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

Endpoint summary

Description of key information

Skin Irritation

REACH_not corrosive | EpiDerm | OECD 431 | #key study#

REACH_not irritating | EpiDerm | OECD 439 | #key study#

Eye Irritation

REACH_not irritating | BCOP | OECD 437 | #key study#

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

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

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 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

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

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

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

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%).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-26 to 2019-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

Version 07-Nov-2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco’s phosphate buffered saline

Details on test system:

This in vitro method is designed to predict and classify the skin irritation potential of a chemical by assessment of its effect on EpiDermTM, a reconstituted three-dimensional human epidermis model. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

1. Negative control: 30 µL Dulbecco’s phosphate buffered saline
2. Positive control: 30 µL 5% sodium dodecyl sulfate solution
3. Test Item: 25 mg + 25 µL Dulbecco’s phosphate buffered saline

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Details on study design:

The test was performed on EpiDerm, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (30 µL DPBS) and the positive control (30 µL 5% SDS), respectively. After 60 ± 1 min treatment period at 37°C (35 min) and room temperature (25 min) the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 2 h. Then the tissues are stained via MTT for 3 hours. The extracts are measured photometrically at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

60 min treatment

Value:

87.1

Vehicle controls validity:

valid

Remarks:

100.0

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

3.7

Other effects / acceptance of results:

Test Acceptance Criteria
The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

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

The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.Therefore, NSC equalled 0%.

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 irritant effects. The mean relative tissue viability (% negative control) was >50% (87.1%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (2.226). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 2.4%).

Result of the test item:

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	2.194	2.189	2.240	0.137	0.115	0.127	1.942	1.875	1.996
	2.186	2.264	2.285	0.130	0.122	0.137	1.941	1.911	2.002
Mean Absolute OD570	2.226****			0.128			1.944
OD570(Blank Corrected)	2.148	2.142	2.193	0.091	0.068	0.080	1.896	1.828	1.949
	2.140	2.217	2.239	0.083	0.075	0.090	1.895	1.864	1.956
Mean OD570of the Duplicates (Blank Corrected)	2.144	2.180	2.216	0.087	0.072	0.085	1.895	1.846	1.953
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	2.180*			0.081			1.898
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.036			0.008			0.053
Relative Tissue Viability [%]	98.4	100.0	101.7	4.0	3.3	3.9	86.9	84.7	89.6
Mean Relative Tissue Viability [%]	100.0			3.7**			87.1
SD of Relative Tissue Viability [%]***	1.7			0.4			2.4
CV [% Viabilities]	1.7			10.4			2.8

^*          Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is ≤ 20%.

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the test item was applied topically to the EpiDerm^TM tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

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

The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.Therefore, NSC equalled 0%.

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 irritant effects. The mean relative tissue viability (% negative control) was > 50% (87.1%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (2.226). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 2.4%).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-10 to 2019-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 09 October 2017)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Species:

other: isolated bovine corneas

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was ground to a powder in a mortar with pestle and subsequently suspended with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 1801168, expiry date: 12/2020) to give a 20% concentration.

Duration of treatment / exposure:

After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

Duration of post- treatment incubation (in vitro):

90 min fluorescein incubation

Number of animals or in vitro replicates:

3 per test group

Details on study design:

CALIBRATION OF THE OPACITOMETER
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

TREATMENT OF THE CORNEAS
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

4 h treatment

Value:

0.52

Vehicle controls validity:

valid

Remarks:

0.34

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

84.28

Irritation parameter:

cornea opacity score

Remarks:

mean corrected value

Run / experiment:

4 h treatment

Value:

0.53

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

57.81

Irritation parameter:

other: Permeability

Remarks:

mean OD490

Run / experiment:

4 h treatment

Value:

0.011

Vehicle controls validity:

valid

Remarks:

0.012

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

1.777

Interpretation of results:

GHS criteria not met

Conclusions:

According to the evaluation criteria the test item is classified into UN GHS "No Category".

Executive summary:

Summary Results

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item: The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.

Visual Observation after treatment:  None of the corneas showed any opacity of the tissue.

Mean in vitro irritation score: 0.52

X	UN GHS No Category
	No prediction can be made
	UN GHS Category 1

Classification

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Conclusion

According to the evaluation criteria the test item is classified into UN GHS "No Category".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion

The skin corrosivity potential of DNPT was analysed in vitro in an OECD 431 using the human skin model EpiDerm. The test item showed no corrosive effects. The mean relative tissue viability was ≥ 50% (103.4%) after 3 min treatment and ≥ 15% (100.2%) after 60 min treatment. 

An in vitro skin irritation test (OECD 431) with human skin model EpiDerm was carried out with DPNT. The test item showed no irritant effects. The mean relative tissue viability was > 50% (87.1%) after 60 min treatment and 42 h post-incubation.

According to the CLP criteria the DNPT does not need to be classified for skin irritation/corrosion.

Eye Irritation/corrosion

The eye irritancy potential of DNPT was investigated in the bovine corneal opacity and permeability assay (OECD 437). The mean in vitro irritation score was 0.52. According to the CLP criteria the DNPT does not need to be classified for eye irritation/corrosion.