3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-21 to 2019-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test”, May 30, 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm; Version 07/11/2014

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

EC Number:

202-928-3

EC Name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Cas Number:

101-25-7

Molecular formula:

C5H10N6O2

IUPAC Name:

3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Test material form:

solid

Details on test material:

- Name: 3,7-Dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- CAS: 101-25-7
- Batch: 458231 GDSK
- MW: 186.17 g/mol
- Purity: 95.5 %
- Appearance: beige solid
- Stability: instable after repeated contact to light; undergoes hydrolysis at acidic pH
- Expiry Date: 31 July 2019
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes (NHEK)

Vehicle:

water

Remarks:

test item was moistened to ensure contact

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg / 25 µL H2O

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h MTT incubation

Number of replicates:

2 replicates for each treatment period

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

3 min treatment

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

5.5

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

60 min treatment

Value:

100.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

4.2

Other effects / acceptance of results:

Test Acceptance Criteria
Value Cut off pass/fail
Mean Absolute OD570 nm NK
(3 min Experiment) 1.609 0.8 ≤ NK ≤ 2.8 pass
Mean Absolute OD570 nm NK
(60 min Experiment) 1.670 0.8 ≤ NK ≤ 2.8 pass
Mean Relative Tissue Viability
[%] of PC (60 min experiment) 1.5 < 15% pass
CV [%] (in the range of 20 – 100% viability) 7.8% - 20.1% ≤ 30% pass

Any other information on results incl. tables

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 25 mg test item per 300 μL Aqua dest. and per 90 μL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (103.4%) after 3 min treatment and ≥ 15% (100.2%) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.808, 1.738). The mean relative tissue viability (% negative control) of the positive control was < 15% (4.2%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (1.3% - 2.6%).

Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.768	1.808	0.138	0.148	1.947	1.745
	1.758	1.853	0.133	0.154	1.964	1.803
	1.801	1.857	0.132	0.150	1.953	1.787
Mean Absolute OD570	1.808****		0.143		1.867
OD570- Blank Corrected	1.723	1.763	0.093	0.103	1.902	1.700
	1.713	1.808	0.088	0.108	1.919	1.758
	1.756	1.812	0.087	0.105	1.908	1.742
Mean OD570of 3 Aliquots (Blank Corrected)	1.731	1.794	0.089	0.105	1.909	1.734
SD OD570 of 3 Aliquots	0.022	0.027	0.003	0.003	0.009	0.030
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.762*		0.097		1.821
SD OD570 of 2 Replicate Tissues	0.045		0.011		0.124
Mean Relative Tissue Viability [%]	100.0		5.5		103.4
Coefficient Of Variation [%]^***	2.6		11.7		6.8

Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.684	1.732	0.125	0.107	1.685	1.788
	1.751	1.764	0.122	0.103	1.718	1.788
	1.735	1.764	0.129	0.112	1.684	1.787
Mean Absolute OD570	1.738****		0.116		1.742
OD570- Blank Corrected	1.639	1.687	0.080	0.062	1.640	1.743
	1.706	1.718	0.077	0.057	1.672	1.743
	1.690	1.719	0.084	0.067	1.639	1.741
Mean OD570of 3 Aliquots (Blank Corrected)	1.678	1.708	0.080	0.062	1.650	1.742
SD OD570 of 3 Aliquots	0.035	0.018	0.004	0.005	0.019	0.001
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.693*		0.071		1.696
SD OD570 of 2 Replicate Tissues	0.021		0.013		0.065
Mean Relative Tissue Viability [%]	100.0		4.2**		100.2
Coefficient Of Variation [%]^***	1.3		18.1		3.8

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.

Executive summary:

In the present study the test item was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 25 mg test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (103.4%) after 3 min treatment and ≥ 15% (100.2%) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD_570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.808, 1.738). The mean relative tissue viability (% negative control) of the positive control was < 15% (4.2%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (1.3% - 2.6%).