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

Endpoint summary

Description of key information

REACH_positive | DPRA | OECD 442C | #key study#

REACH_negative | KeratinoSens | OECD 442D | #key study#

REACH_positive | h-CLAT | OECD 442E | #key study#

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-08 to 2019-05-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.
Since activation of the Keap1-Nrf2-ARE pathway addresses only the second key event of the skin sensitisation AOP, information from test methods based on the activation of this pathway is unlikely to be sufficient when used on its own to conclude on the skin sensitisation potential of chemicals. Therefore data generated according to OECD 442D should be considered in the context of integrated approaches, such as IATA, combining them with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2,78 (experiment 1); 3,28 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Remarks:

I max

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 62.50 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

97.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 62.50 µM

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Remarks:

I max

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 62.50 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

98.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 62.50 µM

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

All acceptance criteria were fullfilled proving the validity of the test.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 μM onwards the test item was cytotoxic.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 μM onwards the test item was cytotoxic.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non-sensitiser.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2		Mean	SD
Solvent Control	-	100	100		100	0.0
Positive Control	4.00	99.5	97.1		98.3	1.7
	8.00	103.7	101.6		102.6	1.5
	16.00	106.3	101.9		104.1	3.1
	32.00	104.8	105.6		105.2	0.5
	64.00	107.8	103.5		105.6	3.1
Test Item	0.98	97.4	109.9		103.7	8.9
	1.95	96.4	106.2		101.3	6.9
	3.91	97.1	102.1		99.6	3.5
	7.81	95.0	99.8		97.4	3.4
	15.63	96.0	104.5		100.3	6.0
	31.25	92.1	98.2		95.1	4.3
	62.50	97.8	98.6		98.2	0.6
	125.00	0.3	0.1		0.2	0.2
	250.00	2.6	2.2		2.4	0.3
	500.00	1.7	0.4		1.1	0.9
	1000.00	0.3	0.3		0.3	0.0
	2000.00	-0.1	0.3		0.1	0.3

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.11	1.12	1.13	0.04
	8.00	1.33	1.62	1.22	1.39	0.21
	16.00	1.44	1.36	1.22	1.34	0.11
	32.00	1.70	1.93	1.54	1.72	0.20	*
	64.00	2.79	3.07	2.47	2.78	0.30	*
Test Item	0.98	1.03	1.10	1.14	1.09	0.06
	1.95	0.96	1.07	1.03	1.02	0.05
	3.91	1.00	1.05	0.94	1.00	0.06
	7.81	0.98	1.05	0.98	1.00	0.04
	15.63	1.01	1.00	0.94	0.98	0.04
	31.25	1.11	1.20	0.99	1.10	0.10
	62.50	1.08	1.19	1.18	1.15	0.06
	125.00	0.11	0.12	0.11	0.11	0.01
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.35	1.22	1.94	1.50	0.39
	8.00	1.33	1.49	1.29	1.37	0.10
	16.00	1.66	1.87	1.60	1.71	0.14	*
	32.00	2.29	1.85	2.24	2.13	0.24	*
	64.00	3.57	3.46	2.80	3.28	0.42	*
Test Item	0.98	1.16	1.32	0.88	1.12	0.22
	1.95	1.11	0.91	0.88	0.97	0.12
	3.91	0.88	0.92	0.87	0.89	0.03
	7.81	0.91	0.99	0.82	0.91	0.09
	15.63	0.90	0.90	0.84	0.88	0.04
	31.25	0.93	0.88	0.88	0.90	0.03
	62.50	1.09	1.25	1.04	1.13	0.11
	125.00	0.01	0.00	0.01	0.01	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.13	1.50	1.32	0.26
	8.00	1.39	1.37	1.38	0.01
	16.00	1.34	1.71	1.53	0.26
	32.00	1.72	2.13	1.93	0.28
	64.00	2.78	3.28	3.03	0.35
Test Item	0.98	1.09	1.12	1.11	0.02
	1.95	1.02	0.97	0.99	0.04
	3.91	1.00	0.89	0.95	0.07
	7.81	1.00	0.91	0.96	0.07
	15.63	0.98	0.88	0.93	0.07
	31.25	1.10	0.90	1.00	0.15
	62.50	1.15	1.13	1.14	0.02
	125.00	0.11	0.01	0.06	0.07
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p<0.05

