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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay; OECD 471; Dreher, D. (2018); Positive (mutagenic)

Mammalian cell cytogeneticity assay; OECD 473; Lloyd, M. (2018); Positive (clastogenic)

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

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)

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

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.

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.

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

 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

 

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2017 - 31 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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: 2-8 ºC, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: No
- 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, to give the maximum required treatment concentration. Subsequent dilutions were made using acetone.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/mL, dilutions of which were prepared 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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

A maximum concentration of 5000 µg/plate was selected for Experiment 1, to ensure treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 1997). For Experiment 2 the maximum concentration tested was selected on the basis of toxicity limitations seen in Experiment 1.

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3 per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.

2. Any observed response was reproducible under the same treatment conditions.

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

The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1       Raw plate counts and calculated mutagenicity data, experiment 1 (- S9 mix)

 

Strain	Compound	Conc. level (µg/plate)	Mean revertants per plate	SD	Fold increase	Dunnett’s T-value	Statistic. sig.	Revertant numbers/plate
TA98	Acetone	-	27.3	10.2	-	-	-	23, 39, 20
	Test item	5	31.0	2.6	1.1	0.75	NS	28, 32, 33
		16	29.7	2.5	1.1	0.52	NS	27, 30, 32
		50	30.7	6.0	1.1	0.67	NS	37, 30, 25
		160	42.3	4.5	1.5	2.54	NS	38, 47, 42
		500	45.0	14.7	1.6	2.83	*	61, 42, 32
		1600	33.0	6.1	1.2	1.07	NS	40S, 30S, 29S
		5000	-	-	-	-	-	-T, -T, -T
	2NF	5	334.3	96.1	12.2	-	-	314, 439, 250
TA100	Acetone	-	118.7	1.5	-	-	-	119, 120, 117
	Test item	5	104.0	7.5	0.9	-1.73	NS	96, 111, 105
		16	108.3	2.1	0.9	-1.20	NS	110, 109, 134
		50	112.0	19.7	0.9	-0.83	NS	96, 106, 134
		160	145.7	17.6	1.2	2.88	*	151, 126, 160
		500	122.7	4.9	1.0	0.45	NS	126, 117, 125
		1600	171.7	9.1	1.4	5.46	**	182S, 168S, 165S
		5000	-	-	-	-	-	-T, -T, -T
	NaN3	2	489.7	19.3	4.1	-	-	468, 505, 496
TA1535	Acetone	-	12.3	4.0	-	-	-	8, 16, 13
	Test item	5	17.0	2.6	1.4	1.03	NS	16, 20, 15
		16	26.3	1.2	2.1	2.68	*	27, 27, 25
		50	18.7	4.6	1.5	1.33	NS	24, 16, 16
		160	13.0	3.0	1.1	0.18	NS	10, 13, 16
		500	20.0	6.6	1.6	1.55	NS	14, 27, 19
		1600	14.3	12.7	1.2	0.12	NS	8S, 29S, 6S
		5000	-	-	-	-	-	-T, -T, -T
	NaN3	2	458.0	62.4	37.1	-	-	466, 516, 392
TA1537	Acetone	-	5.0	1.7	-	-	-	6, 3, 6
	Test item	5	5.3	2.5	1.1	0.14	NS	8, 5, 3
		16	9.0	1.0	1.8	1.98	NS	9, 8, 10
		50	8.3	5.8	1.7	1.44	NS	5, 15, 5
		160	7.7	1.5	1.5	1.8	NS	9, 6, 8
		500	6.0	2.0	1.2	0.54	NS	6, 8, 4
		1600	4.7	0.6	0.9	-0.13	NS	5S, 5S, 4S
		5000	-	-	-	-	-	-T, -T, -T
	AAC	50	87.0	15.5	17.4	-	-	102, 71, 88
TA102	Acetone	-	293.3	16.2	-	-	-	312, 284, 284
	Test item	5	282.0	17.6	1.0	-1.00	NS	269, 302, 275
		16	287.7	15.2	1.0	-0.49	NS	274, 285, 304
		50	285.3	7.8	1.0	-0.69	NS	279, 294, 283
		160	308.0	4.6	1.1	1.27	NS	312, 309, 303
		500	399.3	8.5	1.4	8.50	**	399, 391, 408
		1600	401.0	25.1	1.4	8.60	**	430S, 387S, 386S
		5000	-	-	-	-	-	-T, -T, -T
	MMC	0.2	542.3	22.5	1.8	-	-	568, 526, 533

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

* <0.05

**p<0.01

 

Table 2       Raw plate counts and calculated mutagenicity data, experiment 1 (+ S9 mix)

 

