3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

Endpoint summary

Description of key information

Under the conditions of the study, the test material was shown to have no sensitisation potential in the Local Lymph Node Assay.

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:

22 November 2017 to 12 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study conducted for another regulatory purpose.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

temperature was 18-26ºC and relative humidyt was 23-79%; deviations not considered to adversely effect the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

temperature was 18-26ºC and relative humidyt was 23-79%; deviations not considered to adversely effect the study

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.1 - 21.6 g
- Housing: Type II polypropylene/polycarbonate cages
- Diet: ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0 - 26.0°C
- Humidity (%): 23 - 79%
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Vehicle:

dimethylformamide

Concentration:

0 (negative control), 1, 2.5, 5, 10% w/v

No. of animals per dose:

Four females per dose level

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: tested using AOO, DMF, MEK, propylene glycol, DMSO and 1% aqueous Pluronic® PE9200.

Two animals/dose were tested at doses of 25% and 10% w/v test material in DMF. The preliminary test was conducted in a similar experimental manner to the main study but it was terminated on Day 6 and the radioactive proliferation assay was not performed. The maximum concentration of test material in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 25% (w/v).

- Irritation: all animals observed daily. None at the site of application.
- Systemic toxicity: all animals observed daily. No mortality was observed. Test material precipitate or minimal amount of test material precipitate was observed on the ears of both animals of the 25% dose group on Days 1-6 and for both animals of the 10% dose group on Days 1-5.
- Ear thickness measurements: measured using a thickness gauge on Day 1 (pre-dose), Days 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The ear thickness values and ear punch weights were within the acceptable range.
- Erythema scores: both ears of all animals was observed and scored.
- Marked body weight loss (> 5% decrease) was detected for both animals of the 25% dose group and for both animals of the 10% dose group. The mean body weight loss of these dose groups were more than 5 %, the significance of the body weight loss relative to treatment is equivocal at 10%, but at 25% it appears to be unacceptable for a main study.
- The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical application: During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
- Clinical observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Body weights: Individual body weights were recorded on Day 1 and on Day 6 (prior to 3HTdR injection).

PROLIFERATION ASSAY
- Injection of tritiated thymidine (3HTdR): On Day 6, animals were intravenously injected via the tail vein with 250 µL PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR. Once injected the animals were left for 5 hours (± 30 minutes).
- Removal and preparation of draining auricular lymph nodes: Five hours (± 30 minutes) after intravenous injection, the mice were euthanised by asphyxiation with carbon dioxide. The draining auricular lymph nodes were removed from all animals and the nodes of the animals from each test group were pooled.
- Preparation of single cell suspension of lymph node cells: A single cell suspension of pooled lymph node cells (LNCs) was prepared by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS. Pooled LNCs were pelleted be centrifugation. Centrifugation supernatants were discarded and pellets gently resuspended and added to 10 mL PBS. The washing step was repeated twice.
- Determination of Incorporated 3HTdR: After the final washing, supernatants were removed. Pellets were gently agitated, resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes. After overnight (approximately 18 hours) incubation at 2-8°C, precipitates were centrifuged and supernatants were removed. Pellets were resuspended in 1 mL of 5% TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

Positive control substance(s):

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

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (DMF)

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

5% w/v

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

2.5% w/v

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

1% w/v

Parameter:

SI

Value:

10.7

Test group / Remarks:

Positive control (25% w/v HCA)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value. The results were expressed as 'DPN' (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.

CLINICAL OBSERVATIONS:
No mortality was observed during the main study. Test material precipitate or minimal amount of test material precipitate was observed on the ears of all experimental animals at 5% and 2.5% on Days 1-3 and at 1% dose group at Day 3.

BODY WEIGHTS
Treatment related effects were observed on the mean body weight changes at 10% dose group in the main study. The mean body weight loss (-18.3%) was much higher than the limit of 5% given in the relevant OECD guideline used to indicate systemic toxicity. Therefore, the 10% dose group was excluded from the evaluation of cell proliferation on the grounds of systemic toxicity. Marked body weight loss (≥ 5%) was observed for one animal of the 5% dose group and for one animal of the 1% dose group. However, the mean body weight of the 1% dose group were in the acceptable range and this change was considered as individual variability. In the 5% dose group 1 out of 4 animals had more than 5% body weight loss which resulted in a -5.3% group mean. This value is slightly over the limit given in the relevant OECD guideline. However, the other individual body weight data of this group were in the acceptable range. Therefore, it is considered that this is not treatment-related and this change was considered as individual variability.

