Disodium phthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 November 2012 to 29 November 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and EU method. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium phthalate

EC Number:

240-106-6

EC Name:

Disodium phthalate

Cas Number:

15968-01-1

Molecular formula:

C8H6O4.2Na

IUPAC Name:

disodium phthalate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Phthalic acid sodium salt (disodium phthalate)
- Physical state: white powder
- Analytical purity: > 95%
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g.
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet, ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water.

Concentration:

5, 10 and 25% w/w

No. of animals per dose:

Groups of four mice.

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μl of the test item at a concentration of 25% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 μl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the
pipette. A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 μCi to each mouse.

Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.

A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The
lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

After approximately eighteen hours incubation at approximately 4ºC, the precipitates were recover ed by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately
twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

OBSERVATIONS:
All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the
test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Positive control substance(s):

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

Results and discussion

Positive control results:

A group of five animals was treated with 50 μl (25 μl per ear) of α-Hexylcinnamaldehyde, tech., 85% as an emulsion in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is 7.20.

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: Concentration % w/w Stimulation Index Result 5 0.83 Negative 10 1.01 Negative 25 1.41 Negative

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (% w/w) dpm dpm/Node Vehicle 10828.06 1353.51 5 8947.06 1118.38 10 10947.66 1368.46 25 15296.88 1912.11

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing, and 25% was chosen as it was the maximum dose which did not induce systemic toxicity and/or excessive local skin irritation.

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the OECD Guideline No. 429 "Skin Sensitisation: Local Lymph Node Assay" and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission

Regulation (EC) No. 440/2008. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μl (25 μl per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone. The Stimulation Index expressed as the mean radioactive incorporation foreach treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % w/w Stimulation Index Result

5 0.83 Negative

10 1.01 Negative

25 1.41 Negative

The test item was considered to be a non-sensitiser under the conditions of the test.