Strain	Compound	Conc. level (µg/plate)	Mean revertants per plate	SD	Fold increase	Dunnett’s T-value	Statistic. sig.	Revertant numbers/plate
TA98	Acetone	-	27.7	2.5	-	-	-	30, 25, 28
	Test item	5	43.0	5.6	1.6	2.84	*	44, 37, 48
		16	30.7	10.0	1.1	0.52	NS	42, 23, 27
		50	40.0	6.2	1.4	2.32	NS	35, 38, 47
		160	35.3	4.7	1.3	1.49	NS	37, 30, 39
		500	55.3	8.0	2.0	4.77	**	56, 47, 63
		1600	29.0	7.0	1.0	0.22	NS	32S, 34S, 21S
		5000	-	-	-	-	-	-T, -T, -T
	B[a]P	10	344.3	41.0	12.4	-	-	391, 314, 328
TA100	Acetone	-	120.3	9.0	-	-	-	125, 110, 126
	Test item	5	130.3	15.5	1.1	0.85	NS	115, 146, 130
		16	136.0	12.1	1.1	1.34	NS	143, 143, 122
		50	133.7	4.5	1.1	1.16	NS	129, 138, 134
		160	136.3	12.7	1.1	1.37	NS	134, 125, 150
		500	178.0	16.1	1.5	4.60	**	173, 165, 196
		1600	118.3	22.9	1.0	-0.24	NS	129V, 134V, 92V
		5000	-	-	-	-	-	-T, -T, -T
	AAN	5	746.7	139.6	6.2	-	-	875, 767, 598
TA1535	Acetone	-	21.0	3.6	-	-	-	18, 25, 20
	Test item	5	14.0	1.0	0.7	-1.82	NS	13, 15, 14
		16	20.3	4.0	1.0	-0.17	NS	25, 18, 18
		50	17.3	7.1	0.8	-1.02	NS	16, 11, 25
		160	16.0	7.0	0.8	-1.42	NS	8, 19, 21
		500	23.0	4.0	1.1	0.46	NS	27, 19, 23
		1600	9.7	1.2	0.5	-3.20	NS	9S, 9S, 11S
		5000	-	-	-	-	NS	-T, -T, -T
	AAN	5	270.0	19.0	12.9	-	-	251, 289, 270
TA1537	Acetone	-	19.0	5.0	-	-	-	24, 14, 19
	Test item	5	16.0	7.0	0.8	-0.87	-	24, 13, 11
		16	22.3	3.1	1.2	0.85	NS	25, 19, 23
		50	16.7	6.0	0.9	-0.65	NS	16, 11, 23
		160	18.0	2.6	0.9	-0.22	NS	19, 20, 15
		500	14.7	1.5	0.8	-1.12	NS	16, 15, 13
		1600	-	-	-	-	NS	-T, -T, -T
		5000	-	-	-	-	-	-T, -T, -T
	AAN		405.7	44.0	21.4	-	-	421, 356, 440
TA102	Acetone	-	356.7	21.8	-	-	-	376, 361, 333
	Test item	5	381.0	12.5	1.1	1.74	-	371, 377, 395
		16	373.0	3.5	1.0	1.19	NS	377, 371, 371
		50	344.7	9.2	1.0	-0.86	NS	350, 334, 350
		160	375.3	23.5	1.1	1.33	NS	401, 370, 355
		500	448.0	30.3	1.3	6.23	**	467, 464, 413
		1600	395.3	3.1	1.1	2.74	*	398S, 396S, 392S
		5000	-	-	-	-	-	-T, -T, -T
	AAN	20	1250.3	333.5	3.5	-	-	1464, 866, 1421

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

*p<0.05

** p<0.01

 

Table 3       Raw plate counts and calculated mutagenicity data, experiment 2 (- S9 mix)

 

