Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2017 - 08 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Commission Regulation (EC) N0. 440/2008 of 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetrabutylmethylenediamine

EC Number:

243-678-5

EC Name:

N,N,N',N'-tetrabutylmethylenediamine

Cas Number:

20280-10-8

Molecular formula:

C17H38N2

IUPAC Name:

dibutyl[(dibutylamino)methyl]amine

Constituent 2

Reference substance name:

Dibutylamine

EC Number:

203-921-8

EC Name:

Dibutylamine

Cas Number:

111-92-2

Molecular formula:

C8H19N

IUPAC Name:

1-Butanamine, N-butyl-

Test material form:

liquid

Details on test material:

Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 11-25 ºC (23 Dec 2017 - 11 Jan 2018) and 2-8 ºC (11 Jan 2018 onwards), in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 200 mg/mL in acetone which was used to prepare the maximum required treatment concentration.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by formulating the test article under subdued lighting in acetone, with the aid of vortex mixing (where required), to give the maximum required treatment concentration. Subsequent dilutions were made using acetone. The test article solutions were protected from light and used within approximately 1.5 hours of initial formulation.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution prepared at 200 mg/mL in acetone.
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a liquid.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood from three healthy, non-smoking male volunteers from a panel of donors at the testing facility. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes on the day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
- Suitability of cells: Screened as described above
- Cell cycle length, doubling time or proliferation index: The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours
- Sex, age and number of blood donors if applicable: Male; aged 24-34
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37±1 °C) HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1 °C for approximately 48 hours and rocked continuously.
- Number of passages if applicable: 1
- Methods for maintenance in cell culture if applicable: Described above
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 13±2 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HEPES-buffered RPMI medium containing 1 0% (v/v) heat inactivated foetal calf serum and 0.5 2% penicillin / streptomycin. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No
- Periodically 'cleansed' against high spontaneous background: No

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary solubility data indicated that the test article was soluble in acetone at a concentration of at least 600 mg/mL. The solubility limit in culture medium was in the range of 263.6 to
527.1 μg/mL, as indicated by precipitation at the higher concentration which persisted for 20 hours after test article addition, with warming at 37 °C. A maximum concentration of 2000 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the recommended maximum concentration for in vitro chromosome aberration studies according to current regulatory test guidelines (OECD, 2016). Concentrations selected for the Chromosome Aberration Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents
with the Salmonella/Microsome Test. Mutation Res., 88, 343-350.

Controls

Untreated negative controls:

yes

Remarks:

Untreated; culture medium alone

Negative solvent / vehicle controls:

yes

Remarks:

acetone, batch MKBX7488V (Honeywell: expiry date 3 Apr 2018)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:

1. Compared to the concurrent negative control a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) at one or more concentrations is observed (p ≤ 0.05).

2. The incidence of cells with structural aberrations (excluding gaps) at such a concentration exceeds the normal range in both replicate cultures and falls outside the distribution of the historical negative control data.

3. A concentration-related increase in the proportion of cells with structural aberrations (excluding gaps) is observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.

Statistics:

After completion of scoring and decoding of slides the numbers of aberrant cells in each culture were categorised as follows:
1. Cells with structural aberrations including gaps
2. Cells with structural aberrations excluding gaps
3. Polyploid or endoreduplicated cells.
The totals for category 2 in vehicle control cultures were compared with the 95th percentile of the current historical vehicle control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance criteria). The proportion of cells with structural chromosome aberrations excluding gaps were compared with the proportion in vehicle controls by using Fisher’s exact test (Richardson et al., 1989). In addition, a Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical vehicle control range.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p≤0.05 were accepted as significant.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

 Table 1       3 hour treatment in the absence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	7.7
	B	8.6
	Total	8.2	-
UTC	A	8.2	-
7.256	A	8.0	2
12.09	A	5.8	29
20.16	A	5.0	39
33.59	A	3.7	55
55.99	A	3.2	61
93.31	A	1.1	87
155.5	A	0.2	98
259.2	A	0.2	98P
432.0	A	0.2	98P
720.0	A	0.1	99P
1200	A	0.2	98P
2000	A	0.0	-PE

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at the end of the test

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)

 

