Reaction mass of 5,5'-{(phenylmethanediyl)bis[benzene-4,1-diyl-diazene-2,1-diyl]}bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride and 5,5’-[3,4’-(phenylmethanediyl)diphenylene]bis(diazene-2,1-diyl)bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-10-09 to 2008-03-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations, conducted with the analogue substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: dose group 1: 19.9 ± 0.4; dose group 2: 20.8 ± 0.3; dose group 3: 19.8 ± 0.7; dose group 4: 20.3 ± 1.0
- Housing: Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.

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

IN-LIFE DATES: From: 2007-11-06 To: 2008-01-29

Vehicle:

dimethylformamide

Concentration:

The test item in the first main experiment was assayed at 3.13, 6.25, and 12.5%. Accidentally, the highest possible concentration of 25% was not included. Therefore, a second main experiment had to be performed. The test item in the second main experiment was assayed at 25%.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % solution in dimethylformamide.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 3.13, 6.25, 12.5, and 25% on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense colour of the test item local irritation reactions such as ear redness could not be detected.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: no data
- 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:

Treatment prepration:
The test item was placed into a volumetric flask on a tared balance and dimethylformamide was quantitatively added. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 3.13, 6.25, and 12.5% (w/v) in the first experiment and 25 % (w/v) in the second main experiment in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface ( 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 Rl of 79.6 (first experiment) or 79.8 (second experiment) RCi/ml 3HTdR (corresponds to 19.8 or 19.9 RCi 3HTdR per mouse) by intravenous injection via a tail vein.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

Test Group 2: Test item concentration % (w/v) 5, Stimulation Index S.I. = 1.29
Test Group 3: Test item concentration % (w/v) 10, Stimulation Index S.I. = 3.03
EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9% (w/v)

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

1st main experiment: Test item concentration % (w/v) 0: S.I. - Test item concentration % (w/v) 3.13:S.I. = 2.00 Test item concentration % (w/v) 6.25:S.I. = 3.82 Test item concentration % (w/v) 12.5: S.I. = 2.80 2nd main experiment: Test item concentration % (w/v) 0: S.I. - Test item concentration % (w/v) 25: S.I. = 5.22

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

1st main experiment: Test item concentration % (w/v) 0: DPM = 2905 Test item concentration % (w/v) 3.13: DPM = 5800 Test item concentration % (w/v) 6.25: DPM = 11092 Test item concentration % (w/v) 12.5: DPM = 8129 2nd main experiment: Test item concentration % (w/v) 0: DPM = 2556 Test item concentration % (w/v) 25: DPM = 13244 All DPM are DPM minus background

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The analogue substance was found to be a skin sensitiser. Given the applicability of the proposed read across approach (see IUCLID chapter 13) the same classification is likely to apply to the substance registered.

Executive summary:

The possible contact allergenic potential of the analogue substance was being investigated following the testing protocol as given in OECD guideline 429.

In the study the test item dissolved in dimethylformamide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 3.13, 6.25, and 12.5 % in the first experiment and 25 % in the second experiment.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. After the third application onwards the highest dose (25 %) induced loss of hair of the ears of all 4 animals of the group. Due to the intense colour of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 2.00, 3.82, and 2.80 were obtained in the first main experiment at concentrations of 3.13, 6.25, and 12.5 % in dimethylformamide and a Stimulation Index of 5.22 was determined with the test item at a concentration of 25% in dimethylformamide, respectively.

As no conventional dose response was obtained, the SIs at 6.25 % and 12.5 % were selected arbitrarily to calculate the most conservative EC3 value. Using this approach an EC3 value of 4.8 % was derived for the test item.

Based on the result of this study, the analogue substance is found to be a skin sensitiser. The same classification is likely to apply to the substance registered.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A valid LLNA study has been conducted with the analogue chemical (source chemical). Based on the study results indicating an EC3 value of 4.8%, the analogue substance is found to be a skin sensitizer.

Given the applicability of the proposed read across approach (see IUCLID chapter 13), the registration substance (target molecule) is also considered to be a skin sensitizer. For these reasons the same classification as for the analogue substance has to be applied to the registration substance registered.

Migrated from Short description of key information:
Substance registered is considered to be a skin sensitizer.

Justification for selection of skin sensitisation endpoint:
The selected study was performed under GLP and in accordance with OECD TG 429. No other studies are available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The analogue substance was found to be a skin sensitizer. The same classification has to be applied to the registered substance.

The resulting classification is "irritant" Xi; R43 "May cause sensitisation by skin contact", according to the former DSD regulation, and "Sens. Skin 1", H317 "May cause allergic skin reaction" according to the new CLP regulation.