Strain	Compound	Conc. level (µg/plate)	Mean revertants per plate	SD	Fold increase	Dunnett’s T-value	Statistic. sig.	Revertant numbers/plate
TA98	Acetone	-	22.7	6.5	-	-	-	16, 23, 29
	Test item	31.25	20.3	6.1	0.9	-0.54	NS	19, 27, 15
		62.5	16.3	2.5	0.7	-1.50	NS	16, 14, 19
		125	24.3	7.1	1.1	0.37	NS	23, 32, 18
		250	18.7	3.2	0.8	-0.90	NS	20, 21, 15
		400	18.3	2.5	0.8	-0.98	NS	16, 21, 18
		500	21.7	4.7	1.0	-0.19	NS	18, 27, 20
		750	20.7	7.6	0.9	-0.50	NS	19, 29, 14
		1000	33.0	5.2	1.5	2.17	NS	39S, 30S, 30S
		2000	-	-	-	-	-	-T, -T, -T
	2NF	5	423.7	199.1	18.7	-	-	645, 367, 259
TA100	Acetone	-	82.7	10.2	-	-	-	71, 87, 90
	Test item	31.25	97.0	13.1	1.2	1.22	NS	111, 85, 95
		62.5	93.3	3.2	1.1	0.94	NS	92, 97, 91
		125	105.3	15.3	1.3	1.89	NS	88, 111, 117
		250	117.7	12.2	1.4	2.85	*	115, 107, 131
		500	147.3	30.6	1.8	4.89	**	124, 182, 136
		1000	187.7	24.5	2.3	7.46	**	174S, 173S, 216S
		2000	-	-	-	-	-	-T, -T, -T
	NaN3	2	448.0	61.6	5.4	-	-	500, 380, 464
TA1535	Acetone	-	6.7	1.2	-	-	-	6, 6, 8
	Test item	31.25	10.0	1.0	1.5	1.88	NS	10, 9, 11
		62.5	11.0	1.7	1.7	2.37	NS	13, 10, 10
		125	8.0	2.6	1.2	0.72	NS	5, 10, 9
		250	8.7	3.2	1.3	1.06	NS	10, 11, 5
		500	7.0	2.6	1.1	0.11	NS	9, 8, 4
		1000	11.0	0.0	1.7	2.39	NS	11, 11, 11
		2000	-	-	-	-	-	-T, -T, -T
	NaN3	2	352.7	52.7	52.9	-	-	379, 292, 387
TA1537	Acetone	-	4.0	1.0	-	-	-	4MB, 5, 3
	Test item	31.25	4.7	1.5	1.2	0.44	NS	6, 5, 3
		62.5	8.0	2.6	2.0	2.38	NS	10, 5, 9
		125	4.3	0.6	1.1	0.26	NS	4, 4, 5
		250	8.0	3.0	2.0	2.37	NS	8, 5, 11
		500	10.0	1.0	2.5	3.44	*	10, 11, 9
		1000	10.3	4.0	2.6	3.47	**	14S, 6S, 11S
		2000	-	-	-	-	-	-T, -T, -T
	AAC	50	152.7	71.1	38.2	-	-	201, 71, 186
TA102	Acetone	-	299.0	78.6	-	-	-	386, 278, 233
	Test item	31.25	315.0	39.1	1.1	0.30	NS	334, 341, 270
		62.5	297.3	20.6	1.0	0.02	NS	317, 276, 299
		125	344.3	39.1	1.2	0.77	NS	307, 341, 385
		250	355.7	27.2	1.2	0.94	NS	339, 387, 341
		500	342.3	34.6	1.1	0.74	NS	382, 327, 318
		1000	310.3	7.8	1.0	0.24	NS	304, 308, 319
		2000	188.0	147.8	0.6	-2.38	NS	74V, 355V, 135V
	MMC	0.2	954.3	109.0	3.2	-	-	986, 833, 1044

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

* p<0.05

** p<0.01

 

Table 4       Raw plate counts and calculated mutagenicity data, experiment 2 (+ S9 mix)

 