Table 2       3 hour treatment in the presence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	8.5
	B	9.5
	Total	9.0	-
UTC	A	8.3	-
7.256	A	10.0	0
12.09	A	7.3	19
20.16	A	9.2	0
33.59	A	6.2	31
55.99	A	5.6	38
93.31	A	2.9	68
155.5	A	0.4	96
259.2	A	0.2	98P
432.0	A	0.2	98P
720.0	A	0.4	96P
1200	A	0.3	97P
2000	A	0.4	96PE

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at the end of the test

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)UTC: untreated control

 

Table 3       20 hour treatment in the absence of S9 with 0 hour recovery (20+0),– range finder

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	7.6
	B	8.0
	Total	7.8	-
UTC	A	8.4	-
7.256	A	6.6	15
12.09	A	6.2	21
20.16	A	5.0	36
33.59	A	4.1	47
55.99	A	0.0	-
93.31	A	0.0	-
155.5	A	0.0	-
259.2	A	0.0	-P
432.0	A	0.0	-P
720.0	A	0.2	97P
1200	A	0.3	96P
2000	A	0.0	-PH

UTC: untreated control

P: indicates precipitation observed at the beginning of the test

E: indicates precipitation at harvest

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)UTC: untreated control

 

No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (2000 μg/mL), compared to the concurrent vehicle and negative controls.

 

The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Chromosome Aberration Experiment. Marked cytotoxicity (>60 % MIH) was observed at 55.99 μg/mL and above for the 3+17 and 20+0 treatments in the absence of S-9 and at 93.31 μg/mL and above for the 17+3 hour treatment in the presence of S-9. Based on these observations and in order to permit a range of cytotoxicity under each treatment condition, the maximum concentrations selected for the Chromosome Aberration Experiment were 80 μg/mL for the 3+17 hour treatments in the absence of S-9, 120 µg/mL for the 17+3 hour treatment in the presence of S-9 and 80 µg/mL for the 17+3 hour treatment in the absence of S-9.

 

The results of the MI determinations from the Chromosome Aberration Experiment were as follows:

 

Table 4       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	6.6
	B	7.4
	C	8.0
	D	6.8
	Total	7.2	-
UTC	A	6.6
	B	7.4
	Total	7.0	-
5.000	A	6.4
	B	7.4
	Total	6.9	4
10.00	A	7.7
	B	6.6
	Total	7.2	1
15.00	A	8.1
	B	6.8
	Total	7.5	0#
20.00	A	5.4
	B	6.0
	Total	5.7	21
25.00	A	6.3
	B	5.5
	Total	5.9	18#
30.00	A	5.7
	B	7.3
	Total	6.5	10
35.00	A	6.5
	B	6.5
	Total	6.5	10#
40.00	A	5.7
	B	3.5
	Total	4.6	36
45.00	A	5.5
	B	4.1
	Total	4.8	33#
50.00	A	4.2
	B	2.8
	Total	3.5	51#
60.00	A	2.1
	B	2.3
	Total	2.2	69
80.00	A	2.0
	B	1.3
	Total	1.7	77
MMC, 0.30	A	5.2
	B	5.5
	Total	5.4	26
MMC, 0.40	A	3.8
	B	3.0
	Total	3.4	53#

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)

 

Table 5       3 hour treatment in the presence of S9 with 17 hour recovery (3+17),– range finder

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	8.0
	B	6.8
	C	7.9
	D	7.3
	Total	7.5	-
UTC	A	8.4
	B	8.2
	Total	8.3	-
10.00	A	6.8
	B	9.1
	Total	8.0	0
20.00	A	6.0
	B	7.8
	Total	6.9	8#
30.00	A	5.3
	B	5.5
	Total	5.4	28#
40.00	A	6.1
	B	4.5
	Total	5.3	29
50.00	A	6.9
	B	5.4
	Total	6.2	18
60.00	A	5.5
	B	4.9
	Total	5.2	31
70.00	A	3.9
	B	4.0
	Total	4.0	47#
80.00	A	3.4
	B	4.5
	Total	4.0	47
90.00	A	2.7
	B	4.0
	Total	3.4	55#
100.0	A	2.5
	B	2.2
	Total	2.4	69
120.0	A	2.4
	B	2.6
	Total	2.5	67
CPA, 1.00	A	5.5
	B	5.8
	Total	5.7	25#
CPA, 2.00	A	3.0
	B	3.1
	Total	3.1	59

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)

