(6-aminohexyl)carbamic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-19 to 2017-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(6-aminohexyl)carbamic acid

EC Number:

205-581-6

EC Name:

(6-aminohexyl)carbamic acid

Cas Number:

143-06-6

Molecular formula:

C7H16N2O2

IUPAC Name:

(6-aminohexyl)carbamic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: INTERBUSINESS S.r.l. (Via Spartaco, 2520135 Milano, Italy); batch no. 6061
- Expiration date of the lot/batch: 2018-03-01
- Purity test date: 2016-03-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: prepared immediately before use in culture medium. No assay of test item stability, nor its concentration and homogeneity in solvent were under-taken, as not requested by the Sponsor

OTHER SPECIFICS:
Identity of the test material: INTERCURE®1

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: For the first Main Experiment, two batches of human whole blood, provided by Biopredic International (France), were pooled. For the second Main Experiment, one batch of human whole blood, provided by Biopredic International (France) was used
- Suitability of cells: Not specified
- Sex, age and number of blood donors if applicable: First Experiment: 1) Male (n=1); Age = 23 years; 2) Female (n = 1); Age: 27 Years. Second Experiment: 1) Female (n = 1); Age: 22 Years
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: culture medium (composition shown below) was used for lymphocytes:

RPMI 1640 1x (Dutch modification) 500 mL
Foetal Calf Serum 100 mL
L-Glutamine (200 mM) 6.25 mL
Antibiotic solution 1.25 mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiationof the cultures, medium with the addition of phytohaemagglutin (PHA) was used in thefollowing proportion: 10 mL of PHA was added to 500 mL of medium.
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital – 5,6-Benzoflavone induced Rat liver

Test concentrations with justification for top dose:

General dose selection procedure:
Cytotoxicity was determined for each of the treatment levels and the highest dose level for genotoxicity assessment was selected as a dose which produces approximately 55 ± 5% cytotoxicity compared to solvent control. If the test item does not induce toxicity at any dose level, then the highest treatment level is selected as the highest dose level for scoring.

Two lower dose levels were also selected for the scoring of micronuclei. The lowest dose level for scoring should be on the borderline of cytotoxicity. The intermediate dose level are evenly spaced between the two.

The test item was found to be soluble in culture medium at the concentration of 16.0 mg/mL after few minutes of vortex stirring. This concentration was chosen since, when added to the treatment medium in the ratio 1:10, it gave a maximum dose level of 1600 µg/mL corresponding to 10.0 mMwhich is lower than 2000 µg/mL.

Two main experiments were performed. Based on the preliminary solubility assay, dose levels of 1600, 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 µg/mL were used for the first main experiment. Based on these results, the dose for the main experiment were 1600, 1070 and 711 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test material was solubilized in the culture medium
- Justification for choice of solvent/vehicle: not specified

Controls

Untreated negative controls:

yes

Remarks:

Untreated cultures were included in the experimental scheme and acted as reference contro

Negative solvent / vehicle controls:

yes

Remarks:

culture medium (RPMI 1640, Dutch modification, ob-tained from Gibco, supplemented with fetal calf serum, antiobiotic solution and L-glutamine)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours followed by further incubation for 28 hours (recovery period)
- Expression time (cells in growth medium): 31 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin-B

STAIN (for cytogenetic assays): Acridine Orange in PBS

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease-free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per cell culture (solvent; negative; and positive controls); For cultures treated with Colchicine 1000 mononucleated cells per cell culturewere scored

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for identifying micronuclei were as follows:

1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI)

Rationale for test conditions:

According to OECD Guideline 487.

Evaluation criteria:

Acceptance criteria:

The experiment is considered valid when:

- The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
- Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
- Adequate cell proliferation is observed in solvent control cultures.
- The appropriate number of doses and cells are analysed

Criterion for outcome:

The assay is considered as clearly positive if:

- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
- The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
- There is a significant dose effect relationship.

The assay is considered as clearly negative if:

- None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
- There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
- All the results are inside the distribution of the historical control data.

Statistics:

A modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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, a slight dose related increase of the pH was observed at the highest dose levels in the absence and presence of S9 metabolism
- Effects of osmolality: No remarkable variation of osmolality was observed at any dose level
- Precipitation: Following treatment with the test item, no precipitation or opacity of the medium was observed at beginning or end of treatment, in the absence or presence of S9 metabolism.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See Tables 1-3.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
- Indication whether binucleate or mononucleate where appropriate: Yes, See Tables 1-3.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Data were included in the statistical analysis
- Negative (solvent/vehicle) historical control data: Data were included in the statistical analysis

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of mononucleated, binucleated cells, polynucleated cells