Strain	Compound	Conc. level (µg/plate)	Mean revertants per plate	SD	Fold increase	Dunnett’s T-value	Statistic. sig.	Revertant numbers/plate
TA98	Acetone	-	30.5	3.5	-	-	-	28, -T, 33
	Test item	31.25	22.0	1.7	0.7	-1.30	NS	23, 20, 23
		62.5	23.7	3.1	0.8	-1.04	NS	27, 23, 21
		125	33.3	4.7	1.1	0.39	NS	37, 35, 28
		250	43.0	11.5	1.4	1.57	NS	39, 56, 34
		400	47.0	7.8	1.5	2.08	NS	43, 42, 56
		500	24.3	10.0	0.8	-1.04	NS	14, 34, 25
		750	15.3	2.3	0.5	-2.53	NS	18, 14, 14
		1000	16.0	10.4	0.5	-2.59	NS	11S, 9S, 28S
		2000	-	-	-	-	-	-T, -T, -T
	B[a]P	10	373.3	48.3	12.2	-	-	380, 322, 418
TA100	Acetone	-	101.0	18.3	-	-	-	85, 121, 97
	Test item	31.25	107.0	4.0	1.1	0.17	NS	111, 107, 103
		62.5	125.3	7.5	1.2	0.63	NS	121, 121, 134
		125	130.0	39.6	1.3	0.69	NS	165, 138, 87
		250	181.3	18.1	1.8	1.85	NS	162, 184, 198
		500	269.7	173.2	2.7	3.14	*	462, 221, 126
		1000	52.5	27.6	0.5	-1.40	NS	-T, 72V, 33V
		2000	-	-	-	-	-	-T, -T, -T
	AAN	5	1143.3	78.4	11.3	-	-	1088, 1109, 1233
TA1535	Acetone	-	14.7	3.5	-	-	-	18, 15, 11
	Test item	31.25	13.7	2.5	0.9	-0.34	NS	16, 11, 14
		62.5	12.0	1.7	0.8	-0.96	NS	10, 13, 13
		125	16.0	1.7	1.1	0.50	NS	15, 15, 18
		250	18.3	7.6	1.3	1.12	NS	13, 15, 27
		500	12.7	1.5	0.9	-0.70	NS	14, 11, 13
		1000	-	-	-	-	-	-T, -T, -T
		2000	-	-	-	-	-	-T, -T, -T
	AAN	5	179.7	35.7	12.3	-	-	139, 194, 206
TA1537	Acetone	-	3.7	1.2	-	-	-	3, 3, 5
	Test item	31.25	6.3	4.2	1.7	1.07	NS	3, 11, 5
		62.5	6.0	2.0	1.6	1.06	NS	4, 8, 6
		125	5.7	2.5	1.5	0.88	NS	3, 6, 8
		250	10.0	5.6	2.7	2.36	NS	11, 4, 15
		500	7.3	1.2	2.0	1.62	NS	8V, 8V, 6V
		1000	-	-	-	-	-	-T, -T, -T
		2000	-	-	-	-	-	-T, -T, -T
	AAN	5	249.3	180.7	68.0	-	-	74, 239, 435
TA102	Acetone	-	233.7	19.1	-	-	-	254, 231, 216
	Test item	31.25	218.0	40.8	0.9	-0.43	NS	197, 192, 265
		62.5	281.7	51.0	1.2	1.14	NS	281, 231, 333
		125	328.0	13.5	1.4	2.21	NS	339, 332, 313
		250	264.3	14.3	1.1	0.76	NS	280, 261, 252
		500	302.0	67.6	1.3	1.58	NS	237, 372, 297
		1000	104.7	53.5	0.4	-4.16	NS	133V, 43V, 138V
		2000	-	-	-	-	-	-T, -T, -T
	AAN	20	745.0	85.8	3.2	-	-	791, 798, 646

S: slight thinning of background bacterial lawn

T: toxic, no revertant colonies

V: very thin background bacterial lawn

 

 

 

 

Conclusions:

It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines)

Executive summary:

OECD 471 (2018) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimuriumwere exposed to Reaction mass of N,N,N’,N-tetrabutylmethylenediamine and dibutylamine in acetone at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 31.25, 62.5, 125, 250, 500, 100 and 200 µg/plate (TA98 also tested at 400 and 750 µg/plate) in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix.

 

The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test article at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 1600 and 5000 μg/plate in all strains in the absence and presence of S-9. A two-fold increase in revertant numbers was observed in strain TA98 at 500 μg/plate in the absence of S-9. This change was statistically significant at p≤0.01 using Dunnett’s test.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains, the maximum test concentration was reduced to 2000 μg/plate based on strain specific toxicity observed in Experiment 1. A narrowed concentration range of 31.25, 62.5, 125, 250, 400, 500, 750, 1000, 2000 μg/plate was used to examine more closely those concentrations of the test article approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. All treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. This modification increases sensitivity of the assay to potentially increase the range of mutagenic chemicals that can be detected in the assay.

To investigate the reproducibility of the increase in revertant numbers seen in TA98 at 500 μg/plate in Experiment 1, additional concentrations at 400 and 750 μg/plate were included in the treatments for TA98 in the second experiment using a pre-incubation method and plate-incorporation in the presence of S9.

Following these treatments in Experiment 2, evidence of toxicity was observed partially at1000 and at 2000 μg/plate in all strains in the absence and presence of S-9.

The greater than 2-fold increase in revertant numbers seen in strain TA98 in Experiment 1 was reproduced in Experiment 2. The increase in revertant numbers seen in Experiment 2 was concentration-related and statistically significant (Dunnett’s test (p≤0.01)).

Experiment 2 also showed an increase in revertant numbers greater than 2-fold in strain TA100 at the highest non-toxic concentration. The increase was concentration-dependent and statistically significant (p≤0.01 in the absence of S9 and p≤0.05 in the presence of S-9).

 

It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus screen OECD 474 incorporated into OECD 442; N Barraclough (2018) - Negative

In vivo Comet assay in the rat OECD 489; I Eurlings (2021) - Positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 December 2020 - 09 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 489 and in compliance with GLP with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Suspensions of 500 mg/mL are stable in corn oil for 16 days at room temperature (15 to 25°C)

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) Outbred, SPF-Quality

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean. In the main study, the mean body weights were for males 185 ± 8.9 g and the range for males 163 – 212 g.
- Females were nulliparous and non-pregnant.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together. The animals were housed in room number A0.12 and A0.04.
- Diet (e.g. ad libitum): SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany (pellets). Ad libitum, except during designated procedures. Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 hours light and 12 hours dark (except during designated procedures)
- Photoperiod (hrs dark / hrs light): Ten or more air changes per hour
The actual daily mean temperature during the study period was 18.0 to 19.7°C with an actual daily mean relative humidity of 46 to 52%.