 

Table 6       20 hour treatment in the absence of S9 with 0 hour recovery (20+0),– range finder

 

Treatment (µg/mL)	Replicate	Mitotic Index (%)	MIH* (%)
Vehicle	A	3.7
	B	4.8
	C	5.0
	D	4.5
	Total	4.5	-
UTC	A	6.0
	B	4.9
	Total	5.5	-
5.000	A	3.5
	B	4.2
	Total	3.9	14#
10.00	A	2.7
	B	4.1
	Total	3.4	24
15.00	A	3.6
	B	2.8
	Total	3.2	29#
20.00	A	2.5
	B	3.4
	Total	3.0	34#
25.00	A	3.1
	B	2.3
	Total	2.7	40
27.50	A	2.6
	B	2.3
	Total	2.5	46#
30.00	A	2.7
	B	3.8
	Total	3.3	28
32.50	A	3.1
	B	2.7
	Total	2.9	36
35.00	A	2.2
	B	2.2
	Total	2.2	51#
40.00	A	2.8
	B	2.1
	Total	2.5	46
45.00	A	1.4
	B	1.3
	Total	1.4	70
50.00	A	0.6
	B	0.9
	Total	0.8	83
MMC, 0.05	A	3.3
	B	4.8
	Total	4.1	10#
MMC, 0.10	A	3.5
	B	3.2
	Total	3.4	26

UTC: untreated control

# concentration selected for chromosome aberration analysis

* mitotic inhibition (%)= [1-(mean MI_T/ mean MI_C)] x 100 %

(where T = treatment and C = negative control)

 

Study validity 

1. The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.

2. The proportions of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal ranges.

3. At least 300 cells were suitable for analysis at each concentration, unless 15 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis.

4. The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated structural aberration cell frequencies (excluding gaps) that clearly exceeded the current historical vehicle control ranges


5. The maximum concentration analysed under each treatment condition was a concentration inducing approximately 50% cytotoxicity.


 

Structural aberrations 

Treatment of cells with the test article for 3+17 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were significantly higher (p≤0.001, using Fisher’s exact test), compared to the concurrent vehicle controls at the highest three concentrations analysed (35, 45 and 50 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in both cultures at 35 and 50 μg/mL and in one culture at 45 μg/mL and the Cochran-Armitage linear trend test was statistically significant (p≤0.001).

 

Treatment of cells for 3+17 hours in the presence of S-9 resulted in frequencies of structurally aberrant cells that were significantly higher (p≤0.05), compared to the concurrent vehicle controls at the highest two concentrations analysed (70 and 90 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in one culture at 20 μg/mL and in both cultures at 70 and 90 μg/mL, with a statistically significant Cochran-Armitage linear trend test (p≤0.001).

 

Treatment of cells for 20+0 hours in the absence of S-9 resulted in frequencies of structurally aberrant cells that were significantly higher (p≤0.001), compared to the concurrent vehicle controls at the highest two concentrations analysed (27.5 and 35 μg/mL). The aberration frequencies (excluding gaps) exceeded the normal range in one culture at 5 μg/mL and in both cultures at 27.5 and 35 μg/mL, with a statistically significant Cochran-Armitage linear trend test (p≤0.001).

 

Table 7       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)	Replicate	Cells scored	Cells with aberrations inc. gaps	Cells with aberrations exc. gaps	Significance^§	MIH* (%)
Vehicle	A	150	0	0
	B	150	0	0
	Total	300	0 (0 %)	0 (0 %)	-	-
15	A	150	0	0
	B	150	0	0
	Total	300	0 (0 %)	0 (0 %)	NS	0
25	A	150	1	1
	B	150	0	0
	Total	300	1 (0.33 %)	1 (0.33 %)	NS	18
35	A	150	6#	6#
	B	150	7#	7#
	Total	300	13 (4.33 %)	13 (4.33 %)	p≤0.001	10
45	A	150	10#	10#
	B	150	4	4
	Total	300	14 (4.67 %)	14 (4.67 %)	p≤0.001	33
50	A	150	15#	14#
	B	150	17#	16#
	Total	300	32 (10.67 %)	30 (10.00 %)	p≤0.001	51
MMC 0.4	A	150	21#	20#
	B	145	22#	20#
	Total	295	43 (14.58 %)	40 (13.56 %)	p≤0.001	53

