N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

EC Number:

227-563-7

EC Name:

N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

Cas Number:

5888-87-9

Molecular formula:

C20H34N4O4

IUPAC Name:

N,N'-hexane-1,6-diylbis(2-oxoazepane-1-carboxamide)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): BRUGGOLEN M12 / N, N’-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)
- Substance type: organic
- Physical state: solid
- Analytical purity: > 99.5%
- Impurities (identity and concentrations): not specified
- Lot/batch No.: 12030701
- Expiration date of the lot/batch: 30 November 2013
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1970, 1130, 643, 368, 210, 120, 68.6, 39.2, 22.4 and 12.8 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Evaluation criteria:

A test item will be considered as clearly positive in this assay, if the following criteria are met:
(i)
Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
(ii)
The proportion of micronucleated cells at such data points exceeds the normal range. If the increases fall within the range of values, normally observed in the negative control cultures, the test item cannot be classified as positive. Any significant increases over the concurrent negative controls will therefore be compared with historical control values derived from recent studies.
(iii) There is a significant dose-relationship.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses, seen only at high levels of cytotoxicity, will require careful interpretation when assessing their biological significance.

Statistics:

The numbers of binucleated cells with micronuclei in the control and treated cultures will be compared using an appropriate statistical method. The solvent controls will be used as the reference point for comparison in the statistical evaluation and the evaluation of the results.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity above 253 ug/mL in the absence and presence of S9 metabolism.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment with the test item, no remarkable variation of pH over the control values was observed in the absence or presence of S9 metabolism.
- Effects of osmolality: Following treatment with the test item, no remarkable variation of osmolality over the control values was observed in the absence or presence of S9 metabolism.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 

		Presence of S9 metabolism				Absence of S9 metabolism
Treatment	Dose level
	µg/mL	%Mn cells		Sig.	%Cytotoxicity	%Mn cells		Sig.	%Cytotoxicity
Untreated	0.0	1.00			15	0.60			3
Solvent 1%	0.0	1.05			0	0.60			0
Test item	176	-			-	0.65	NS		22
Test item	211	1.35	NS		18	1.10	NS		22
Test item	253	11.0	***		35	3.60	***		58
Test item	311	13.1	***		64	-	-		-
Mitomycin-C	0.300	-	-		-	5.70	***		19
Colchicine	2.50	-	-		-	8.90	***		§
Cyclophosphamide	10.0	5.80	***		52	-			-

Key:

 

%Mn cells                                                 : Percentage of cells bearing micronuclei

Sig.          : Statistical significance

NS           : Not significant

-              : Not tested or not selected for scoring

*            : Statistically significant at p<0.05

**           : Statistically significant at p<0.01

***         : Statistically significant at p<0.001

§             : not calculated - induction of multinucleation was attributable to multipolar division as a

                 consequence of induction of multipolar spindles

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

annex VIII, 8.4

Executive summary:

On the basis of the results obtained, it is concluded that BRUGGOLEN M12 induces micronuclei in Chinese hamster V79 cells after in vitro treatment in the absence and presence of S9 metabolic activation, under the reported experimental conditions.

Therefore, according to ECHA's guidance R.7a "Endpoint specific guidance", no second in vitro testing has to be performed but a testing proposal for an in vivo testing (REACH Annex IX) has to be submitted (see section 7.6.2).