IN-LIFE DATES: From: 04 Jan 2021 To: 11 Feb 2021

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item was dissolved (clear yellow solution) in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands).
- Amount of vehicle (if gavage or dermal): not reported
- Lot/batch no.: #MKLM3364, Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands
- Purity: not reported

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item.
Test item concentrations were dosed within 3 hours after preparation. Any residual volumes were discarded.

ADMINISTRATION
A limited quantity of food was supplied after the second dose (approximately 7 g/rat).
The first day of dosing was designated as Day 1. The doses were given using a plastic feeding tube.
The dosing volume was 10 mL/kg body weight.

Duration of treatment / exposure:

2 exposures (0 and 21 hours)

Frequency of treatment:

Twice

Post exposure period:

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Number of Males: 4 (dose range finding), 34 (main study). Main study = 5 per dose for Comet + 3 spares in group 4; 3 per dose for bioanalysis (Table 1)
Number of Females: 4 (dose range finding)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulphonate (#BCB28402EMS, Sigma Aldrich, Steinheim, Germany)
- Justification for choice of positive control(s): not reported, but common positive control according to OECD TG 489
- Route of administration: oral
- Doses / concentrations: 200 mg/kg body weight dissolved in physiological saline. The dosing volume was 10 mL/kg body weight.

Tissues and cell types examined:

- Tissues collected: Liver, Stomach, Duodenum
- Histology processing and microscopic evaluation: Stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the dose-range finding study (see results).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

- Liver
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

- Isolation of glandular stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The fore- stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

- Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study.
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodenum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded.
The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

DETAILS OF SLIDE PREPARATION:

- Preparation of Slides
Slides were prepared immediately after single cell preparation and within 3 hours after necropsy. Everything was kept on ice during this time with exception of the collagenase step for liver. To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 11-27 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

- Lysis, Electrophoresis and Staining of the Slides
The cells on the slides were overnight (approximately 17-19 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator (2-8°C in the dark). After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then immersed in freshly prepared alkaline solution for 20 or 30 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 (for duodenum and stomach) or 30 (liver) minutes under constant cooling (actual temperature 4.5°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (≥99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 10 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

- Sampling, fixation and storage of tissue for histotechnology and histopathology
Part of the liver, stomach, duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

METHOD OF ANALYSIS: COMET SCORING

To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.

The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
• Scoring at the edge of slides should be avoided
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. For animal 24 in liver only 118 cells were scored, however as other animals in the group were scored 150 cells this has no impact. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

HISTOPATHOLOGY

- Histology
Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

- Microscopic Evaluation
Tissues were evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

ACCEPTABILITY CRITERIA
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data was analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg/day
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Evaluation criteria:

A test item is considered positive in the Comet assay if all of the following criteria are met:

a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Welch t test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Linear regression (p < 0.05) was performed to test whether there is a significant trend in the induction.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 mg/kg bw and 350 mg/kg bw (MTD)
- Clinical signs of toxicity in test animals: cf. Table 2
- Evidence of cytotoxicity in tissue analysed: None


RESULTS OF DEFINITIVE STUDY (Mean values: Cf. Table 3 to 10 / Individual data: Cf. Attachments)

- Appropriateness of dose levels and route:
Based on the results of the dose-range finding study dose levels of 87.5, 175 and 350 mg/kg body weight were selected as appropriate doses for the main test. Five male animals were used in each treatment group. The animals of the groups treated with 87.5 and 175 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Ataxia, tremors, lethargy, hunched posture and ventral recumbency were observed in the groups treated with 350 mg test item /kg body weight.

- Comet slide analysis:
LIVER / DUODENUM: No statistically significant increase in the mean Tail Intensity (%) was observed in liver and duodenum cells of test item treated male animals compared to the vehicle treated animals.
STOMACH: A statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals in all dose-groups and significant trend was present. The values for the high and intermediate dose- groups were outside the historical control database of the negative controls.

