Barbituric acid

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:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or no or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2005

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:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

EC Number:

253-256-2

EC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Cas Number:

36888-99-0

Molecular formula:

C16H9N5O6

IUPAC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dipyrimidine-2,4,6(1H,3H,5H)-trione

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- strain: CBA/CaOlaHsd
- source: Harlan Netherlands
- Age at study initiation: 8-12 weeks (beginning of acclimatization)
- Weight at study initiation: 16.9 g to 20.2 g (mean 18.8 g, SD 1.0 g)
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0 (control), 7.5, 15 and 30 % (w/v)

No. of animals per dose:

4 animals per treatment group, 4 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice with concentrations of 3.75, 7.5, 15 and 30 %. The data showed that the test item could be suspended in acetone:olive oil, 4:1 (v/v) up to 30 % (w/v). The test item could be suspended better in this vehicle than in DMSO or DMF.

- Irritation:
At this concentration the treated mouse did not show any signs of systemic toxicity. Due to the intense yellow colour of the test item local irritation reactions of the ear skin such as redness could not be detected. No swelling of the ears was observed.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled (4 animals/group) LLNA

- Criteria used to consider a positive response:

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:
Four female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. the application volume, 25 µL, was spread oer the entire dorsal surface of each ear lobe once daily. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

-

Results and discussion

Positive control results:

Stimulation indices of 2.0, 3.0 and 4.9 were determined in a separate test with the positive control substance at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil. An EC3 value of 9.9% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

7.5 % dose group

Results 2

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

15 % dose group

Results 3

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

30 % dose group

Any other information on results incl. tables

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were noted.

Due to the intense yellow colour of the test item local irritation reactions such as ear redness could not be examined. However, no swelling of the ears was observed.

The body weights of the animals, recorded prior to the first application and prior to necropsy, were within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The test item has not to be classified for skin sensitisation according to Regulation (EC) No 1272/2008.

Conclusions:

The test item (Pigment Yellow 139) was found to be not a skin sensitiser in the LLNA when tested at concentrations up to 30.0 % (w/v) in acetone:olive oil, 4:1 (v/v). Therefore the test item has not to be classified for skin sensitisation according to Regulation (EC) No 1272/2008.

Executive summary:

In the study the test item suspended in acetone:olive oil, 4:1 (v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed according to OECD TG 429 using test item concentrations of 7.5, 15 and 30 % (w/v). The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.4, 1.1, and 1.1 were determined with the test item at concentrations of 7.5, 15 and 30 % (w/v) acetone:olive oil, 4:1 (v/v). The EC3 value could not be calculated since all SI#s are below 3. The test item was found to be not a skin sensitiser in this assay.

Therefore the test item has not to be classified for skin sensitisation according to Regulation (EC) No 1272/2008.