Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May - 11 Jun 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 Jul 2010

Deviations:

yes

Remarks:

ear thickness measurements in pre-test not performed

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

EC Number:

224-580-1

EC Name:

Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Cas Number:

4418-26-2

Molecular formula:

C8H7O4.Na

IUPAC Name:

sodium 1-(6-methyl-2,4-dioxo-2H-pyran-3(4H)-ylidene)ethanolate

Specific details on test material used for the study:

Dehydroacetic acid

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, Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.2 ± 0.5
- Housing: Individual in Makrolon Type I with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10 and 20% (w/v)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 20 % suspension in propylene glycol.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 20 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation.
- Systemic toxicity: During the pre-test, no systemic toxicity could be observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation.
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, 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:
25 µl of the test compound was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on days 2 and 3. On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 19.7 µCi of 3H-methylthymidine (3H-TdR) into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells was prepared from each mouse and pooled per group (8 nodes per group).The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um 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.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations of the body weights were calculated.

Results and discussion

Positive control results:

The positive control substance hexyl cinnamic aldehyde (25% (w/v) induced positive reactions and a SI of 4.87 could be determined, thus meeting the reliability criteria for the LLNA.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

5% (w/v)

Results 2

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

10% (w/v)

Results 3

Key result

Parameter:

SI

Value:

2.36

Test group / Remarks:

20% (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Please refer to table 1 under "any further information on results".

EC3 CALCULATION
The EC3 value could not be calculated, since all SI values were below 3.

CLINICAL OBSERVATIONS
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 first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Table 1 Calculation and results of individual data

Test item concentration % (w/v)	Measurement DPM	Calculation			Result
		DPM-BG	number of lymph nodes	DPM per lymph node	SI
BG I	35	-
BG II	33	-
Vehicle control	3816	3782	8	472.8	-
5	4098	4064	8	508.0	1.07
10	7963	7929	8	991.1	2.10
25	8959	8925	8	1115.6	2.36

BG = Background (1 mL 5% trichloracetic acid) in duplicate

Vehicle = propylene glycol

SI = Stimulation index

Table 2 Individual body weights

Animal No.	Dose group	Initial Weight (g)		weight prior to treatment with 3HTdR (g)
		Individual	Mean± SD	Individual	Mean± SD
1	1	19.9	20.3 ± 0.9	21.2	21.2 ± 1.1
2	1	19.3		19.8
3	1	21.3		21.2
4	1	20.8		22.4
5	2	18.6	19.4 ± 1.1	20.3	21.1 ± 1.2
6	2	18.9		19.9
7	2	21.0		22.7
8	2	19.0		21.3
9	3	20.9	20.3 ± 0.5	21.0	20.6 ± 0.5
10	3	20.4		19.8
11	3	20.0		20.6
12	3	19.8		20.9
13	4	21.5	20.8 ± 1.5	22.1	21.0 ± 1.4
14	4	22.4		22.2
15	4	18.9		19.6
16	4	20.3		20.0

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

The test material was not a skin sensitiser.
CLP: not classified

Executive summary:

In a LLNA three groups each of four female mice were treated daily with the test item at concentrations of 5, 10, and 20% (w/v) in propylene glycol by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle (propylene glycol) only. All treated animals survived the scheduled study period and no signs of toxicity were

observed. The test material was found not to be a skin sensitiser.