- Historical control data & Validity criteria
The mean Tail Intensity in liver, glandular stomach and duodenum cells of vehicle-treated rats was 4.13 ± 0.95% (mean ± SD), 5.21 ± 1.02% (mean ± SD) and 11.96 ± 3.01% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Liver: -0,9 – 6,1% / Glandular stomach: 0,3- 7,1%) , except in the duodenum cells (0,6-8,2%). Since the mean Tail Intensity was above the upper limit of the acceptability criteria and clearly no effect was observed by the test item, therefore this has no effect on the validity of the results (Table 25). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 81.74 ± 2.35% (mean ± SD; p<0.001 Students t test), 60.74 ± 2.91% (mean ± SD; p<0.001 Students t test) and 56.65 ± 4.67% (mean ± SD; p<0.001 Students t test) in male animals in liver, glandular stomach and duodenum cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database
Adequate numbers of cells and doses were analysed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

- Histopathology:
On Day 2 of the study five animals of each group were sacrificed and subjected to necropsy. Histopathologic examination was performed on the stomach of the animals from Groups 1, 2, 3 and 4.
There were no test item-related microscopic findings.
There were no morphologic alterations in the stomach following two administrations of O7017- Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine by oral gavage to male Wistar (Han) rats up to 350 mg/kg.

Table 2 - Mortality and Toxic Signs in the Dose-range Finding Study

Group	Sex	Animal Number	Dose mg/kg	Toxic signs**
				day 1	day 2		day 3
				2 hrs^*	21 hrs^*	2 hrs^*	21 hrs^*
1	Male	101	250	B	B	B
1	Female	102	250	B	B^1)	B
2	Male	103	350	CEF	J	EFW	B
2	Female	104	350	B	B^1)	FN	B
2	Male	105	350	B	B	B	B
2	Male	106	350	F	B	F	B
2	Female	107	350	B	B^1)	CEF	B
2	Female	108	350	B	B^1)	B	B

*  Within … hrs after dosing

** Legend 'Mortality and toxic signs':

      B = showed no abnormalities; C = ataxia; E = tremors; F = lethargy; J = hunched posture; N = rough coat; W = ventral recumbency.

1) Did not finish food.

 

Table 3 - Mortality and Toxic Signs after Treatment in the Main Study

Group	Sex	Animal Number	Dose mg/kg	Toxic signs*
				day 1 within … hours after dosing		day 2 within … hours after dosing
				2 hrs		21 hrs	2 hrs
1	Male	1	0	B		B	B
1	Male	2	0	B		B	B
1	Male	3	0	B		B	B
1	Male	4	0	B		B	B
1	Male	5	0	B		B	B
2	Male	6	87.5	B		B	B
2	Male	7	87.5	B		B	B
2	Male	8	87.5	B		B	B
2	Male	9	87.5	B		B	B
2	Male	10	87.5	B		B	B
3	Male	11	175	B		B	B
3	Male	12	175	B		B	B
3	Male	13	175	B		B	B
3	Male	14	175	B		B	B
3	Male	15	175	B		B	B
4	Male	16	350	B		B^1)	EFW
4	Male	17	350	B		B^1)	EFW
4	Male	18	350	B		B^1)	FJ
4	Male	19	350	B		B^1)	FJ
4	Male	20	350	B		B^1)	EFW
5	Male	24	200 EMS	B		B	B
5	Male	25	200 EMS	B		B	B
5	Male	26	200 EMS	B		B	B
5	Male	27	200 EMS	B		B	B
5	Male	28	200 EMS	B		B	B
Add. 4	Male	21	350	B		B	FJ
Add. 4	Male	22	350	B		B	CFJ
Add. 4	Male	23	350	B		B	A
TK1	Male	29	0	B		B
TK1	Male	30	0	B		B
TK1	Male	31	0	B		B
TK4	Male	32	350	B		B^1)
TK4	Male	33	350	B		B^1)
TK4	Male	34	350	B		B^1)

* Legend 'Mortality and toxic signs':

A = died; B = showed no abnormalities; C = ataxia; E = tremors; F = lethargy; J = hunched posture; W = ventral recumbency.

1) Did not finish food.

 

Table 4 - Mean Body Weight Immediately Prior to Dosiing

Group code	Dose (mg/kg/bw)	Day 1 Body weight gram (Mean ± S.D.)			Day 2 Body weight gram (Mean ± S.D.)
1	0	190.4	±	5.8	189.0	±	7.0
2	87.5	187.0	±	8.3	183.8	±	10.3
3	175	182.8	±	6.9	176.0	±	5.1
4	350	181.2	±	5.1	179.2	±	5.6
5	200 (EMS)	184.8	±	18.0	175.0	±	15.9
Add. 4	350	181.3	±	5.5	175.7	±	9.8
TK1	0	187.0	±	9.8	179.0	±	11.5
TK4	350	189.7	±	2.1	174.3	±	5.1

 

Table 5 - Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.13	0.95
Test Item 87.5 mg/kg	3.23	0.77
Test Item 175 mg/kg	2.66	0.56
Test Item 350 mg/kg	2.93	0.51
EMS 200 mg/kg	81.74	2.35

 

Table 6 - Overview Tail Intensity in Duodenuml Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	11.96	3.01
Test Item 87.5 mg/kg	9.15	2.18
Test Item 175 mg/kg	9.56	1.14
Test Item 350 mg/kg	8.93	2.78
EMS 200 mg/kg	56.65	4.67

