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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic, ReH CZ 21760118
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 17.1 to 21.6 g (at start of dosing)
- Housing: in groups of maximum six
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

- Temperature (°C): 22 ±3°C
- Humidity (%): 30 - 70 %,
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: 23.2.2009 To: 25.2.2009
other: DAE 433 - mixture of40% dimethylacetamide, 30% acetone and 30% ethanol
0.1, 1 and 10%
No. of animals per dose:
Details on study design:
- Compound solubility: The test item is a pigment and insoluble in organic or inorganic solvents. A suspension of up to 10% could be tested.
- Irritation: Not irritating
- Lymph node proliferation response: No data

- Name of test method: non-radiolabelled LLNA
- Criteria used to consider a positive response: The results of the LLNA were evaluated according to the following criteria.
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05
LN weight: 1.2
LN cell count: 1.3

1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear
concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.
2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a clear
concentration dependence.

TREATMENT PREPARATION AND ADMINISTRATION: Before start of application the suspension was mixed for 5 minutes with a
magnetic stirrer and then were still mixed during application.
Positive control substance(s):
other: Dinitrochlorobenzene
Key result
Test group / Remarks:
negative control
Key result
Test group / Remarks:
0.1% test substance
Key result
Test group / Remarks:
1% test substance
Key result
Test group / Remarks:
10% test substance
Key result
Test group / Remarks:
positive control

No animal died during the main experiment.

No symptoms of toxicity were observed in animals fram the negative contral group and in groups administered by the test substance at the concentration 0.1 %, 1% and 10%. All animals in the positive contral group showed these symptoms: hyperaemia of skin and


Interpretation of results:
GHS criteria not met
In a GLP-compliant study according to OECD TG 429, the substance did not induce any skin sensitization up to the maximum testable concentration (in suspension) of 10%.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The potential for skin sensitization was tested in two independent assay.

One study was performed in 1980 using the Maurer Optimization procedure (Maurer Th., Thomann P., Weirich E.G., Hess R., The Optimization test in the guinea pig Agents & Actions 5 ( 2 ) , 174-179, 1975 and Predictive evaluation in animals of the contact allergenic potential of medically important substances Contact Dermatitis 4, 321-333, 1978, Contact Dermatitis 5, 1-10, 1979). The test was performed on groups of 10 male and 10 female guinea pigs of the Pirbright White strain. During the induction period the animals received one injection every second day (except weekends) to a total of 10 intracutaneous injections of a freshly prepared 0.1 % suspension in propylene glycole. One control group was treated with the vehicle alone. Fourteen days after the last sensitizing injection, a challenge injection of 0.1 ml of a freshly prepared 0.1 % suspension in propylene glycole was administered into the skin of the left flank.

Twenty-four hours after of the induction period injection the reactions each injection during the first week and 24 hours after the challenge were recorded. Erythema and edema were scored according to the Draize system. Both for the vehicle control and the test item, three of the 20 animals showed a skin reaction. Ten days later a subirritant dose of the test substance was applied for epicutaneous challange for 24h. No skin reactions were observed in any of the 20 animals.

For the second study, the local lymph node assay in its non-radioactive version was applied (Synthesia 2009a). This testing was conducted according to EU method B.42 with modifications as described in publications of Ulrich P, Streich J, Suter W, 2001; Ehling et al., 2005; Ehling et al.,2005A. In this GLP-compliant study the contact allergenic potential of C.I. Pigment Red 177 was evaluated after topical application to female BALB/c mice. Six mice per group were exposed by test and control substances on the dorsum of both ears once a day during three consecutive days. Draining lymph nodes were taken off at 24 hours after the last application. Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Pigment Red 177: 10%, 1%, 0.1% (w/v) in DAE 433. Endpoints: ear weight, auricular (ear-draining) lymph node weights and cell counts = lymph node (LN) hyperplasia. The animals exposed to the test substance were no clinical observations attributable to the treatment with test substance in any dose level. There was no difference in body weight increment of all groups in comparison to the vehicle control. The positive contral substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and LN hyperplasia, which was in congruence with his expected mode of action as a contact allergen. The comparison of values between treated groups and control group revealed that the test substance did not cause statistically significant increase in LN cell count or in LN weight. Also index of LN weight and LN cell count were not exceeded in any dose level. In conclusion, at the given experimental conditions the test substance Pigment Red 177 elicited a negative response in LLNA test.

Migrated from Short description of key information:
The substance was found to be non sensitizing upon testing in the local lymph node assay in mice according to OECD testing guideline 429 (Synthesia 2009a) and in the guinea pig maximization test according to OECD testing guideline 406 (Ciba-Geigy Ltd 1980)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

Classification, Labelling, 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, as amended for the second time in Directive (EC 286/2011).