NS: not significant

^§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 8       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – cells with structural aberrations

 

Treatment (µg/mL)	Replicate	Cells scored	Cells with aberrations inc. gaps	Cells with aberrations exc. gaps	Significance^§	MIH* (%)
Vehicle	A	150	1	1
	B	150	0	0
	Total	300	1 (0.33 %)	1 (0.33 %)	-	-
20	A	150	4#	4#
	B	150	0	0
	Total	300	4 (1.33 %)	4 (1.33 %)	NS	8
30	A	150	3	3
	B	150	2	2
	Total	300	5 (1.67 %)	5 (1.67 %)	NS	28
70	A	150	15#	13#
	B	150	22#	20#
	Total	300	37 (12.33 %)	33 (11.00 %)	p≤0.001	47
90	A	136	30#	30#
	B	150	23#	22#
	Total	286	53 (18.53 %)	52 (18.18 %)	p≤0.001	55
CPA, 1.00	A	150	8#	8#
	B	150	11#	9#
	Total	300	19 (6.33 %)	19 (5.67 %)	p≤0.001	25

NS: not significant

^§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 9       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – cells with structural aberrations

 

Treatment (µg/mL)	Replicate	Cells scored	Cells with aberrations inc. gaps	Cells with aberrations exc. gaps	Significance^§	MIH* (%)
Vehicle	A	150	1	1
	B	150	2	2
	Total	300	3 (1.00 %)	3 (1.00 %)	-	-
UTC	A	150	1	1
	B	150	3	3
	Total	300	4 (1.33 %)	4 (1.33 %)	NS	-
5	A	150	6#	5#
	B	150	2	2
	Total	300	8 (2.67 %)	7 (2.33 %)	NS	14
15	A	150	3	3
	B	150	4	2
	Total	300	7 (2.33 %)	5 (1.67 %)	NS	29
27.5	A	150	11#	10#
	B	150	10#	10#
	Total	300	21 (7.00 %)	20 (6.67 %)	p≤0.001	46
35	A	150	25#	23#
	B	150	24#	24#
	Total	300	49 (16.33 %)	47 (15.67 %)	p≤0.001	51
MMC, 0.05	A	150	17#	17#
	B	150	15#	14#
	Total	300	32 (10.67 %)	31 (10.33 %)	p≤0.001	10

NS: not significant

^§statistical significance

# numbers exceed historical vehicle control range

* mitotic inhibition

 

Table 10       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	G	Chr del.	Chr exch.	Ctd del.	Ctd exch.	Other	Abs +g	Abs -g
Vehicle	A	150	0	0	0	0	0	0	0	0
	B	150	0	0	0	0	0	0	0	0
	Total	300	0	0	0	0	0	0	0	0
15.0	A	150	0	0	0	0	0	0	0	0
	B	150	0	0	0	0	0	0	0	0
	Total	300	0	0	0	0	0	0	0	0
25.0	A	150	0	0	0	1	0	0	1	1
	B	150	0	0	0	0	0	0	0	0
	Total	300	0	0	0	1	0	0	1	1
35.0	A	150	0	0	0	6	0	0	6	6
	B	150	0	2	0	5	2	0	9	9
	Total	300	0	2	0	11	2	0	15	15
45.0	A	150	0	1	0	5	4	0	10	10
	B	150	0	0	0	4	0	0	4	4
	Total	300	0	1	0	9	4	0	14	14
50.0	A	150	1	3	0	12	3	0	19	18
	B	150	1	4	1	18	3	0	17	16
	Total	300	2	7	1	120	6	0	36	34
MMC, 0.40	A	150	2	5	0	11	6	0	24	22
	B	145	4	3	0	17	3	0	27	23
	Total	295	6	8	0	28	9	0	51	45

