5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-12 to 2014-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

EC Number:

276-344-2

EC Name:

5-[(2,3-dihydro-6-methyl-2-oxo-1H-benzimidazol-5-yl)azo]barbituric acid

Cas Number:

72102-84-2

Molecular formula:

C12H10N6O4

IUPAC Name:

5-[(1E)-2-(6-methyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)diazen-1-yl]-1,3-diazinane-2,4,6-trione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 01256BJ7
- Expiration date of the lot/batch: 24 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 9 - 10 weeks, main study: 8 - 9 weeks
- Weight at study initiation: pre-test: 19.2 and 21.1 g, main test: mean weight 19.4 g (+/-0.7 g)
- Housing: group
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 45-65
- Photoperiod: 12 hours darknes/ 12 hours light

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

2, 5, and 10 % in DMSO

No. of animals per dose:

5

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: First, 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, 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.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test was used for detection of possible outliers (performed with Microsoft Excel 2007).

Results and discussion

Positive control results:

The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2013.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

No deaths

Test group / Remarks:

Vehicle Control Group (DMSO)

Results 2

Key result

Parameter:

SI

Value:

0.87

Variability:

No deaths

Test group / Remarks:

Dose group 2%

Results 3

Key result

Parameter:

SI

Value:

1.23

Variability:

No deaths

Test group / Remarks:

Dose group 5%

Results 4

Key result

Parameter:

SI

Value:

1.08

Variability:

No deaths

Test group / Remarks:

Dose group 10%

Any other information on results incl. tables

Redness of the ear skin could not be determined due to the colour of the test item. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Table 1: Mean DPM +/- SD

	Group Calculation
Test item concentration	Mean DPM/ animal (2 lymphnodes)*	SD
Vehicle Control Group (DMSO)	2166.6	945.0
2 % test item	1884.7	741.7
5 % test item	2656.9	770.4
10% test item	2330.5	818.5

* Mean DPM/ animal 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met