Phthalonitrile

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 guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Phthalonitrile

EC Number:

202-044-8

EC Name:

Phthalonitrile

Cas Number:

91-15-6

Molecular formula:

C8H4N2

IUPAC Name:

benzene-1,2-dicarbonitrile

Details on test material:

Identity: ortho-Phtalodinitrile
Batch No.: Laufprobe 26.11.2009
CAS No.: 91-15-6
Purity: 99.51 area-% (exact value is being currently analysed, BASF study code 09L00445)
Storage: At room temperature
Expiration Date: November 26, 2011

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system
Mice, CBA/CaOlaHsd

Source
Harlan Laboratories B.V. (Postbus 61745960 AD Horst / The Netherlands)

Number of animals for the pre-test
2 females

Number of animals for the main study
30 females (reserve animals will be used as needed and listed in raw data and report)

Number of animals per group
5 females (nulliparous and non-pregnant)

Number of test groups 3

Number of control (vehicle) groups 2
(If possible, concurrent controls will be used for several Harlan CCR projects performed in parallel to save animals).

Number of positive control groups 1
(If possible, concurrent controls will be used for several Harlan CCR projects performed in parallel to save animals).

Age
8 - 12 weeks (beginning of treatment)

Body weight
15 - 25 g (beginning of treatment),

Identification
Single caging.

Acclimatisation
At least 5 days prior to the start of dosing under test conditions after health examination.

Housing:
Single

Cage Type:
Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen / Germany)

Bedding:
Granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg / Germany)

Feed:
Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst /Netherlands)

Water:
Tap water, ad libitum, (Gemeindewerke, 64380 Roßdorf / Germany)

Environment:
Temperature 22 + 2°C

Relative humidity
approx. 45-65%

Artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 0.5, 1, 2.5

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: solouble in Dimethylformamide
- Irritation: no but dermal toxicity. mortality obseved in the preexperiment


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals will be distributed into the test groups at random and identified by cage number.
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared balance and dimethylformamide was quantitatively added. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion and used directly after preparation.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with test item at concentrations of 0.5, 1, and 2.5% (w/v) in dimethylformamide or with the positive control item at 25% (w/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear once daily for three consecutive days. Two further groups of mice (control animals) were treated with an equivalent volume of the respective vehicle for the test item (dimethylformamide) or for the positive control item (acetone:olive oil (4:1)) alone. Five days after the first topical application, all mice were administered with 250 µl of 79.7 µCi/ml 3HTdR (corresponds to 19.9 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a cintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Additionally: After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group and also to assess if there is a dose response relationship. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test.
However, the primary point of consideration was the biological relevance of the results.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Stimulation Indices (S.I.) of 1.29, 1.36, and 1.18 were determined with the test item at concentrations of 0.5, 1, and 2.5% (w/v) in dimethylformamide, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. The S.I. of the positive control group was 7.48.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: A statistically significant difference in DPM/animal was not observed in any of the groups treated with different concentrations of the test item in comparison to the vehicle control group (see table below).

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group

Test item concentration	Mean DPM per Group (5 animals)^a)	SD^b)	S.I.
Vehicle for the Test Item (dimethylformamide)	758.9	396.7	1.00
5% ortho-Phthalodinitrile	978.7	223.4	1.29
10% ortho-Phthalodinitrile	1029.5	217.9	1.36
25% ortho-Phthalodinitrile	898.1	454.7	1.18
Vehicle for the Positive Control (acetone:olive oil (4+1))	857.7	196.7	1.00
25% Positive Control	6416.1	1837.4	7.48

a)      Mean DPM/Group was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

b)      SD=Standard deviation

The EC3 value could not be calculated, since all S.I.´s are below 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met