2,2'-azobis[2-methylbutyronitrile]

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done according to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dimethyl-2,2'-azodipropiononitrile

EC Number:

201-132-3

EC Name:

2,2'-dimethyl-2,2'-azodipropiononitrile

Cas Number:

78-67-1

Molecular formula:

C8H12N4

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylpropanenitrile)

Details on test material:

Identity: 2,2‘-dimethyl-2,2’-azodipropiononitrile
Chemical name: 2,2‘-dimethyl-2,2’-azodipropiononitrile

AZDN Batch No.: 6867
Aggregate state at room temperature: solid (powder)
Color: white
Purity: 99.9 %
Stability in solvent: not determined by the sponsor.
Log Kow: 1.1 (at 25°C)
Storage: 2-8 °C in the fridge, light protected
Molecular weight: 164.2 g/mole
Expiry date: December, 2010

Radiolabelling:

no

Test animals

Species:

other: human skin

Strain:

other:

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not Applicable; in vitro study

Administration / exposure

Type of coverage:

other: non occluded

Vehicle:

unchanged (no vehicle)

Duration of exposure:

24 hours

Doses:

5 mg of the prepared test item were applied on the skin in the respective donor chamber and were also used as the reference value for calculating the mass balance and the percentage of penetration.

No. of animals per group:

Not Applicable; in vitro study

Control animals:

no

Details on study design:

Design of the Study

The test item was analysed in two independent experiments with 6 replicates each under static conditions. The volume of each sample was determined by measuring the weight difference of the vessels used for sampling. After checking the skin integrity, the test item was left on the skin for 24 hours under non-occluded conditions in a practice relevant manner. Then the test item was washed off with ACN. After the penetration experiment, some of the skin samples were stripped 10 (2 x 5 times) with a 1 cm2 piece of Tesa® film (see 8.11, page 18). The stripes of Tesa® film were extracted. The remaining skin samples were separated by heat separation in epidermis and dermis and each skin compartment was extracted separately.

The amount of AZDN was determined in the receptor solutions of the different time points, in the different pipette washing solutions, in the combined washing solutions used to remove the test item formulation together with the solutions removed from the donor chambers after 24 hours (WL+SN), and in the skin membrane extracts as weil as in the reference solutions (REF, one per chamber).

Controls with benzoic acid and 2-ethylhexyl trans-4-methoxycinnamate were used to check the performance of the skin penetration system at least once a year. For the data of the latest control run see annex 5.

Details on in vitro test system (if applicable):

Human skin was provided dermatomized by Biopredic, Rennes (France). According to the guidelines cited whole skin free of any adipose tissue was used. The surface of the skin in contact to the test item was 1.0 cm². For two chambers the skin of one donor was used in order to assure that the study has included four donors.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Percutaneous absorption

Dose:

5 mg

Remarks on result:

other: 24h

Remarks:

AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480 ± 0.220 µg/cm2 (0.010 ± 0.005 % of applied dose).

Any other information on results incl. tables

Summary of the Method Validation

 The following limits and aspects were tested :

 Linear Range:    From 0.505 up to 1004 ng/mL for AZDN (reference material),validated by analysing calibration series in receptor solution and in extraction solution.

 Accuracy:  The accuracy expressed as recovery was between 89.6% and 106% in receptor solution and between

91.3% and 101% in extraction solution.

 Precision:  The intra-day precision in receptor solution expressed as  CV  was between 1.85%  and 8.80%  and in extraction solution between 1.35% and 12.1% over the whole calibration range.

Lower Limit of Quantitation:    0.5 ng/mL

Limit of Detection:    0.25 ng/mL

Specificity/Selectivity:  The chromatographic system showed no injection carry- over and no change in retention time.

Stability in Matrix:  The stability for AZDN is granted up to 24 hours in both receptor solution and extraction solution and AZDN can be stored by freezing (determination after 24 hours at room temperature, in the auto sampler (24 hours at 8°C) and after one freeze/thaw cycle (at-20°C)).

