CMP

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:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

-

EC Number:

435-030-7

EC Name:

-

Cas Number:

26452-81-3

Molecular formula:

C5H5N2OCl

IUPAC Name:

4-chloro-6-methoxypyrimidine

Test material form:

other: solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Test system: Mice CBA/Ca/Ola/Hsd
- Age at study initiation: young adults
- Housing: max 4/cage, in cages suitable for animals of this strain and weight range
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: acetone

Concentration:

Experiment 1: 1%; 3%; 10% w/v
Experiment 2: 0.1%, 0.3%, 1% w/v

No. of animals per dose:

4

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph node assay
- Criteria used to consider a positive response: one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: Using a variable volume micro-pipette, approximately 25 µL of preparations of test substance in acetone were applied to the dorsal surface of each ear. Vehicle control groups were similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added and after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to beta-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.

Positive control substance(s):

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

Results and discussion

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 1%, 3% and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at the 3% and 10% w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.43

Test group / Remarks:

Exp 2 (1% w/v)

Results 2

Key result

Parameter:

SI

Value:

1.23

Test group / Remarks:

Exp 2 (0.3% w/v)

Results 3

Key result

Parameter:

SI

Value:

1.66

Test group / Remarks:

Exp 2 (0.1% w/v)

Results 4

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

Exp 1 (10% w/v)

Results 5

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Exp 1 (3% w/v)

Results 6

Key result

Parameter:

SI

Value:

1.69

Test group / Remarks:

Exp 1 (1% w/v)

Results 7

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Experiment 1: Vehicle only 94 cpm/node 1% w/v 159 cpm/node 3% w/v 122 cpm /node 10% w/v 95 cpm/node Experiment 2: Vehicle only 96 cpm/node 0.1% w/v 159 cpm/node 0.3% w/v 118 cpm/node 1% w/v 137 cpm/node

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is unlikely to be a moderate or strong skin sensitiser under the conditions of the test.

Executive summary:

In a GLP compliant Local Lymph Node Assay, tested according to OECD guideline 429, 4 male CBA mice per dose were treated daily with the test item by topical application to the dorsum of each ear (left and right) for three consecutive days. Initially concentrations of 1%, 3% and 10% w/v in acetone were applied. A concentration-related reduction in isotope incorporation was seen at 3% and 10%. This can be indicative of a cytotoxic response which could mask a sensitisation reaction. Consequently, additional levels of 0.1%, 0.3% and 1% w/v in acetone were tested. In both experiments, a control group of four mice was treated with the vehicle (acetone) only. Three days after the last topical application the test animals received an intravenous injection of radio labelled thymidine (³H-methyl thymidine) into a tail vein. Approximately five hours thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. In the first experiment, stimulation indices of 1.69, 1.30, 1.01 were determined with the test item at concentrations of 1, 3, and 10% (w/v), respectively in acetone. In the second experiment, stimulation indices of 1.66, 1.23, and 1.43 were determined with the test substance at concentrations of 0.1, 0.3 and 1% w/v in acetone, respectively. Based on the described study, it is concluded that the test substance is unlikely to be a moderate to strong skin sensitizer under the conditions of the test.No classification for skin sensitisation is warranted.