N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24. 10. – 6. 11. 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

EC Number:

252-021-1

EC Name:

N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Cas Number:

34432-92-3

Molecular formula:

C22H31N3O2

IUPAC Name:

N-ethyl-N-[2-(1-isobutoxyethoxy)ethyl]-4-(phenyldiazenyl)aniline

Test material form:

liquid: viscous

Details on test material:

Name of test material (as cited in study report): Solvent Yellow 124
Substance type: organic
Physical state: liquid
Appearance: dark yellow viscous liquid

Composition of test material, percentage of components:
- Analytical purity: 90.0 % (w/w)
- Impurities (identity and concentrations):
4´-[2-((hydroxy)ethyl)ethylamino]azobenzene 3.0 % (w/w)
1,1-bis(N-ethyl[4-(phenylazo)phenyl]aminoethan-2-oxy)ethan 2.5 % (w/w)
- Unknown impurities 4.5 % (w/w)

Lot/batch No.: S2408
Expiration date of the lot/batch: Unlisted
Stability under test conditions: stable
Storage condition of test material: During the study the test substance was stored in glass bottle at laboratory temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 16.38 – 19.66 g
- Housing: maximum six in macrolon cages with sterilized softwood shavings
- Diet (e.g. ad libitum): ST 1 BERGMAN - pelleted standard diet for experimental animals ad libitum
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3°C
- Relative humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

STUDY TIME SCHEDULE
Animal arrival/ start of acclimatization: 16. 10. 2012
Pilot experiment: 24.- 30. 10 2012
First day of administration: 31. 10. 2012
End of treatment period: 02. 11. 2012
Application of radionuclide and necropsy: 05. 11. 2012

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

The test substance was administered in the form of suspension in DAE 433. Concentration in formulation:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

No. of animals per dose:

Exposed groups - 15 females (5 animals per concentration)

Details on study design:

PILOT EXPERIMENT
The highest concentration 30% was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During the pilot experiment no clinical symptoms were observed in all three animals and no macroscopic changes (after necropsy) were detected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were subjected to a clinical examination (health check) shortly after arrival.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.

Stimulation index
The SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI.

Cell proliferation
The response towards the test substance is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship).
The response is considered negative, if the stimulation index (SI) is < 3 without the dose–response relationship.
The response is considered ambiguous if the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.

Validity criteria:
The test is considered valid, if the positive control substance DNCB produces a positive LLNA response, and if the stimulation index SI is > 3 over the negative control group.

Ear weight – irritation effect
If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.
A positive result in cell proliferation reveals that the test substance could be a contact allergen, but an irritation effect of test substance (increased ear weight) does not rule out the possibility that it can be a false positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
- Dosage volume: 25μL / ear / animal
- Preparation for administration:
All solutions were prepared by mixing an appropriate amount test substance and the vehicle to obtain the application form at concentrations of 30%, 3% or 0.3% (w/v).
The solutions were prepared before the start of application by mixing on a magnetic stirrer and then were still mixed during application.
- Frequency of preparation: each day before administration.
- Application: The volume of the application form was constant at all groups of animals - 25μL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette to avoid losses caused by draining from the ear.
- In vivo examination
Health and mortality: at least twice daily during the dosing period
Clinical observations: at least twice daily during the dosing period
Body weight: on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide

- Post mortem investigations:
Immediately after death, both ears of one animal were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (= 0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.

Lymph node cell counts:
Incorporation of 3H-methyl thymidine
The tissues of both of the lymph nodes were prepared by gentle mechanical disaggregation through 100 µm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes were analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 oC for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).

Positive control substance(s):

other: dinitrochlorbenzene

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Results and discussion

Positive control results:

Examination of cell proliferation in lymph nodes: in the positive control group, the SI was ≥ 3 (7.89) – test LLNA was efficient.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: At the highest dose level of the test article, the SI was 8.4, at the middle dose level the SI was 2.7, and at the lowest dose level, the mean SI was 1.81. The stimulation indexes of all of the test groups were below the threshold (see Table 1).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM for the test groups treated with the test substance was decreased in dose-related manner (see Table 1).

Any other information on results incl. tables

Table 1.  Summary table

Group	Radioisotope incorporation		Ear weight
	Mean DPM	SI	Mean (mg)
NC	112.89	1.00	24.10
PC	890.38	7.89+	34.84*
30%	948.33	8.40+	37.74*
3%	304.32	2.70	26.78*
0.3%	204.55	1.81	24.80

Figures with asterisk * = values statistically significant on probability level < 0.05 (Mann-Whitney test)

Figures with cross + = values ≥ 3  

NC – Negative control group  

PC – Positive control group

Applicant's summary and conclusion

Interpretation of results:

other: Positive results in cell proliferation revealed that the test substance could be a contact allergen in mice.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the given test conditions, the test substance, Solvent Yellow 124, elicited sensitising response in LLNA assay. Positive results in cell proliferation revealed that the test substance Solvent Yellow 124 could be a contact allergen in mice.

Executive summary:

The test substance, Solvent Yellow 124, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012.

In this study the contact allergenic potential of Solvent Yellow 124 was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The evaluation of ear weight was also performed for elimination of false positive findings with certain skin irritants.

Concentrations of test substance in vehicle (DAE 433): 30%, 3%, 0.3% (w/v)

During the pilot experiment the manifestation of high toxicity was recorded in animal No. 1. The female No. 1 (50%) died after 2nd application therefore the animal No. 4 was added and applied by the concentration of 30% dose level.

According to the results of pilot experiment these doses were chosen for the main experiment:

The test substance was administered in the form of suspension in DAE 433.

Concentration in formulation:

30% (w/v) - 300 mg/mL

3% (w/v) - 30 mg/mL

0.3% (w/v) - 3 mg/mL

During the main study the animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the main experiment. The test substance caused marked colouration of skin on dorsum of both ears in animals at the dose levels 30% and 3% so the assessment of erythema and other skin reactions could not be evaluated. At the lowest dose level (0.3%) no erythema was detected.

During the main experiment slight reduction of body weight was recorded in females at the highest dose level.

The positive control substance DNCB (concentration 0.5% (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

The test substance Solvent Yellow 124 showed a tendency to increased ear weight in the highest and middle of concentrations tested. Residues of the test substance on the ears could cause this weight increase. The result of skin irritation effect was considered as negative – it means the test substance probably did not cause irritation of skin.    

Comparison of Stimulation Indexes between all treated groups and control vehicle group revealed that the test substance Solvent Yellow 124 caused a significant and dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated group (30% v/v) was 8.40.

 

In conclusion, at the given experimental conditions the test substance Solvent Yellow 124 provided a positive result in LLNA study.

Positive results in cell proliferation revealed that the test substance Solvent Yellow 124 could be a contact allergen in mice.