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5	n.a.	n.a.	n.a.	n.a.
Imax	1.15	1.13	1.14	0.02
IC30	80.31	80.64	80.48	0.24
IC50	93.14	93.33	93.23	0.14
IC70	105.96	106.02	105.99	0.04

n.a. = not applicable

Interpretation of results:

other: Negative in the KeratinoSens Assay

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 186.17 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. From the concentration 125 µM onwards the test item was cytotoxic.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated. From the concentration 125 µM onwards the test item was cytotoxic.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-15 to 2019-06-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
In the present study 3,7-Dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane was dissolved in acetonitrile, based on the results of the pre-experiments.
Based on a molecular weight of 186.17 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.24%.

Key result

Run / experiment:

other: Prediction Model 1 (Cysteine/Lysine Peptide)

Parameter:

other: Mean Peptide Depletion [%]

Value:

21.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

66.24

Remarks on result:

positive indication of skin sensitisation

Remarks:

Low Reactivity

Run / experiment:

other: Cysteine run

Parameter:

other: Mean Peptide Depletion [%]

Value:

39.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

69.79

Run / experiment:

other: Lysine run

Parameter:

other: Mean Peptide Depletion [%]

Value:

4.37

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

62.70

Other effects / acceptance of results:

Acceptance Criteria for Cysteine Peptide
- coefficient of determination R² > 0.99 0.9999 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5069 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.5038 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.5038 pass
- CV of the peak area of RC B < 15% 0.54 pass
- CV of the peak area of RC C (PC) < 15% 0.65 pass
- CV of the peak area of RC C (TI) < 15% 0.65 pass
- mean peptide depletion of the PC 60.8% < x < 100% 69.79 pass
- SD of peptide depletion of the PC replicates < 14.9% 0.24 pass
- SD of peptide depletion of the TI replicates < 14.9% 0.92 pass

Acceptance Criteria for Lysine Peptide
- coefficient of determination R² > 0.99 1.0000 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5001 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.4977 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.4977 pass
- CV of the peak area of RC B < 15% 0.34 pass
- CV of the peak area of RC C (PC) < 15% 0.44 pass
- CV of the peak area of RC C (TI) < 15% 0.44 pass
- mean peptide depletion of the PC 40.2% < x < 69.0% 62.70 pass
- SD of peptide depletion of the PC replicates < 11.6% 0.18 pass
- SD of peptide depletion of the TI replicates < 11.6% 0.16 pass

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RCCacetonitrile).

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (21.91%). Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.24%.

The controls confirmed the validity of the study for both, the cysteine and lysine run

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.7390	0.5340	16.2710	0.5340
STD2	9.0140	0.2670	8.1100	0.2670
STD3	4.5420	0.1335	4.0200	0.1335
STD4	2.2270	0.0667	2.0340	0.0667
STD5	1.0700	0.0334	1.0090	0.0334
STD6	0.5090	0.0167	0.4970	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.1220	0.1534	69.51	69.79	0.24	0.35
	5.0570	0.1515	69.90
	5.0470	0.1512	69.96
Test Item	10.2960	0.3086	38.71	39.45	0.92	2.33
	10.2210	0.3064	39.16
	9.9990	0.2997	40.48

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.6490	0.1858	62.72	62.70	0.18	0.29
	5.6810	0.1869	62.51
	5.6270	0.1851	62.87
Test Item	14.4670	0.4752	4.53	4.37	0.16	3.62
	14.5150	0.4768	4.21
	14.4910	0.4760	4.37

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	21.91	Low Reactivity	positive	39.45	Moderate Reactivity	positive
Positive Control	66.24	High Reactivity	positive	69.79	Moderate Reactivity	positive

Interpretation of results:

other: Positive in the DPRA Assay

Conclusions:

In this study under the given conditions the test item showed low reactivity towards both peptides. The test item is considered as “sensitiser”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study the test item was dissolved in acetonitrile, based on the results of the pre-experiments.

