2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. October 2016 - 28 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

EC Number:

239-593-8

EC Name:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

Cas Number:

15545-97-8

Molecular formula:

C16H28N4O2

IUPAC Name:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

impurity 1

Reference substance name:

IUPAC-name not available, the reference substance may consist of one or more impurities

Molecular formula:

not applicable

IUPAC Name:

IUPAC-name not available, the reference substance may consist of one or more impurities

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TY2013
- Expiration date of the lot/batch: 13.01.2017
- Purity test date: not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the freezer (-15 - -25 °C) protected from light
- Stability under test conditions: not stated
- Solubility and stability of the test substance in the solvent/vehicle: not stated by sponsor,
a solubility experiment was performed to determine vehicle and dose selection
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was placed into an appropriate container on a tared balance and PG (propylene glycol) was added (weight per weight). The test item was slightly crushed with a spatula, since sonicating alone could not formulate the test item into a fine suspension.
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not determined
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.7 - 20.1 g
- Housing: group
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none reported, Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5% 10%, 25%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: tested and used for dose selection
- Irritation: tested and used for dose selection
- Systemic toxicity: tested and used for dose selection
- Ear thickness measurements: tested and used for dose selection
- Erythema scores: determinde and used for dose selection

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin Sensitisation: Local Lymph Node Assay
- Criteria used to consider a positive response: 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and PG (propylene glycol) was added (weight per weight). The test item was slightly crushed with a spatula, since sonicating alone could not formulate the test item into a fine suspension.
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter ca. 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).

Positive control substance(s):

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

Statistics:

none applied

Results and discussion

Positive control results:

Results of the GLP Positive Control (October 2016): EC3 = 8.2% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

all tested concentrations resulted in SI values below 3

Results 2

Key result

Parameter:

SI

Value:

1

Variability:

lymph nodes were pooled, no individual values available

Test group / Remarks:

1: vehicle control (0%)

Results 3

Key result

Parameter:

SI

Value:

1.69

Variability:

lymph nodes were pooled, no individual values available

Test group / Remarks:

2: low dose (5%)

Results 4

Key result

Parameter:

SI

Value:

1.44

Variability:

lymph nodes were pooled, no individual values available

Test group / Remarks:

3: medium dose (10%)

Results 5

Key result

Parameter:

SI

Value:

1.46

Variability:

lymph nodes were pooled, no individual values available

Test group / Remarks:

4: high dose (25%)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

see table in "any other information on results incl. tables"

DETAILS ON STIMULATION INDEX CALCULATION

The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION

Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot (S.I. against concentration).

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were observed during the study period. From day 2 to 5, the animals treated with test item concentrations of 10 and 25% showed an erythema of the ear skin (Score 1). Animals treated with 5% test item concentration did not show any signs of local skin irritation.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG	number of lymph nodes	DPM per lymph node	S.I.
-	BG I	18	-	-	-	-
-	BG II	17	-	-	-	-
0	1	4027	4009.5	8	501.2	1.00
5	2	6799	6781.5	8	847.7	1.69
10	3	5780	5762.5	8	720.3	1.44
25	4	5870	5852.5	8	731.6	1.46

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item 2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) formulated in propylene glycol (PG) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25%. The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 to 5, the animals treated with a test item concentration of 10 and 25% showed an erythema of the ear skin (Score 1). Animals treated with 5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 1.69, 1.44, and 1.46 were determined with the test item at concentrations of 5, 10, and 25% in PG, respectively.

The test item 2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) was not a skin sensitiser under the test conditions of this study.