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Table 11       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	G	Chr del.	Chr exch.	Ctd del.	Ctd exch.	Other	Abs +g	Abs -g
Vehicle	A	150	0	0	0	1	0	0	1	1
	B	150	0	0	0	0	0	0	0	0
	Total	300	0	0	0	1	0	0	1	1
20.0	A	150	0	1	0	3	0	0	4	4
	B	150	0	0	0	0	0	0	0	0
	Total	300	0	1	0	3	0	0	4	4
30.0	A	150	0	0	0	3	1	0	4	4
	B	150	0	1	0	1	0	0	2	2
	Total	300	0	1	0	4	1	0	6	6
70.0	A	150	3	3	0	6	4	0	16	13
	B	150	3	1	0	18	3	0	25	22
	Total	300	6	4	0	24	7	0	41	35
90.0	A	136	2	8	0	28	2	0	40	38
	B	150	3	2	0	17	6	0	28	25
	Total	286	5	10	0	45	8	0	68	63
CPA, 1.00	A	150	3	3	0	8	0	0	14	11
	B	150	2	2	0	9	0	0	13	11
	Total	300	5	5	0	17	0	0	27	22

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Table 12       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of structural aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	G	Chr del.	Chr exch.	Ctd del.	Ctd exch.	Other	Abs +g	Abs -g
Vehicle	A	150	0	0	0	1	0	0	1	1
	B	150	0	1	0	1	0	0	2	2
	Total	300	0	1	0	2	0	0	3	3
UTC	A	150	0	1	0	0	0	0	1	1
	B	150	0	0	0	3	0	0	3	3
	Total	300	0	1	0	3	0	0	4	4
5	A	150	1	1	0	5	0	0	7	6
	B	150	1	1	0	1	0	0	3	2
	Total	300	2	2	0	6	0	0	10	8
15	A	150	0	1	0	3	0	0	4	4
	B	150	2	2	0	1	0	0	5	3
	Total	300	2	3	0	4	0	0	9	7
27.5	A	150	1	0	0	19	0	0	20	19
	B	150	0	3	0	10	0	0	13	13
	Total	300	1	3	0	29	0	0	33	32
35	A	150	2	6	0	26	3	0	37	35
	B	150	2	2	0	32	1	2	39	37
	Total	300	4	8	0	58	4	2	76	72
MMC, 0.05	A	150	0	2	0	14	3	0	19	19
	B	150	1	1	0	12	2	0	16	15
	Total	300	1	3	0	26	5	0	35	34

* total cells examined for structural aberrations

G: gaps

Chr del: chromosome deletion

Chr exch: chromosome exchanges

Ctd del: chromatid deletion

Ctd exch: chromatid exchanges

Abs: aberrations

 

Numerical Aberrations

 

Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and normal ranges were observed under all treatment conditions. There were no concentration-related responses but numerical aberrations were not analysed quantitatively. Increases in numerical aberration frequency in chromosome aberration assays in vitro may be attributable to aneugenicity, but polyploidy alone does not indicate aneugenic potential and may simply indicate cell cycle perturbation or cytotoxicity. As such, although numerical aberrations can be detected, this assay is not specifically designed for quantitative evaluation of aneugens.

 

Table 13       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	E	P	Total abs.	% with numerical abs.
Vehicle	A	150	0	0	0	0
	B	150	0	0	0	0
	Total	300	0	0	0	0
15	A	150	0	0	0	0
	B	151	0	1	1	0.7
	Total	301	0	1	1	0.3
25	A	150	0	0	0	0
	B	152	0	2#	2	1.3
	Total	302	0	2	2	0.7
35	A	154	0	4#	4	2.6
	B	159	0	9#	9	5.7
	Total	313	0	13	13	4.2
45	A	154	0	4#	4	2.6
	B	151	0	1	1	0.7
	Total	305	0	5	5	1.6
50	A	150	0	0	0	0
	B	151	0	1	1	0.7
	Total	301	0	1	1	0.3
MMC 0.4	A	150	0	0	0	0
	B	145	0	0	0	0
	Total	295	0	0	0	0

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 14       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	E	P	Total abs.	% with numerical abs.
Vehicle	A	150	0	0	0	0
	B	150	0	0	0	0
	Total	300	0	0	0	0
20	A	150	0	0	0	0
	B	150	0	0	0	0
	Total	300	0	0	0	0
30	A	156	0	6#	6	3.8
	B	150	0	0	0	0
	Total	306	0	6	6	2.0
70	A	155	0	5#	5	3.2
	B	154	1#	3#	4	2.6
	Total	309	1	8	9	2.9
90	A	136	0	0	0	0
	B	151	0	1	1	0.7
	Total	287	0	1	1	0.3
CPA, 1.0	A	150	0	0	0	0
	B	152	0	2#	2	1.3
	Total	302	0	2	2	0.7