 

Table 7 - Overview of Tail Intensity in Glandular Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	5.21	1.02
Test Item 87.5 mg/kg	6.78	1.32
Test Item 175 mg/kg	8.17	1.44
Test Item 350 mg/kg	7.65	0.90
EMS 200 mg/kg	60.74	2.91

 

Table 8 - Mean Tail Intensity in Liver Cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	3.32
	Rat 2	3.72
	Rat 3	3.47
	Rat 4	4.55
	Rat 5	5.60
	Mean	4.13
	S.D.	0.95
Test Item 87.5 mg/kg		Tail Intensity (%)
Group 2	Rat 6	2.22
	Rat 7	3.71
	Rat 8	2.68
	Rat 9	3.44
	Rat 10	4.10
	Mean	3.23
	S.D.	0.77
Test Item 175 mg/kg		Tail Intensity (%)
Group 3	Rat 11	2.61
	Rat 12	3.58
	Rat 13	2.48
	Rat 14	2.06
	Rat 15	2.58
	Mean	2.66
	S.D.	0.56
Test Item 350 mg/kg		Tail Intensity (%)
Group 4	Rat 16	2.58
	Rat 17	2.49
	Rat 18	2.99
	Rat 19	3.78
	Rat 20	2.82
	Mean	2.93
	S.D.	0.51
EMS 200 mg/kg		Tail Intensity (%)
Group 5	Rat 24	85.77
	Rat 25	81.05
	Rat 26	79.67
	Rat 27	80.83
	Rat 28	81.37
	Mean	81.74
	S.D.	2.35

 

Table 9 - Mean Tail Intensity in Duodenum Cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	10.75
	Rat 2	9.98
	Rat 3	13.76
	Rat 4	16.32
	Rat 5	9.02
	Mean	11.96
	S.D.	3.01
Test Item 87.5 mg/kg		Tail Intensity (%)
Group 2	Rat 6	7.55
	Rat 7	10.96
	Rat 8	12.03
	Rat 9	7.38
	Rat 10	7.82
	Mean	9.15
	S.D.	2.18
Test Item 175 mg/kg		Tail Intensity (%)
Group 3	Rat 11	7.83
	Rat 12	9.55
	Rat 13	10.32
	Rat 14	9.29
	Rat 15	10.81
	Mean	9.56
	S.D.	1.14
Test Item 350 mg/kg		Tail Intensity (%)
Group 4	Rat 16	5.48
	Rat 17	10.12
	Rat 18	12.91
	Rat 19	7.66
	Rat 20	8.47
	Mean	8.93
	S.D.	2.78
EMS 200 mg/kg		Tail Intensity (%)
Group 5	Rat 24	52.24
	Rat 25	59.05
	Rat 26	50.98
	Rat 27	60.15
	Rat 28	60.83
	Mean	56.65
	S.D.	4.67

 

Table 10 - Mean Tail Intensity in Glandular Stomach cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	3.95
	Rat 2	4.53
	Rat 3	6.61
	Rat 4	5.34
	Rat 5	5.60
	Mean	5.21
	S.D.	1.02
Test Item 87.5 mg/kg		Tail Intensity (%)
Group 2	Rat 6	4.89
	Rat 7	7.00
	Rat 8	8.43
	Rat 9	7.36
	Rat 10	6.24
	Mean	6.78
	S.D.	1.32
Test Item 175 mg/kg		Tail Intensity (%)
Group 3	Rat 11	7.65
	Rat 12	5.94
	Rat 13	9.50
	Rat 14	8.53
	Rat 15	9.26
	Mean	8.17
	S.D.	1.44
Test Item 350 mg/kg		Tail Intensity (%)
Group 4	Rat 16	6.25
	Rat 17	7.23
	Rat 18	8.22
	Rat 19	8.14
	Rat 20	8.40
	Mean	7.65
	S.D.	0.90
EMS 200 mg/kg		Tail Intensity (%)
Group 5	Rat 24	59.17
	Rat 25	59.71
	Rat 26	65.93
	Rat 27	59.63
	Rat 28	59.25
	Mean	60.74
	S.D.	2.91

 

Conclusions:

In conclusion, the test is valid, and Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine is genotoxic in glandular stomach cells and not genotoxic in liver and duodenum cells in the Comet assay when sampled approximately 3-4hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 350 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report.

Executive summary:

In an Alkaline in vivo Comet Assay performed according to the OECD TG 489 and in compliance with GLP, males Wistar Han rats were treated twice (at 0 and 21 hours) by gavage administration with Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine  at doses of 0, 87.5, 175 and 350 mg/kg bw.  The vehicle was corn oil.

