3-hydroxy-3'-nitro-2-naphthanilide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-3'-nitro-2-naphthanilide

EC Number:

205-209-2

EC Name:

3-hydroxy-3'-nitro-2-naphthanilide

Cas Number:

135-65-9

Molecular formula:

C17H12N2O4

IUPAC Name:

3-hydroxy-N-(3-nitrophenyl)-2-naphthamide

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C, relative humidity approx. 45-65%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0.25, 0.5, 1, and 2.5%

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
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 DMF. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. On days 6, both animals showed slight eschar formation.
Therefore, a second pre-test was performed using test item concentrations of 2.5 and 5%. On day 3, 4 and, 6, the animals showed an erythema of ear skin (score 1) and scaly ears. Slight slight eschar formation and visible swelling of ears (see Appendix 1 for details) was observed in the animal treated with 5% of the test item. It was unclear if the animal treated with a test item concentration of 2.5% showed very slight eschar formation or only scaly ears on day 6, therefore it was decided to test 4 concentrations in the subsequent main study, in order to achieve a valid assay even if the highest dose level would turn out to be invalid due to excessive local skin irritation.
Thus, the test item in the main study was assayed at 0.25, 0.5, 1, and 2.5%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.


Experimental Design and Study Conduct

Determination of Ear Thickness
In the pre-test prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.25, 0.5, 1, and 2.5% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.5 µCi of 3H-methyl thymidine (equivalent to 81.8 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).

Preparation of Single Cell Suspensions
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Determination of Ear Weights
In the pre-test and main experiment after the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Observations

Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Ear thickness: In the pre-test, the ear thickness was determined prior to the first application (day 1), on day 3, and prior to sacrifice on day 6.

Ear weights: In the pre-test and main experiment, the ear weight was determined after sacrifice (biopsy punches will be taken from each ear).

Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Positive control substance(s):

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

Results and discussion

Positive control results:

October 2016 (study 1792800): S.I. of 1.50, 3.84, and 11.76 were derived at tested concentrations of 5, 10, 25%, respectively and an EC3 value of 8.2% was calculated.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

3.28

Test group / Remarks:

0.25% test item concentration

Results 2

Key result

Parameter:

SI

Value:

4.04

Test group / Remarks:

0.5% test item concentration

Results 3

Key result

Parameter:

SI

Value:

4.16

Test group / Remarks:

1% test item concentration

Results 4

Key result

Parameter:

SI

Value:

4.8

Test group / Remarks:

2.5 % test item concentration

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: DMF

Test item concentration	Group	Measurement DPM	Calculation			Result
%			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	17	---	---	---	---
---	BG II	23	---	---	---	---
0	1	10206	10186	8	1273.2	1.00
0.25	2	33388	33368	8	4171.0	3.28
0.5	3	41166	41146	8	5143.2	4.04
1	4	42432	42412	8	5301.5	4.16
2.5	5	48931	48911	8	6113.9	4.80

1    =  Control Group

2-5=  Test Group

^a)   =  The mean value was taken from the figures BG I and BG II

^b)      =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

 Calculation of the EC3 value

The EC3 value could not be calculated, since all S.I.´s are above the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. The animals treated with the mid and high dose of the test item showed scaly ears on test day 6.

Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

 Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The test item Naphtol AS-BS was found to be a skin sensitiser under the test conditions of this study.

The lowest dose tested (0.25%) caused an SI value of 3.28, which meets criteria for classification according to Regulation (EC) No 1272/2008: Category 1A, H317: May cause an allergic skin reaction

Executive summary:

In order to study a possible skin sensitising potential of Naphtol AS-BS, four groups each of four female mice were treated once daily with the test item at concentrations of 0.25, 0.5, 1, and 2.5% in DMF by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. In the pre-experiments, it remained unclear if the animal treated with a test item concentration of 2.5% showed very slight eschar formation or only scaly ears on day 6, therefore it was decided to test 4 concentrations in the main study, in order to achieve a valid assay if the highest dose level would turn out to be invalid due to excessive local skin irritation.

A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ß-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. The animals treated with the mid and high dose of the test item showed scaly ears on test day 6. A statistically relevant increase in ear weights was not observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 3.28, 4.04, 4.16, and 4.80 were determined with the test item at concentrations of 0.25, 0.5, 1, and 2.5% in DMF. A dose response was observed. The EC3 value could not be calculated, since all obtained SI´s were above the threshold value of 3.