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

Endpoint summary

Description of key information

Based on the results of a local lymph node assay conducted on mice, the test substance was considered to be not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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)

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

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

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).

Positive control results:

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

Key result

Parameter:

SI

Value:

1

Variability:

No deaths

Test group / Remarks:

Vehicle Control Group (DMSO)

Key result

Parameter:

SI

Value:

0.87

Variability:

No deaths

Test group / Remarks:

Dose group 2%

Key result

Parameter:

SI

Value:

1.23

Variability:

No deaths

Test group / Remarks:

Dose group 5%

Key result

Parameter:

SI

Value:

1.08

Variability:

No deaths

Test group / Remarks:

Dose group 10%

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.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Local Lymph node assay

The test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. The study was conducted in compliance with GLP and according to OECD Guideline 429. Test item concentrations of 2, 5, and 10% (w/w) in DMSO were used. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.87, 1.23, and 1.08 were determined with the test item at concentrations of 2, 5, and 10% (w/w) in DMSO, respectively. A dose response was not observed. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and cell count was not observed in any of the dose groups in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not exceeded in any dose group. Thus, the test item was not a skin sensitiser under the test conditions of this study.

Optimization Test

In a skin sensitization study in guinea pigs (Optimization test (Maurer Th.) and Draize) 10 Pirbright White guinea pigs per sex were injected 10 times intracutaneaus, every second day (except weekends), (0.1% solution of test substance in saline 0.9%). During the second and third week of the induction period the test material was incorporated in a mixture of the normal vehicle with complete Bacto Adjuvant. Fourteen days after the last sensitizing injection, a challenge injection of 0.1 mL 0.1 % solution of test substance in saline 0.9% was administered into the skin. 24 Hours after the challenge injection reactions were recorded. Ten days after the intracutaneous challenge injection a subirritant dose of the test compound was applied epicutaneously under occlusive dressings which were left in place for 24 hours. The skin irritation was recorded according to Draize. Significant differences between the test group and the vehicle-treated controls were only seen after intradermal challenge application of the test substance, i.e. when the skin barrier was intentionally by-passed. An epidermal challenge was carried out and found to be negative, but the assessment was difficult since the compound had a reddish colour. No differences between the test and the control group was seen after epidermal challenge application. The negative results upon epidermal challenge demonstrate that, in artificially sensitizied guinea-pigs, exposure of the intact skin to the test compound does not provoke contact dermatitis. However, the study is performed quite some time ago and does not comply with the current guidelines and with guidelines which were in place from 1992 on. The method applied uses intradermal injections only for induction. Furthermore, the topical challenge was performed with a very low test concentration (0.1%) and at a rather late date after induction (10 days after the intradermal challenge and 24 days after the final induction treatment). Normal test concentrations of powders to be examined in sensitization studies by topical application are usually 10-50%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.