Individual body weights for all animals with group means

Animal number	Identity number	Test group name	Initial body weight (g)	Terminal body weight (g)	Change (%) ^#
5140	1	Negative (vehicle) control	21.2	20.8	-1.9
5146	2		21.0	20.5	-2.4
5150	3		20.6	21.6	4.9
5145	4		20.9	21.2	1.4
		Mean	20.9	21.0	0.5
5152	5	10% w/v test material	21.6	17.6	-18.5
5151	6		21.0	17.0	-19.0
5138	7		20.7	17.7	-14.5
5156	8		20.4	16.1	-21.1
		Mean	20.9	17.1	-18.3
5141	9	5% w/v test material	21.4	18.4	-14.0
5147	10		21.0	20.0	-4.8
5157	11		20.6	20.5	-0.5
5159	12		21.5	21.1	-1.9
		Mean	21.1	20.0	-5.3
5139	13	2.5% w/v test material	21.5	21.2	-1.4
5158	14		21.1	20.7	-1.9
5148	15		20.4	19.6	-3.9
5136	16		20.1	20.3	1.0
		Mean	20.8	20.5	-1.6
5153	17	1% w/v test material	21.0	19.4	-7.6
5142	18		21.3	21.5	0.9
5155	19		20.3	21.9	7.9
5144	20		20.8	21.3	2.4
		Mean	20.9	21.0	0.9
5143	21	Positive control	21.5	21.0	-2.3
5154	22		21.4	21.7	1.4
5137	23		20.6	21.0	1.9
5149	24		20.2	21.4	5.9
		Mean	20.9	21.3	1.7

# = (terminal body weight - initial body weight) / initial body weight x 100

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of the study, the test material was shown to have no sensitisation potential in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test material was assessed following dermal exposure in mice in line with standardised guidelines OECD 429 and EU Method B.42, under GLP conditions. Formulations at 25% (w/v), 10% (w/v), 5% (w/v), 2.5% (w/v) and 1% (w/v), using DMF, as a vehicle were used in the test; the formulations appeared to be solutions by visual examination. The preliminary irritation/toxicity test was performed in mice using two doses (2 animals/dose) of 25% and 10%. Based on the observations recorded in the preliminary test, the 10% dose was selected as the top dose for the main test. In the main test, 24 female mice were allocated to six groups of four animals each: four groups received the test material at concentrations of 10%, 5%, 2.5% and 1%, the negative control group received the vehicle (DMF) only and the positive control group received 25% w/v HCA (dissolved in DMF). The test material was applied on the dorsal surface of the animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality was observed during the main study. Test material precipitate or minimal amount of test material precipitate was observed on the ears of all experimental animals at 5% and 2.5% on Days 1-3 and at 1% on Day 3. Marked body weight loss (≥ 5%) was observed for one animals of the 5% dose group and for one animal of the 1% dose group. The mean body weight loss was slightly higher than 5% in case of the 5% dose group. However, these changes were considered as individual variability. Excessive body weight loss in the 10% dose group was observed in the main study. Therefore, the 10% dose group was excluded from the evaluation of cell proliferation in this study. The stimulation index values were 1.4, 2.3 and 2.5 and concentrations of 5%, 2.5% and 1%, respectively. The result of the positive control substance confirmed the validity of the study.

Under the conditions of the study, the test material was shown to have no sensitisation potential in the Local Lymph Node Assay and the substance does not require classification as a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the test material was assessed following dermal exposure in mice in line with standardised guidelines OECD 429 and EU Method B.42, under GLP conditions. Formulations at 25% (w/v), 10% (w/v), 5% (w/v), 2.5% (w/v) and 1% (w/v), using DMF, as a vehicle were used in the test; the formulations appeared to be solutions by visual examination. The preliminary irritation/toxicity test was performed in mice using two doses (2 animals/dose) of 25% and 10%. Based on the observations recorded in the preliminary test, the 10% dose was selected as the top dose for the main test. In the main test, 24 female mice were allocated to six groups of four animals each: four groups received the test material at concentrations of 10%, 5%, 2.5% and 1%, the negative control group received the vehicle (DMF) only and the positive control group received 25% w/v HCA (dissolved in DMF). The test material was applied on the dorsal surface of the animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality was observed during the main study. Test material precipitate or minimal amount of test material precipitate was observed on the ears of all experimental animals at 5% and 2.5% on Days 1-3 and at 1% on Day 3. Marked body weight loss (≥ 5%) was observed for one animals of the 5% dose group and for one animal of the 1% dose group. The mean body weight loss was slightly higher than 5% in case of the 5% dose group. However, these changes were considered as individual variability. Excessive body weight loss in the 10% dose group was observed in the main study. Therefore, the 10% dose group was excluded from the evaluation of cell proliferation in this study. The stimulation index values were 1.4, 2.3 and 2.5 and concentrations of 5%, 2.5% and 1%, respectively. The result of the positive control substance confirmed the validity of the study.

Under the conditions of the study, the test material was shown to have no sensitisation potential in the Local Lymph Node Assay and the substance does not require classification as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin sensitisation.