Disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)

Endpoint summary

Description of key information

Not 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:

From the 18th of May to the 30th of May, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Tes conducted according to an internationally accepted test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Species and strain: Mice, CBA/JN
Sex: Females (nulliparous and non-pregnant)
Age at order: 7 to 8 weeks old
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
Date of arrival: 12 May 2016
Weight range at arrival: 19 to 22 grams
Acclimatisation period: At least 5 days
Veterinary health check: During acclimatisation period

ANIMAL HUSBANDRY
Animals per cage: Up to 5 during acclimatisation; 1/cage during the study
Housing :Polysulphone solid bottomed cages measuring 35.5 23.5 19 cm with nesting material
Cage control: Daily inspected and changed as necessary (at least twice/week)
Water: drinking water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply: ad libitum throughout the study
ENVIRONMENTAL CONDITIONS
Temperature range: 22 °C±2 °C
Relative humidity range 55 % ± 15 %
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour

Vehicle:

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

Remarks:

negative control

Concentration:

Preliminary test
Test item: 1, 2.5, 5, 10, 25 dose level w/w in terms of test item corrected for purity content 77.1%
Vehicle 0

Main test
Test item: 5, 10, 25 dose level w/w in terms of test item corrected for purity content 77.1%
Vehicle 0

No. of animals per dose:

Preliminary test: 1 per dose, negative and positive control
Main test: two per dose, negative and positive control

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
Animal identification was permanent, following arrival, by ink marking on the tail. Animals were identified by odd numbers. Animals were randomised at arrival. Healthy animals without observable skin lesions were chosen. At the time of treatment (for preliminary test and main assay), the animals aged approximately 9-12 weeks.

PRELIMINARY TEST
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations, depending on the animal
A dose volume of 25 µL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 µL/animal/day), using a micropipette and/or a sirynge.

In vivo observations
Mortality and morbidity: Throughout the study, all animals were checked twice daily.
Clinical signs: The animals were observed for clinical signs on:
Day 1: before and 1 hour after dosing
Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable)
Body weight: The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

Ear thickness measurement
The ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
Termination: Euthanasia method The animals were sacrificed on Day 6 by carbon dioxide narcosis.
Necropsy procedure: After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together. No necropsy was performed on the animals.

Selection of dose levels
In the main assay, the test item was used at the higher concentrations that were systemically tolerated and judged not to cause excessive local skin irritation. In particular, concentrations are considered irritant if:
– erythema grading (score) is 3 at any day of measurement and/or
– ear thickness is 25% with respect to Day 1 and
– ear punch weight is 25% with reference to the negative control group

MAIN TEST
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test or control item formulations.
A dose volume of 25 µL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 µL/animal/day), using a micropipette and/or a sirynge.

Day 1, 2, 3: Dermal application of the test item, the positive control or vehicle at a dose volume of 25 µL/ear (50 µL/animal).
Day 4: no application
Day 5: Intraperitoneal administration of BrdU
Day 6: Sacrifice, processing of lymph nodes and determination of cell proliferation.

Frequency of treatment
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test or control item formulations.Dosing method
A dose volume of 25 µL/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 µL/animal/day), using a micropipette and/or a sirynge.

5-bromo-2-deoxyuridine (BrdU) treatment
The animals were treated intraperitoneally, on Day 5 (once only), with 0.5mL/animal of a solution of BrdU at a concentration of 10mg/mL in physiological saline 0.9% NaCl), using a plastic graded syringe.

In vivo observations
Mortality and morbidity
Throughout the study, all animals were checked twice daily.
Clinical signs
The animals were observed for clinical signs before dosing commenced and daily up to sacrifice (approximately 1 hour after dosing on Days 2, 3 and 5).
Body weight
The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

Terminal phase
Euthanasia method and lymph node collection
The animals were killed on Day 6, approximately 24 hours after BrdU injection, by carbon dioxide narcosis.
No necropsy was performed on the sacrificed animals. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2% BSA-PBS [2% bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation at lymph node as detailed below.

Determination of cellular proliferation
Preparation of single cell suspension
A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 µmnylon mesh. The suspensions thus obtained were centrifuged and each supernatant resuspended in 20mL of 2% BSA-PBS.

