cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-25 to 2005-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

: 1) no data on the purity of the test substance was provided. 2) the relative humidity in the animal room was between 30-96%.

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

EC Number:

262-765-9

EC Name:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Cas Number:

61397-56-6

Molecular formula:

C18H15BrCl2O4

IUPAC Name:

[(2R,4R)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl]methyl benzoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-16479671-AAA (T001095)
- Physical state: solid (powder)
- Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00452023 ZT001095PUA121
- Expiration date of the lot/batch: no data
- Purity: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle:
solubility in vehicle: 210 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMF) was quantitatively added. The test item concentrations were prepared by serious dilution with DMF using a magnetic stirrer as homogeniser. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 6-7 weeks (beginning of acclimatization)
- Weight at study initiation: mean: 19.8 +/- 1.3 g (range of 18.4 to 23.5 g)
- Housing: single in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D33178 Borchen)
- Diet: pelleted standard diet, ad libitum (Harlan Winkelman GmbH, D-33178 Borchen)
- Water: tap water, ad libitum (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: No data on period; however, under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 96 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. to 6:00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Pre-test: 1.125, 4.25, 8.5 and 17% (w/v)
Main study: 4.25, 8.5, and 17 % (w/v)

No. of animals per dose:

Pre-test: 2 females (in total)
Main study: 4 females per dose (nulliparous and non-pregnant)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
- Irritation: in a pre-test in two mice, test substance concentrations of 1.125, 4.25, 8.5, and 17% (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.
- Systemic toxicity: no data
- Ear thickness measurements: no data
- Erythema scores: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
A test substance is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
1) That exposure to at least one concentration of the test substance resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2) That the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMF) was quantitatively added. The test substance concentrations were prepared by serious dilution with DMF using a magnetic stirrer as homogeniser. Homogeneity of the test substance in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion. The top dose was the highest technically achievable concentration.

- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test substance concentrations of 4.25, 8.5 and 17% (w/v) in DMF. The application volume, 25 µL, was spread over the entire dorsal surface (~ 8 mm diameter) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of the test substance applied.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weights.
A statistical analysis was conducted for assignment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c)[(3-d)/(b-d)]+c.
EC3 is the estimated concentration of the test substance required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the local lymph node assay response plot.

Results and discussion

Positive control results:

Measured DPM/node: 728.5, 769.3, 1315.8 and 2914.5 respectively for vehicle, test item concentrations 5, 10 and 25% (w/v)
S.I. : 1.06, 1.81 and 4.00, respectively for test item concentrations of 5, 10 and 25% (w/v)
EC3 = 18.2 % (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.05

Test group / Remarks:

4.25% w/v group

Results 2

Parameter:

SI

Value:

1.16

Test group / Remarks:

8.5% w/v group

Results 3

Parameter:

SI

Value:

1.42

Test group / Remarks:

17% w/v group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM per lymph node (8 lymph nodes in total):
vehicle: 535.8
4.25% w/v: 560.1
8.5% w/v: 623.0
17% w/v: 759.6

DETAILS ON STIMULATION INDEX CALCULATION
see results table above

EC3 CALCULATION
An EC value was not calculated as the SI values were below 3 in all test groups.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to a concentration of 17%, it was not considered to be a skin sensitiser. Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact according to the criteria of the GHS and the CLP Regulation.