Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Endpoint summary

Description of key information

Macrolex Rot B is a red powder with a molecular weight of 500 g/mole. The substance has a very low water solubility of < 3 µg/L at 20°C (Currenta, 2017) and a high log Kow (octanol/water) of +6.1 at 25°C (Currenta, 2016). No information is available on dermal bioavailability of the substance. However, based on the physical-chemical parameters given above dermal absorption can be expected to be very low if any (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

Macrolex Rot B was tested for skin irritating effects in vivo in an acute dermal irritation test on rabbits (BASF, 1978). No irritation was reported on intact skin after 24 h exposure to 0.5 g of the compound moistened with water. In occupational settings no skin irritating or skin sensitizing effects were reported regarding human skin.

Based on this information no significant skin irritation was expected under the conditions of the Local Lymph Node Assay (LLNA) and it was assumed that the highest technically feasible compound suspension would be considered appropriate for this investigation.

However, in the mouse LLNA performed with the test item (OECD 429, radioactive method) it appeared that 5% of the test item in acetone/olive oil (4:1, v/v) was the highest applicable concentration avoiding local irritation determined in a pre-test on two mice. The 10% concentration led to slight ear swelling. No detailed information is given in the report on the effects seen in this pre-test.  The main study was thus performed with the test item at concentrations of 1%, 2.5%, and 5% (w/v) in acetone/olive oil on 4 mice per group (Wang-Fan, 2004). No clinical signs or indications of local effects were seen after treatment. Lymph nodes were pooled and ³H-methyl thymidine activity was measured by beta-scintillation. Stimulation indices (S.I.) of 1.2, 1.4, and 1.4 were determined with the test item at the concentrations 1, 2.5 and 5%. The test item did thus not show a skin sensitizing potential when tested up to the concentration of 5%.

To clarify the discrepancy between the information gained via physical-chemical properties/skin irritation studies on the one side and the irritating effects observed in the LLNA on the other side, another pre-test for a LLNA was initiated (Dony, 2016). In this pre-test all tested concentrations (25, 10, 5 and 2.5%) induced excessive local skin irritation under the conditions of the LLNA using DMSO as vehicle. Ear thickness was increased by ≥ 23% on day 6 at 10, 5, and 2.5%. It is assumed that these irritant effects are related to the formulation of the test substance in DMSO and do not indicate irritating effects of the compound per se. Consequently, the study was stopped after the pre-test since testing the substance in the LLNA as suspension in DMSO will lead to artificial results and is not appropriate for human risk assessment.

Testing of Macrolex Rot B in chemico in the Direct Peptide Reactivity Assay (DPRA; OECD 442C) was technically not feasible. The substance was soluble in dimethyl sulfoxide/acetonitrile (1:1) but was insoluble in all other solvents that are acceptable for the DPRA. However, the dimethyl sulfoxide / acetonitrile 1:1 mixture itself had significant impact on the percent peptide depletion. The mean cysteine concentration of the reference control C (solvent control) was reduced by > 40%. Thus, the acceptance criteria for the DPRA were not fulfilled and no DPRA result for Macrolex Rot B could be obtained.

Testing of Testing of Macrolex Rot B in vitro for skin sensitization in the ARE-Nrf2 Luciferase Test (OECD TG 442D) and in the h-CLAT (OECD TG 442E) was also technically not feasible. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for both tests. It was thus concluded that the test article is outside of the applicability domain for these studies and the tests could thus not be performed.

In conclusion, based on the available phys-chem data and the LLNA results of the test item in acetone/olive oil Macrolex Rot B is considered to exert no skin sensitizing potential. This conclusion is in line with in silico results obtained via OECD QSAR Toolbox (version 4.0) that show no structural alert for skin sensitization for Macrolex Rot B (Schlecker, 2017).