Measurement of BrdU content in lymphocytes DNA
BrdU was measured by ELISA using a commercial kit (Roche Applied Science,Mannheim, Germany, Catalogue No. 11 647 229 001, batch No. 11417600), according to manufacturer instructions. Briefly, 100 µL of the LNC suspension were added to the wells of a flat-bottom 96-well microplate in triplicate. Two replicate microplates were prepared. After fixation and denaturation of the LNC, 100 µL of anti-BrdU antibody labelled with peroxidase were
added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and 100 µL of the substrate solution were thenadded and allowed to produce chromogen. The reaction was finally stopped by adding 25 µL of stop solution (1MH2SO4).
Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).
The replicate plate was destroyed after the evaluation of the results obtained in the first plate, since the objective of the study had been achieved.

Calculation
The BrdU labelling index is defined as follows:
BrdU labelling index = (OD450–ODblank450 )–(OD690–ODblank690 )
The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated.
Results for each treatment group were expressed as the mean Stimulation Index (SI).
The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

Positive control substance(s):

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

Statistics:

Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous, a Modified test (Cochran and Cox) was applied.

Positive control results:

In the group treated with the positive control item, a Stimulation Index of 4.15 was calculated. As it was greater than 2, the study was regarded as valid.

Parameter:

SI

Value:

1.13

Remarks on result:

other: 5% dose

Parameter:

SI

Value:

1

Remarks on result:

other: 10% dose

Parameter:

SI

Value:

1.27

Remarks on result:

other: 25% dose

Cellular proliferation data / Observations:

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group.

Criteria of interpretation for test item induced response

The test item is considered to induce sensitisation when the SI for any single treatment dose group is 1.6.

It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 - SI - 1.9).

The assay is considered satisfactory if the Stimulation Index (SI) of the positive control group is higher than 2.0.

PRELIMINARY TEST RESULTS .

No clinical signs were observed at the tested concentrations. Several animals showed a body weight loss, but not dose correlated.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated [25, 10, 5, 2.5 and 1% (w/w)].

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that no increase was found at any dose level investigated.

Based on the results described above, the highest concentration selected for the main assay was 25% (w/w).

MAIN ASSAY, IN VIVO PHASE

No mortality or clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5% (w/w)].

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Interpretation of results:

other: not classified

Remarks:

Classification criteria according to the CLP Regulation 1272/2008 and its amendments

Conclusions:

No increase in cell proliferation of draining lymph nodes was observed in any treatment group.
The calculated Stimulation Indices were 1.13, 1.00 and 1.27 at low, mid- and high dose levels, respectively (5, 10, 25 %)

Executive summary:

Method

The potential of the test item to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals No. 442b.

Preliminary test

Five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1% w/w in acetone:olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. No clinical signs were

observed at the tested concentrations. Several animals showed a body weight loss, but not dose correlated. According to the results of the irritation screening, the concentration of 25% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in acetone:olive oil 4:1 (v/v).

Observations

No mortality nor clinical signswere recorded in any animal. Changes in bodyweight observed during the study were within the expected range for this strain and age of animals. No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.13, 1.00 and 1.27, respectively at the low, mid- and high dose levels [5, 10 and 25 %, respectively]. No correlation with the doses nor statistical significance was observed.

Conclusion

The above results indicate that the test item does not elicit any sensitisation response in mice following dermal exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test item to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals No. 442b, in GLP.

A preliminary test was performed before the main assay, where the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in acetone:olive oil 4:1 (v/v).

No mortality nor clinical signswere recorded in any animal. No increase in cell proliferation of draining lymph nodes was observed in any treatment group and the SI of 1.13, 1.00, 1.27, indicate that the test item does not elicit any sensitisation response in mice following dermal exposure.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

According to the CLP Regulation n. 1272/2008, substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub- categorisation (1A and 1B) in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test (according to 3.4.2.2.4.1).

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test- ≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Under the experimental conditions employed, 10% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings.

As conclusion, according to the paragraph 3.4 of the CLP Regulation n. 1272/2008, table 3.4.3, the registered substance is not classified as skin sensitizer.