Sodium 3-[[4-[(2-ethoxy-5-methylphenyl)azo]-1-naphthyl]azo]benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Study period:

From April 15 to May 23, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

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)

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

In vivo test system

Test animals

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: 14 April 2016
Weight range at arrival: 19 to 20 grams
Acclimatisation period: at least 5 days
Veterinary health check: during acclimatisation period

ANIMAL HUSBANDRY
Animals per cage: ip 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 ± 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

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

negative control

Concentration:

Preliminary test
Group number 1
Test item: 25, 10, 5, 2.5, 1 dose level w/w in terms of test item corrected for purity content 73.15 %
Vehicle: 0 dose level w/w

Group number 2
Test item: 0.5, 0.25, 0.1, 0.05 dose level w/w in terms of test item corrected for purity content 73.15 %
Vehicle: 0 dose level w/w

No. of animals per dose:

Preliminary test: 1 per dose, negative and positive control
Main test: 2 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
Animals were treated for three consecutive days (days 1, 2, 3) with the vehicle or test item formulations, depending on the animal.
Dosing method: 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: 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: animals were weighed at allocation (day 1) and on sacrifice (day 6).

Ear thickness measurement
Ear thickness was measured by a suitable micrometer on day 1 (before dosing), on day 3 (before dosing) and on day 6.
Termination: 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 animals.

Selection of dose levels
In the main assay, test item was used at the higher concentrations, 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
Day 1, 2, 3: dermal application of test item, 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
Animals were treated for three consecutive days (days 1, 2, 3) with 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
Animals were treated intraperitoneally, on day 5 (once only), with 0.5 ml/animal of a solution of BrdU at a concentration of 10 mg/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: 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: animals were weighed at allocation (day 1) and on sacrifice (day 6).

Euthanasia method and lymph node collection: animals were killed on day 6, approximately 24 hours after BrdU injection, by carbon dioxide narcosis. No necropsy was performed on sacrificed animals. Shortly after sacrifice of each animal, 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].

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 µm nylon mesh. Such suspensions 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 then added and allowed to produce chromogen. The reaction was finally stopped by adding 25 µl of stop solution (1M H2SO4).
Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).
The replicate plate was destroyed after the evaluation of results obtained in the first plate, as 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 mean Stimulation Index (SI).
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 corresponding vehicle group.

Positive control substance(s):

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

Statistics:

Differences between each treated group and concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. Data homogeneity 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.

Results and discussion

Positive control results:

Stimulation Index of 3.35 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.14

Remarks on result:

other: 0.1% dose

Results 2

Parameter:

SI

Value:

1.07

Remarks on result:

other: 0.25%dose

Results 3

Parameter:

SI

Value:

1.01

Remarks on result:

other: 0.5% dose

Cellular proliferation data / Observations:

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

Any other information on results incl. tables

PRELIMINARY TEST RESULTS

Initially, five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1 % w/w in acetone:olive oil 4:1 (v/v)] of the test item were selected to be used in the preliminary phase.

Several animals showed signs of toxicity: on day 3 of study, one animal (no. 3) showed decreased activity and hunced posture and was sacrificed for humane reasons. On day 4 of study, another animal (no. 1) showed decreased activity, hunched posture, cold to touch and decreased reactivity and was sent to necropsy for humane reasons.

The other treated animals showed semiclosed eyes and hunched posture from day 4 up to day 6 with the exception of one animal (no. 9), that recovered from these signs on day 6 of study. Animal nos. 5 and 7 showed also a marked body weight loss.

Consequently, it was decided to test other 4 concentrations (0.5, 0.25, 0.1 and 0.05% w/w).

No signs of toxicity (significant clinical signs or body weight losses) were observed at these concentrations.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated [25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1 and 0.05 % (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 these results, the highest concentration selected for the main assay was 0.5 % (w/w).

MAIN ASSAY, IN VIVO PHASE

Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [0.5, 0.25, 0.1 % (w/w)].

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

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

No increase in cell proliferation of draining lymph nodes was observed in any treatment group.
Stimulation Indices were calculated as 1.14, 1.07 and 1.01 at low, mid and high dose levels, respectively (0.1, 0.25 and 0.5 %).

Executive summary:

Method

The potential of 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 OECD guideline 442b.

In the preliminary test, 5 concentrations, i.e. 25, 10, 5, 2.5 and 1 % w/w in acetone:olive oil 4:1 (v/v), were tested, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. Adverse effects on treated animals were noted: 2 animals were sacrificed due to decreased activity/reactivity and hunched posture on day 3 and 4; the other treated animals showed semiclosed eyes and hunched posture from day 4 up to day 6 with the exception of one animal, that recovered from these signs on day 6; 2 animals showed also a marked body weight loss.

Consequently, other 4 concentrations, i.e. 0.5, 0.25, 0.1 and 0.05 %, were tested; the concentration of 0.5 % w/w resulted as not toxic and not irritant.

Main assay

In the main assay, test item was topically administered at concentrations of 0.5, 0.25 and 0.1 % (w/w), in acetone:olive oil 4:1 (v/v).

Results

No mortality nor clinical signs were 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.

Stimulation Indices (SI) were 1.14, 1.07 and 1.01, respectively at the low, mid- and high dose levels of 0.1, 0.25 and 0.5 % w/w.

No correlation with the doses nor statistical significance was observed.

In conclusion, test item did not elicit any sensitisation response in mice following dermal exposure.