.1, 0.25, 0.5 and 1 % in DMSO on groups of 4 mice per dose level. At the request of the UK Competent Authority (HSE), the test material was tested in a second experiment at higher concentrations of 5, 10 and 25 % in propylene glycol. Concurrent positive (HCA) and negative (vehicle) controls were carried out.

In each experiment, the test material, vehicle or positive control was applied over the ears (25 μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test material was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

In the first experiment, no mortality and no clinical signs were observed during the study. Almost all the treated animals showed a dryness of the skin of the ears on day 6. A very slight increase in ear thickness was observed in the animals at the concentrations of 0.1, 0.25 and 0.5 %, without clear evidence of dose-response relationship. No lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with HCA at 25 %. The SI values were 0.63, 0.78, 0.82, 0.86 and 0.86 at concentrations of 0.05, 0.1, 0.25, 0.5 and 1 %, respectively.

In the second experiment, no mortality and no clinical signs were observed during the study. No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups. No noteworthy lymphoproliferation was noted at any of the tested concentrations, while significant lymphoproliferation was observed with HCA at 25 %. The SI values were 1.13, 1.13 and 1.29 at 5, 10 and 25 %, respectively.

Under the conditions of the test, the test material does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and the material is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.