Any other information on results incl. tables

Table 1. Main Experiment 1 – Proliferation Index Treatment: 3 hours - without S9 Sampling Time: 32 hours
Treatment (µg/mL)	Culture No.	MonoN	BiN	PoliN	Total Cells	CBPI	Mean Value	Cytotoxicity
Untreated	1	160	260	80	500	1.840
	2	166	270	64	500	1.796	1.818	0
Test Item 316	11	162	258	80	500	1.836
	12	186	253	61	500	1.750	1.793	3
Test Item 474	9	180	223	97	500	1.834
	10	168	240	92	500	1848	1.841	-3
Test Item 711	7	193	220	87	500	1.788
	8	207	233	60	500	1.706	1.747	9
Test Item 1070	5	182	251	67	500	1.770
	6	182	244	74	500	1.784	1.777	5
Test Item 1600	3	166	238	96	500	1.860
	4	182	237	81	500	1.798	1.829	-1

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control cultures

 

Table 2. Main Experiment 1 – Proliferation Index Treatment: 3 hours - with S9 Sampling Time: 32 hours
Treatment (µg/mL)	Culture No.	MonoN	BiN	PoliN	Total Cells	CBPI	Mean Value	Cytotoxicity
Untreated	23	160	244	96	500	1.872
	24	178	233	89	500	1.822	1.847	0
Test Item 316	33	128	259	113	500	1.970
	34	151	240	109	500	1.916	1.943	-11
Test Item 474	31	137	258	105	500	1.936
	32	135	256	109	500	1.948	1.942	-11
Test Item 711	29	166	223	111	500	1.890
	30	136	240	124	500	1.976	1.933	-10
Test Item 1070	27	176	236	88	500	1.824
	28	146	232	122	500	1.952	1.888	-5
Test Item 1600	25	132	258	110	500	1.956
	26	129	262	109	500	1.960	1.958	-13
Cyclophosphamide 20.0	45	350	143	7	500	1.314
	46	320	167	13	500	1.386	1.350	59
Cyclophosphamide 15.0	47	338	155	7	500	1.338
	48	311	168	21	500	1.420	1.379	55

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control culure

Table 3. Main Experiment 2 – Proliferation Index Treatment: 31 hours - without S9 Sampling Time: 31 hours
Treatment (µg/mL)	Culture No.	MonoN	BiN	PoliN	Total Cells	CBPI	Mean Value	Cytotoxicity
Untreated	49	188	229	83	500	1.790
	50	137	285	78	500	1.882	1.836	0
Test Item 316	59	136	278	86	500	1.900
	60	153	265	82	500	1.858	1.879	-5
Test Item 474	57	138	265	97	500	1.918
	58	147	277	76	500	1.858	1.888	-6
Test Item 711	55	146	270	84	500	1.876
	56	157	263	80	500	1.846	1.861	-3
Test Item 1070	53	203	248	49	500	1.692
	54	178	258	64	500	1.772	1.732	12
Test Item 1600	51	261	202	37	500	1.552
	52	263	220	17	500	1.508	1.530	37
Colchicine 0.080	71	449	41	10	500	1.122
	72	468	27	5	500	1.074	1.098	88
Colchicine 0.040	73	477	20	3	500	1.052
	74	490	10	0	500	1.020	1.036	96
Colchicine 0.020	75	355	122	23	500	1.336
	76	387	93	20	500	1.266	1.301	64

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control cultures

 

Table 4. Main Experiment 1 – Scoring of Micronuclei Treatment: 3 hours - without S9 Sampling Time: 32 hours
Treatment (µg/mL)	Culture No.	Cells scored (BiN cells)	BiN cells with 1 Mn	BiN cells with 2 Mn	BiN cells with >2 Mn	BiN cells with Mn
Untreated	1	1000	2	0	0	2
	2	1000	2	1	0	3
Test Item 711	7	1000	3	0	0	3
	8	1000	1	1	0	2
Test Item 1070	5	1000	5	0	0	5
	6	1000	2	0	0	2
Test Item 1600	3	1000	0	0	0	0
	4	1000	4	0	0	4

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 5. Main Experiment 1 – Scoring of Micronuclei Treatment: 3 hours - with S9 Sampling Time: 32 hours
Treatment (µg/mL)	Culture No.	Cells scored (BiN cells)	BiN cells with 1 Mn	BiN cells with 2 Mn	BiN cells with >2 Mn	BiN cells with Mn
Untreated	23	1000	3	0	0	3
	24	1000	4	0	0	4
Test Item 711	29	1000	2	0	0	2
	30	1000	5	0	0	5
Test Item 1070	27	1000	2	0	0	2
	28	1000	2	0	0	2
Test Item 1600	25	1000	1	0	0	1
	26	1000	3	0	0	3
Cyclophosphamide 20.0	45	1000	22	2	0	24
	46	1000	24	1	1	26