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:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

no quantitatative data is given for the effects in the pre-test;

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Name of test material given in the report: Sandoplast Red BB
appearance: red solid
purity: 98.5%

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL-5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at beginning of acclimatization: 8-12 weeks
- Weight: 16 - 24 g
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (plus/minus 3°C)
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12h
- IN-LIFE DATES: From: 15.09.2004 To: 29.09.2004

Vehicle:

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

Concentration:

non-GLP pre-test: 1, 2.5, 5, and 10%
GLP main test: 1, 2.5, and 5%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Irritation: in a non-GLP pretest in two mice test item concentrations of 1, 2.5, 5, and 10% (w/v) were tested on one ear each; after a single application no irritation effects were observed up to the concentration of 5%; slight ear swelling was noted 24 hours after treatment at the local site dosed at 10%
- Ear thickness measurements: slight ear swelling was noted 24 hours after treatment at the local site dosed at 10%; thus, 5% was determined as the highest applicable concentration while avoiding systemic toxicity and excessive local irritation in the chosen vehicle.

MAIN STUDY
Test item preparation: The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. The test item formulation were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: radioactive LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- first, exposure to at least one concentration results in ³HTdR incorporation of at least 3-fold over control mice as indicated by the Stimulation Index (S.I.)
- second, 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:
Treamtent: Mice were treated topically on the dorsal surface of each ear lobe with the test item concentrations 1, 2.5 and 5% (w/v) in acetone/olive oil (4:1). The application volume (25 µL) was spread over the entire dorsal surface of the ear lobes once daily for 3 consecutive days. A hair dryer was passed briefly over the ear's surface to prevent the loss of any test item applied.
Administration of ³H-Methly Thymidine: (specific activity 2Ci/mmol, concentration 1 mCi/mL) five days after the first topical application all mice were administered with 250 µL of 76.6 µCi/mL ³HTdR (equal to 19.1 µCi) by intravenous injection via a tail vein.
Determination of ³HTdR: approximately 5 hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2. Draining lymph nodes were excised and pooled for each experimental group (8 nodes per group). The level of ³hThR incorporation and background activity were measured on a beta-scintillation counter (disintegration per minute, DPM).

Positive control substance(s):

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

Statistics:

mean values and standard deviations were calculated for the body weights

Positive control results:

Stimulation indices of 2.7, 3.4, and 12.4 were determined for the positive control in concentrations of 5, 10, and 25%. The positive control was thus found to be a skin sensitizer and an EC3 value of 7.1% (w/v) was derived.

Parameter:

SI

Value:

1.2

Test group / Remarks:

1%

Parameter:

SI

Value:

1.4

Test group / Remarks:

2.5%

Parameter:

SI

Value:

1.4

Test group / Remarks:

5%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
no data given on cellular proliferation

DETAILS ON STIMULATION INDEX CALCULATION
DPM were calculated for pooled lymph nodes (8 per group) and the DPM per lymph node were calculated by dividing the measured value by the number of lymph nodes pooled.
The DPM per lymph node is given with 351 for the control and 417, 484, and 508 for the treatment groups 1%, 2.5%, and 5%.

EC3 CALCULATION
Calculation of EC3 could not be done because no test concentration produced a S.I. of 3 or higher

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears and no systemic findings were observed during the study.

BODY WEIGHTS
Body weights were within the range of the control with the exception of two mice (one in the 1% and 5% group each) that lost weight during the study. This was considered to be incidental.

Executive summary:

A radioactive mouse LLNA (OECD 429) was performed with the test item at concentrations of 1%, 2.5%, and 5% (w/v) in acetone/olive oil (4:1, v/v) on 4 mice per group. 5% was the highest applicable concentration avoiding local irritation determined in pre-test in two mice. Treatment occurred on 3 consecutive days. ³H-methyl thymidine was injected intravenously on day 5.

No clinical signs or indications of local effects were seen after treatment. Lymph nodes were pooled and ³H-methyl thymidine activity was measured by beta-scintillation. Stimulation indices (S.I.) of 1.2, 1.4, and 1.4 were determined with the test item at the concentrations 1, 2.5 and 5%. The test item did thus not show an allergenic potential when tested up to the concentration of 5%.

Justification for classification or non-classification

Based on the available phys-chem data and the LLNA results of the test item in acetone/olive oil Macrolex Rot B is considered to exert no skin sensitizing potential. This conclusion is in line with in silico results obtained via OECD QSAR Toolbox (version 4.0) that show no structural alert for skin sensitization for Macrolex Rot B (Schlecker, 2017). Thus, no classification is warranted for skin sensitization.