Based on a molecular weight of 186.17 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_acetonitrile).

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (21.91%). Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.24%.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-10 to 2019-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The h-CLAT is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP) for skin sensitisation, which is the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers.
However, as DC activation represents only one key event of the skin sensitisation AOP, information generated with test methods measuring markers of DC activation alone may not be sufficient to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore, data generated with the test methods described in the Guideline are proposed to support the discrimination between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated Approaches to Testing and Assessment (IATA).

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments.
The threshold of 150% for CD86 (380% experiment 1; 293% experiment 2) and 200% for CD54 (577% experiment 1; 471% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1 [Concentration: 73.48 µg/mL]

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

335

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1 [Concentration: 73.48 µg/mL]

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2 [Concentration: 51.03 µg/mL]

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

192

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2 [Concentration: 42.53 µg/mL]

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

110

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were fullfilled proving the validity of the test.

Severe cytotoxic effects were observed for the cells treated with the highest test item concentrations. Relative cell viability at the highest test item concentration was reduced to 18.8% (CD86), 17.2% (CD54) and 15.4% (isotype IgG1 control) in the first experiment and to 20.1% (CD86), 17.7% (CD54) and 14.9% (isotype IgG1 control) in the second experiment. At a concentration of 73.48 μg/mL for main experiment 1 and concentration of 51.03 μg/mL for main experiment 2 the cells were viable.

The expression of the cell surface marker CD86 was upregulated to 335% in the first and 192% in the second experiment. The upregulation above the threshold of 150% was observed at a concentration of 73.48 μg/mL in the first experiment and at a concentration of 51.03 μg/mL in the second experiment. The expression of the cell surface marker CD54 was upregulated to 200% in the first experiment, it was not upregulated in the second experiment.

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Results of the Cell Batch Activation Test (batch 6)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	85.4	327	>150	85.6	287	>200	yes	pass
NiSO4	100 µg/mL	86.7	251	>150	86.5	269	>200	yes	pass
LA	1000 µg/mL	96.5	76	≤150	96.6	77	≤200	no	pass

Results of the Cell Batch Activation Test (batch 7)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	86.9	422	>150	85.4	647	>200	yes	pass
NiSO4	100 µg/mL	89.1	254	>150	90.0	454	>200	yes	pass
LA	1000 µg/mL	97.9	53	≤150	98.0	70	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	96.30	--	94.70
Solvent Control	DMSO	--	96.80	--	94.50
3,7-Dinitroso-1,3,5,7-tetraacabicyclo[3.3.1]nonane	C8	3.59	96.70	3.59	94.50
	C7	7.19	96.70	7.19	94.80
	C6	14.38	96.50	14.38	94.30
	C5	28.75	96.80	28.75	94.50
	C4	57.50	95.60	57.50	94.10
	C3	115.00	37.30	115.00	40.20
	C2	230.00	44.80	230.00	52.10
	C1	460.00	44.70	460.00	48.40
Calculated CV75 [µg/mL]		73.46		73.51
Mean CV75 [µg/mL]		73.48
SD CV 75 [µg/mL]		0.04

CD54 and CD86 Expression Experiment 1

	Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluores-cence Intensity		Relative Fluores-cence Intensity (RFI)		Ratio Isotype IgG1 to [%]
			CD86	CD54	Iso-type IgG1	CD86	CD54	Iso-type IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Exp. 1	Medium Control	-	96.8	96.2	96.2	1330	1028	611	719	417	83	104	218	168
	Solvent Control	0.20%	96.2	95.8	96.3	1508	1046	645	863	401	100	100	234	162
	DNCB	4.00	82.3	79.9	78.9	3955	2992	679	3276	2313	380	577	582	441
	3,7-Dinitroso-1,3,5,7-tetraaza-bicyclo-[3.3.1] nonane	88.18	18.8	17.2	15.4	2986	1694	1106	1880	588	218	147	270	153
		73.48	75.1	75.3	70.8	3574	1482	681	2893	801	335	200	525	218
		61.24	80.2	79.5	79.3	3363	1289	705	2658	584	308	146	477	183
		51.03	95.0	93.9	93.8	1823	1126	605	1218	521	141	130	301	186
		42.53	95.0	94.8	94.1	1609	1135	590	1019	545	118	136	273	192
		35.44	95.5	95.2	94.1	1576	1117	714	862	403	100	101	221	156
		29.53	95.2	95.5	95.1	1673	1151	568	1105	583	128	145	295	203
		24.61	95.5	95.3	94.5	1621	1166	673	948	493	110	123	241	173