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 15       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – numbers and types of numerical aberrations observed

 

Treatment (µg/mL)	Rep	Cells*	E	P	Total abs.	% with numerical abs.
Vehicle	A	151	1	0	1	0.7
	B	150	0	0	0	0
	Total	301	1	0	1	0.3
UTC	A	150	0	0	0	0
	B	151	0	1	1	0.7
	Total	301	0	1	1	0.3
5	A	150	0	0	0	0
	B	150	0	0	0	0
	Total	300	0	0	0	0
15	A	151	0	1	1	0.7
	B	150	0	0	0	0
	Total	301	0	1	1	0.3
27.5	A	155	0	5#	5	3.2
	B	157	0	7#	7	4.5
	Total	312	0	12	12	3.8
35	A	158	0	8#	8	5.1
	B	156	0	6#	6	3.8
	Total	314	0	14	14	4.5
MMC 0.05	A	150	0	0	0	0
	B	152	0	2#	2	1.3
	Total	302	0	2	2	0.7

* total cells examined for numerical aberrations

# number exceeds historical vehicle control range

E: endoreduplicated

P: polyploid (> 68 chromosomes)

 

Table 16       3 hour treatment in the absence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)	Cells scored	Aberrant cells	Proportion	Fisher’s exact test	Significance
Vehicle	300	0	0.000	-	-
15	300	0	0.000	0.500	NS
25	300	1	0.003	0.250	NS
35	300	13	0.043	0.000	p≤0.001
45	300	14	0.047	0.000	p≤0.001
50	300	30	0.100	0.000	p≤0.001
MMC 0.4	295	40	0.136	0.000	p≤0.001

Binomial dispersion testc²: 3.929            DF: 6               p-value: 0.686                Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance: p≤0.001

NS: not significant

DF: degrees of freedom

 

Table 17       3 hour treatment in the presence of S9 with 17 hour recovery (3+17), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)	Cells scored	Aberrant cells	Proportion	Fisher’s exact test	Significance
Vehicle	300	1	0.003	-	-
20	300	4	0.013	0.109	NS
30	300	5	0.017	0.062	NS
70	300	33	0.110	0.000	p≤0.001
90	286	52	0.182	0.000	p≤0.001
CPA, 1.0	300	17	0.057	0.000	p≤0.001

Binomial dispersion testc²: 9.459           DF: 5               p-value: 0.089              Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance:p≤0.001

NS: not significant

DF: degrees of freedom

 

Table 18       20 hour treatment in the absence of S9 with 0 hour recovery (20+0), chromosome aberration experiment – statistical analysis

 

Treatment (µg/mL)	Cells scored	Aberrant cells	Proportion	Fisher’s exact test	Significance
Vehicle	300	3	0.010	-	-
UTC	300	4	0.013	0.363	NS
5	300	7	0.023	0.112	NS
15	300	5	0.017	0.253	NS
27.5	300	20	0.067	0.000	p≤0.001
35	300	47	0.157	0.000	p≤0.001
MMC 0.05	300	31	0.103	0.000	p≤0.001

Binomial dispersion testc²: 1.882            DF: 5               p-value: 0.865                Significance: NS

Cochran-Armitage Linear Trend                                     p-value: 0.000                Significance:p≤0.001

NS: not significant

DF: degrees of freedom

 

 

Applicant's summary and conclusion

Conclusions:

It is concluded that Reaction Mass of N,N,N',N’-tetrabutylmethylenediamine and Dibutylamine induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.

Executive summary:

OECD 473 (2018) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine at concentrations of 0, 15, 20, 25, 30, 35, 45, 50, 70 and 90 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 5, 15, 27.5 and 35 µg/mL.

 

Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was tested up to precipitating and cytotoxic concentrations of 55.99 and 93.31 µg/mLfor the 3 h exposure, in the absence and presence of S9 mix, respectively and 93.31 µg/mL for the 20 h exposure with S9 mix. Positive controls induced the appropriate response.

 

There was evidence that the test item induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9 and is therefore considered to have clastogenic potential in this chromosome aberration test. Sporadic increases in the frequencies of cells with numerical aberrations, particularly polyploidy, which marginally exceeded the concurrent controls and the normal ranges, were observed under all treatment conditions, but this assay is not specifically designed for the quantitative evaluation of polyploidy.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.