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 6. Main Experiment 2 – Scoring of Micronuclei Treatment: 31 hours - without S9 Sampling Time: 31 hours
Treatment (µg/mL)	Culture No.	Cells scored (BiN cells)	BiN cells with 1 Mn	BiN cells with 2 Mn	BiN cells with >2 Mn	BiN cells with Mn
Untreated	49	1000	3	0	0	3
	50	1000	2	0	0	2
Test Item 711	55	1000	2	0	0	2
	56	1000	3	0	0	3
Test Item 1070	53	1000	3	0	0	3
	54	1000	3	0	0	3
Test Item 1600	51	1000	5	0	0	5
	52	1000	4	0	0	4
Treatment (µg/mL)	Culture No.	Cells scored (MonoN cells)	MonoN cells with 1 Mn	MonoN cells with 2 Mn	MonoN cells with >2 Mn	MonoN cells with Mn
Colchicine 0.040	73	1000	17	4	0	21
	74	1000	22	2	0	24

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 7. Main Experiment 1 – Statistical Analysis Treatment: 3 hours - without S9 Sampling Time: 32 hours
Treatment (µg/mL)	Cells scored (BiN cells)	BiN cells with Micronuclei	χ^2	Significance
Untreated	2000	5
711	2000	5	0.100	N.S.
1070	2000	7	0.084	N.S.
1600	2000	4	0.000	N.S.

BiN: Binucleated cells

N.S. Not significant

 

Table 8. Main Experiment 1 – Statistical Analysis Treatment: 3 hours - with S9 Sampling Time: 32 hours
Treatment (µg/mL)	Cells scored (BiN cells)	BiN cells with Micronuclei	χ^2	Significance
Untreated	2000	7
711	2000	7	0.072	N.S.
1070	2000	4	0.365	N.S.
1600	2000	4	0.365	N.S.
Cyclophosphamide 20.0	2000	50	31.395	***

BiN: Binucleated cells

N.S. Not significant

*** Significant atp<0.001

 

Table 9. Main Experiment 2 – Statistical Analysis Treatment: 31 hours - without S9 Sampling Time: 31 hours
Treatment (µg/mL)	Cells scored (BiN cells)	BiN cells with Micronuclei	χ^2	Significance
Solvent 1%	2000	5
711	2000	5	0.100	N.S.
1070	2000	6	0.000	N.S.
1600	2000	9	0.645	N.S.
Treatment (µg/mL)	Cells scored (MonoN cells)	MonoN cells with Micronuclei	χ^2	Significance
Colchicine 0.040	2000	45	30.805	***

BiN: Binucleated cells

N.S. Not significant

*** Significant atp<0.001

 

Table 10. Main Experiment 1 – Summary Table Treatment: 3 hours Sampling Time: 32 hours
Treatment	Presence of S9 Metabolism				Absence of S9 Metabolism
	Dose Level (µg/mL)	% Mn cells	Sig.	% Cytotoxicity	Dose Level (µg/mL)	% Mn cells	Sig.	% Cytotoxicity
Untreated	0.00	0.35		0	0.00	0.25		0
Test Item	711	0.35	N.S.	-10	711	0.25	N.S.	9
Test Item	1070	0.20	N.S.	-5	1070	0.35	N.S.	5
Test Item	1600	0.20	N.S.	-13	1600	0.20	N.S.	-1
Cyclophosphamide	20.0	2.50	***	59		-		-

%Mn cells Percentage of cells bearing micronuclei

N.S. Not significant

*** Significant atp<0.001

 

Table 11. Main Experiment 2 – Summary Table Treatment: 31 hours Sampling Time: 31 hours
Treatment	Absence of S9 Metabolism
	Dose Level (µg/mL)	% Mn cells	Sig.	% Cytotoxicity
Untreated	0.00	0.25		0
Test Item	711	0.25	N.S.	-3
Test Item	1070	0.30	N.S.	12
Test Item	1600	0.45	N.S.	37
Colchicine	0.040	2.25	***	96

%Mn cells Percentage of cells bearing micronuclei

N.S. Not significant

*** Significant atp<0.001

Applicant's summary and conclusion

Conclusions:

Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.

Executive summary:

In a key Guideline (OECD 487) in vitro micronucleus test, the test material was assayed to evaluate its ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

Two main experiments were performed in this study. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test material were prepared in culture medium. For both experiments, the highest dose level of 1600 μg/mL was used (corresponding to10 mM), the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 μg/mL. Each experiment included appropriate negative and positive controls and two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test material treatments calculated by the cytokinesis-block proliferation index (CBPI). For both experiments, the following dose levels were selected for scoring: 1600, 1070 and711 μg/mL. One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

 

Following treatment with the test material, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicin and all results were within the distribution of historical negative control data.

 

Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.