3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-20 to 2005-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study, performed according to OECD Guideline 429 and Guideline ISO 10993 Biological Evaluation of Medical Devices Part 12: Sample preparation and reference materials, 2002. No deviations were recorded.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

other: Guideline ISO 10993 Biological Evaluation of Medical Devices Part 12: Sample preparation and reference materials, 2002

Deviations:

no

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

EC Number:

225-795-3

EC Name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

Cas Number:

5081-87-8

Molecular formula:

C10H9ClN2O2

IUPAC Name:

3-(2-CHLOROETHYL)QUINAZOLINE-2,4(1H,3H)-DIONE

Details on test material:

- Name of test material (as cited in study report): T1201 (CAS no. 5081-87-8)
- Substance type: no data
- Physical state: white to pale-beige solid
- Analytical purity: no data
- Purity test date: no data
- Lot/batch No.: 00464885 RT001201G1A251
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature, protected from moisture
- Other:
- Solubility - 10 g/L in methanol; 7.9 g/L in ethanol; 10.2 g/L in dichloromethane; 7.6 g/L in 2-propanol; 26.7 g/L in acetone; 290 g/L in N,n-dimethyl formamide.
- Solubility in water: 0.09 g/L
- Molecular weight: 224.65 g/mol

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: nulliparous and non-pregnant female animals; Harland Netherlands
- Age at beginning of acclimatization: 6-7 weeks
- Weight at study initiation: 17.2-21.7 g (mean = 18.9 g)
- Housing: Individually in Makrolon type 1 cages with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Local tap water, ad libitum
- Acclimation period: under test conditions after health examination; no further data


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3 °C
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2005-10-10 To: 2005-10-11 for main experiment

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

8.25, 16.5, and 33% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: no data
- Irritation: A pre-test in 2 mice, and test substance concentrations of 4.125, 8.25, 16.5, 0.63, and 33% (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Exposure to at least one test concentration must result in incorporation of 3HTdR at least three times or greater than that recorded in the control mice, as indicated by the SI, and the data must be compatable with a conventional dose reponse, although allowances must be made (especially at higher concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Each test group of mice was treated by topical application to the dorsal surface of each ear lobe wth the test substance at concentrations of 8.25%, 16.5%, and 33% in DMSO. 25 uL was spread over the entire surface of each ear lobe once daily for three days. Another group of mice was treated with an equal volume of DMSO alone. A hair dryer was used to dry the ear to avoid loss of test substance.
- Five days after the first application, all mice were administered 250 uL of PBS containing 80 uCi/uL 3HTdR (equal to 20 uCi 3HTdR) by IV injection via a tail vein.
- Approximately five hours after treatment with 3HTdR, all mice were euthanized by intraperitoneal injection of Na-thiopental.
- The draining lymph nodes were rapidly excised and pooled for each group (8 nodes per group).
- Cell suspensions were washed and prepared in phosphate buffered saline.
-The level of HTdR incorporation was measured on a B-scintillation counter. Background levels were also measured.
- HTdR incorporation is expressed as the number of disintegrations per minute.

Positive control substance(s):

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

other: 2,4 dinitrobenzene sulphonic acid, sodium salt (DNBS) in 1% pluronic L92 in distilled water

Statistics:

The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted to assess the dose-response relationship. The EC3 could not be calculated.

Results and discussion

Positive control results:

5% (w/v)- Stimulation index = 1.06
10% (w/v)- Stimulation index = 1.81
25% (w/v) - Stimulation index = 4.00
EC3=18.2%
Considered to be a skin sensitizer.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 8.25% - 1.18 16.5% - 1.38 33% - 1.34

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 8.25% (w/v) - 4645.25 16.5% (w/v) - 5410.22 33% (w/v) - 5268.12 per lymph node: 8.25% (w/v) - 580.66 16.5% (w/v) - 676.28 33% (w/v) - 658.52

Any other information on results incl. tables

Observations:

-No mortality occurred during the study period.

-No symptoms of local toxicity and no systemic findings were observed during the study period.

-The body weights of the animals were within normal ranges for this strain and age.

Positive Control Local Lymph Node Assay in the Mouse (2005)

Project Number	Start Date	Finish Date	Test Material	Concentration	Vehicle	Stimulation Index^a	Classification^b
039/747·	04/03/2005	10/03/2005	α‑Hexylcinnamaldehyde, tech., 85%	5%, 10%, 25% v/v	acetone/olive oil 4:1	2.76, 3.34, 8.91	Positive
039/750*	20/04/2005	26/04/2005	α‑Hexylcinnamaldehyde, tech., 85%	5%, 10%, 25% v/v	ethanol/distilled water 7:3	2.64, 8.36, 12.94	Positive
039/769A*	14/07/2005	20/07/2005	α‑Hexylcinnamaldehyde, tech., 85%	5%, 10%, 25% v/v	1% pluronic L92 in distilled water	0.86, 1.50, 6.17	Positive
039/769B*	14/07/2005	20/07/2005	2,4 Dinitrobenzene sulphonic acid, sodium salt (DNBS)	1%, 10%, 20% v/v	1% pluronic L92 in distilled water	1.16, 9.59, 20.71	Positive

a=         Ratio of test to control lymphocyte proliferation

b=         Stimulation index greater than 3.0 indicates a positive result

* =         Standard Test Method 595 (Pooled nodes)

·=          Standard Test Method 599 (Individual nodes)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was not considered to be a skin sensitizer and therefore is considered not to be classified.