CD54 and CD86 Expression Experiment 2

	Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluores-cence Intensity		Relative Fluores-cence Intensity (RFI)		Ratio Isotype IgG1 to [%]
			CD86	CD54	Iso-type IgG1	CD86	CD54	Iso-type IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Exp. 2	Medium Control	-	97.5	97.9	97.6	1194	902	556	638	346	82	88	215	162
	Solvent Control	0.20%	98.0	97.4	97.9	1348	966	571	777	395	100	100	236	169
	DNCB	4.0	84.9	86.5	87.3	2903	2486	624	2279	1862	293	471	465	398
	3,7-Dinitroso-1,3,5,7-tetraaza-bicyclo-[3.3.1] nonane	88.18	20.1	17.7	14.9	2171	1409	928	1243	481	160	122	234	152
		73.48	18.9	18.6	15.1	2698	1190	873	1825	317	235	80	309	136
		61.24	24.4	22.8	21.4	2922	1245	814	2108	431	271	109	359	153
		51.03	94.3	94.8	94.8	2057	980	562	1495	418	192	106	366	174
		42.53	95.8	96.4	96.8	1406	978	544	862	434	111	110	258	180
		35.44	96.8	96.5	96.4	1432	915	532	900	383	116	97	269	172
		29.53	96.5	96.8	96.2	1377	834	562	815	272	105	69	245	148
		24.61	97.2	97.1	97.0	1360	812	511	849	301	109	76	266	159

Interpretation of results:

other: Positive in the h-CLAT Test

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.

The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 230 mg/mL to 1.80 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 73.48 ± 0.04 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

88.18, 73.48, 61.24, 51.03, 42.52, 35.44, 29.53, 24.61 µg/mL

In the second dose-finding assay, little precipitation of the test item was observed for the highest concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Severe cytotoxic effects were observed for the cells treated with the highest test item concentrations. Relative cell viability at the highest test item concentration was reduced to 18.8% (CD86), 17.2% (CD54) and 15.4% (isotype IgG1 control) in the first experiment and to 20.1% (CD86), 17.7% (CD54) and 14.9% (isotype IgG1 control) in the second experiment. At a concentration of 73.48 µg/mL for main experiment 1 and concentration of 51.03 µg/mL for main experiment 2 the cells were viable.

The expression of the cell surface marker CD86 was upregulated to 335% in the first and to 192% in the second experiment.The upregulation above the threshold of 150% was observed at a concentration of 73.48 µg/mL in the first experiment and at a concentration of 51.03 µg/mL in the second experiment. The expression of the cell surface marker CD54 was upregulated to 200% in the first experiment, it was not upregulated in the second experiment.

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DNPT was submitted to in vitro sensitization testing in an OECD 442C (DPRA), OECD 442D (Keratinosens) and an OECD 442E study (h-CLAT). Positive results were obtained in the DPRA and h-CLAT indicating that there is a potential to covalently interact with proteins and to activate dendritic cells. Furthermore, DNPT is not stable in aqueous solution and at acidic pH, under these conditions DNPT undergoes ring cleavage.

Formation of formaldehyde, ammonia and nitramide is postulated. Under neutral conditions condensation of formaldehyde and ammonia occurs (Scranage J.K., Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/6562/). The so called Mannich reaction between formaldehyde and ammonia results in generation of hexamethylenetetramine (Mannich, C.and Kroesche W.,Archiv der Pharmazie250 (1): 647–667, 1912), which exhibits like formaldehyde skin sensitizing properties. For this reason, DPNT is precautionarily classified as skin sensitizer category 1 based on a Weight of Evidence approach.