Integrity of theSkin

 

The integrity of the skin was demonstrated prior to application and after the last sampling. The conductivity prior to the experiment was in the acceptable range of <900 µS/cm for all skin samples used.

Summary of results for the test item of all chambers measured

 

Experiment 1
	Chamber
	1	2	3	4	5	6
Amount of AZDN applied (µg/cm2)1	4912	4957	4786	4897	4925	5024
Total amount of AZDN measured (µg)	5260	5248	5250	4844	5401	4992
Recovery(%)	105	103	105	96.7	109	97.5
Total absorption of AZDN (µg/cm2)2	0.542	0.373	3.695	0.958	0.498	0.566
Total absorption (%)3	0.011	0.008	0.077	0.020	0.010	0.011
Experiment 2
	Chamber
	1	2	3	4	5	6
Amount of AZDN applied (µg/cm2)1	4948	4752	4680	4737	4901	3483
Total amount of AZDN measured (µg)	4692	4393	4394	2991	4498	4795
Recovery(%)	91.5	87.0	86.7	60.7	88.7	97.1
Total absorption of AZDN (µg/cm2)2	0.423	0.249	0.353	1.689	0.192	0.649
Total absorption (%)3	0.009	0.005	0.008	0.036	0.004	0.019

1: amount of AZDN present in 5 mg test item

2:the value is the sum of the amount of AZDN measured in the receptor solution and in the skin extract

(epidermis and dermis) of each diffusion cell.

3:percent total absorption=(total amount of AZDN penetrated/absorbed (µg/cm2)*100)/ amount of applied

AZDN (µg)

Grey shading indicates chambers not used for further calculations.

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable Human skin layers and the receptor fluid out of the test item dilution with 0.480 ±0.220 µg/cm2 (0.010 ± 0.005 % of applied dose).

Executive summary:

The test item AZDN (CAS No. 78 -67 -1)was assessed for its potential to permeate human skin in a GLP guideline study (OECD 428). AZDN was the measured compound. For the test item 12 replicates were analysed. 5 mg of the test item were applied to each chamber for 24 hours and then removed by washing each human skin sample nine times with 1 mL extraction solution. PBS was used as the receptor fluid. The conductivity across the skin samples of each chamber was measured before treatment and after the sampling. No abrupt change in conductivity, indicating a loss of barrier properties of the skin, occurred in any chamber up to the maximal duration of the experiment, except for chamber 3 in experiment 1, where removal of the upper part of the skin could be observed after the experiment. The samples were analysed by LC-MS/MS for the presence of AZDN. For the test item two experiments with 6 replicates each were performed under non-occluded conditions. Two chambers did not meet the acceptance criteria (chamber 3 in experiment 1; chamber 4 in experiment 2) and, therefore, 10 chambers were used for the analysis of AZDN. In total 4 donors were used in this study. In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480 ± 0.220 µg/cm² (0.010 ± 0.005 % of applied dose).     

 

	AZDN
Amount of AZDN	Expressed as µg/cm^2of skin surface mean±S.D. (n=10)#			Expressed as % of dose mean±S.D. (n=10)#
Amount applied	4748	±	456	100	±
Washing + SN solution	4552	±	624	95.8	±	8.04
Stratum corneum (isolated by stripping) + EXR	0.013	±	0.015	0.000	±	0.000
Epidermis (isolated after 24 hour)	0.004	±	0.005	0.000	±	0.000
Dermis (isolated after 24 hours)	0.026	±	0.023	0.001	±	0.001
Receptor fluid	0.451	±	0.206	0.010	±	0.005
Recovery	4852	±	368	96.2	±	7.69
Bioavailable portion (receptor fluid + epidermis + dermis)	0.480	±	0.220	0.010	±	0.005
SN = Solution of the donor chamber EXR = Exterior region of the skin

 

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480 ± 0.220 µg/cm² (0.01 0± 0.005 % of applied dose).