1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-03-11 - 2011-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Adopted on March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

EC Number:

935-211-5

Cas Number:

1305113-15-8

Molecular formula:

C30H28O10P2

IUPAC Name:

1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

Details on test material:

- Name of test material (as cited in study report): Isosorbid-O,O'-bis(diphenylphosphorsäureester); Lab test item number: 10/0230-4
- Physical state: solid / white
- Analytical purity: 98.2 mol% (1H- and 13C NMR spectroscopy)
- Lot/batch No.: 10264/10/022 B
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed.
- Storage condition of test material: room temperature
- Other: the test item was homogeneous by visual inspection.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/J mouse from Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany.
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 18.9 g – 21.5 g
- Housing: single housed in Makrolon cage, type II
- Diet (ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water (ad libitum): tap water
- Acclimation period: at least 5 days before the first test substance application.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

MEK

Concentration:

50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: a solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration, which can be technically used, was a 50% test-substance preparation in methyl ethyl ketone (MEK).
- Irritation and lymph node proliferation response: to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Two mice were treated with a test substance concentration of 50% on three consecutive days, whereas clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally the weight of the pooled lymph nodes from both sides was determined for each animal.
At the tested concentration (50%) the animals did not show any signs of local irritation as confirmed by the ear weight and lymph node weight measurements. Also, the animals did not show any signs of systemic toxicity. Therefore, a 50% test-substance preparation was chosen for the main test, which was carried out as a limit test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The increase in [3H]-thymidine incorporation by a factor of ≥3 (SI≥3) and/or the increase in cell count by a factor of ≥1.5 (SI≥1.5) as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
In addition the following is taken into consideration for evaluation:
- If a test substance shows a statistically significant and/or biologically relevant increase in [3H]-thymidine incorporation, cell count, and/or lymph node weight as compared to the vehicle control in the presence of statistically significant and/or biologically relevant increased ear weights as indications of skin irritation, it is considered not to be a sensitizer.
- If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in 3H-thymidine incorporation, cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance may be considered to be a sensitizer or subjected to further investigations.
- If statistically significant and/or biologically relevant increases in ear weights are accompanied by an increase in [3H]-thymidine incorporation, cell count and/or lymph node weight, it cannot be ruled out that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values may be performed. If one or a combination of the measured parameters change (with statistical significance), evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response.
- If a test substance does not elicit a statistically significant increase in [3H]-thymidine incorporation, cell count and/or lymph node weight but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
- Application volume: 25 μL per ear (the animals were treated with vehicle or test-substance preparation)
- Form of application: epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site.
- [3H]-Thymidine injection: on study day 5 (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 μCi of [3H]-thymidine in 250 μL of sterile saline.
- Determination of ear weight: the animals were sacrificed on study day 5 (ca. 5 hours after [3H]-thymidine injection) by cervical dislocation under Isoflurane anesthesia. Immediately thereafter, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals and the weight of the pooled punches determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: immediately after removal of the ear punches the left and right auricular lymph nodes were dissected, and the weight of the pooled lymph nodes from both sides determined for each animal.
- Preparation of cell suspension and determination of cell count: after weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) and a single cell suspension was prepared as soon as possible after dissection. For determination of cell counts, an aliquot of each suspension was further diluted in a ratio 1:500, and the cell count determined.
- Measurement of [3]-thymidine incorporation of the lymph node cells: the remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of [3H]-thymidine into the cells was measured in a β-scintillation counter.

Positive control substance(s):

other: Studies using the positive control substance Alpha-Hexylcinnamaldehyde (techn. 85%) are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of [3H]-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
[3H]-thymidine incorporation, cell count, lymph node weight and ear weight were further analysed by WILCOXON test.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

No increase above the cut off Stimulation Index of 3 for [3H]-thymidine incorporation into the cells from the auricular lymph nodes nor increase to 1.5 fold or above for lymph node cell counts or lymph node weight, were observed compared to the vehicle control values (see Table 1).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

[3H]-thymidine incorporation: when applied as 50% solution in MEK the test substance did not induce a biologically relevant or statistically significant increase of [3H]-thymidine incorporation into the cells from the auricular lymph nodes (see Table 1).

Any other information on results incl. tables

ADDITIONAL OBSERVATIONS

- Cell count and lymph node weight: there were no statistically significant or biologically relevant increases in lymph node cell counts and lymph node weights.

- Ear weights: the 50% test-substance solution caused some statistically significant increase in ear weights, probably due to test substance residues.

- Body weights: the expected body weight gain was generally observed in the course of the study.

- Clinical signs: test substance residues were noted on the ear skin of the animals treated with the 50% test substance preparation on study day 2, only. Table 1: [3H]-thymidine incorporation, cell count, lymph node weight and ear weight; test group mean values, standard deviations and stimulation indices

Treatment	³H-thymidine incorporation [DPM/Lymph Node Pair]
	Mean ± Standard deviation	Stimulation index
Vehicle	206 ± 45.2	1.00
50% TS in vehicle	234.9 ± 53.5	1.15
	Cell Counts [Counts/Lymph Node Pair]
	Mean ± Standard deviation	Stimulation index
Vehicle	6150600 ± 443038	1.00
50% TS in vehicle	5827800 ± 995434	0.95
	Lymph Node Weight [mg/Lymph Node Pair]
	Mean ± Standard deviation	Stimulation index
Vehicle	4.9 ± 0.4	1.00
50% TS in vehicle	4.8 ± 0.3	0.99
	Ear Weight [mg/animal]
	Mean ± Standard deviation	Stimulation index
Vehicle	29.8 ± 0.7	1.00
50% TS in vehicle	31.3 ± 1.1	1.05

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information