 

Blood for bioanalysis of Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine in plasma was collected from satellite animals for the 350 mg/kg group (highest dose group) and from satellite animals for the vehicle control group.

 

Blood was sampled 1, 2, 4, 6, and 24 h after the first dose of satellite animals dosed with the vehicle and the highest concentration of the test item.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

 

No statistically significant increase in the mean Tail Intensity (%) was observed in liver and duodenum cells of test item treated male animals compared to the vehicle treated animals.

A statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals in all dose-groups and a significant trend was present. The values for the high and intermediate dose-groups were outside the historical control database of the negative controls.

 

The mean Tail Intensity in liver, glandular stomach and duodenum cells of vehicle-treated rats was 4.13 ± 0.95% (mean ± SD), 5.21 ± 1.02% (mean ± SD) and 11.96 ± 3.01% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control, except in the duodenum cells. Since the mean Tail Intensity was just above the upper limit of the acceptability criteria, this has no effect on the validity of the results. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 81.74 ± 2.35% (mean ± SD), 60.74 ± 2.91% (mean ± SD) and 56.65 ± 4.67% (mean ± SD) in male animals in liver, glandular stomach and duodenum cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

 

Histopathology of the stomach was performed. There were no morphologic alterations in the stomach following two administrations of the test material by oral gavage to male Wistar (Han) rats up to 350 mg/kg.

 

In conclusion, the test is valid, and Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine is genotoxic in glandular stomach cells and not genotoxic in liver and duodenum cells in the Comet assay when sampled approximately 3-4hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 350 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report.

 

This study is classified as acceptable and satisfies the requirement for OECD TG 489 for in vivo Mammalian Alkaline Comet Assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

This assay was incorporated into OECD 422 study design.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2017 - 11 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Similar to OECD 474 guideline - the study was conducted similar to a guideline study, but incorporated into an OECD 422. All validity criteria were met under both guidelines. The study was conducted under GLP.

Justification for type of information:

The assay was incorporated into OECD 422 study design but it meet all the criteria for the OECD 474 guideline.

Reason / purpose for cross-reference:

exposure-related information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Assessment incorporated into OECD 442 - ICH S2(R1) (2011).

Deviations:

no

Principles of method if other than guideline:

An integrated standard genotoxicity assessment into a general toxicology study (OECD, 2016) & ICH S2(R1) (2011).

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Specific details on test material used for the study:

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

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Males: 307.4 and 385.4 g; Females: 174.1 and 218.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of four by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 16 days prior to initiation of dosing (males) or 9 days prior to initiation of smearing (females).

DETAILS OF FOOD AND WATER QUALITY: As described above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): Minimum of 15 hrs
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 15th Feb 2018 To: 13th Apr 2018

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Formulations (excluding the control group) were stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed for up to 57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter. Females were sent to necropsy on LD 14 or 26 days post‑coitum).
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition.
A dose volume of 5 mL/kg was used. Dose volumes were calculated using the most recent recorded body weight for each animal.

Duration of treatment / exposure:

Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43.

Females were dosed for up to 57 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13).

Frequency of treatment:

Once daily

Post exposure period:

None

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4 (High)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

10 mg/kg/day cyclophosphamide (dissolved in saline).

Tissues and cell types examined:

Bone marrow from isolated femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on dose range finder study 8359421



TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Bone marrow at necropsy, no later than 24 hours after the last administration. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum and filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, room temperature).



DETAILS OF SLIDE PREPARATION: The pellet was mixed into small volume of serum using a Pasteur pipette, one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water.

Slides were dried at room temperature. Two male slides per animal were stained on the day of fixation. Female slides (two per animal) were stored at room temperature for subsequent staining. Slides were stained in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, dried and stored protected from light at room temperature prior to analysis.
Male and female positive control slides were stained and coded alongside the study slides


METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification (x400 for ratios of PCE (polychromatic erythrocytes):NCE (normochromatic erythrocytes) and either x600 or x1000 (under oil immersion) for MN PCE).
 


OTHER: The following criteria were applied during slide assessment:
1. Cells were to be of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeds the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) is observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.

Statistics:

Statistical analyses (ANCOVA) were performed.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 2: Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine: Summary and Statistical Analysis of Micronucleus Data – Bone  Marrow – Males.

Treatment (mg/kg/day)	PCE Cell Total	% PCE	MN PCE	% MN PCE†	SD	Heterogeneity		Contingency X^2C
						X^2	S	P-value	Significance
0	20000	47.16	35	0.18	0.07	4.87	NS
30	20000	48.48	36	0.18	0.12	11.80	p≤0.05	0.50	NS
50	20000	50.00	37	0.19	0.06	3.41	NS	0.45	NS
100	20000	50.08	23	0.12	0.09	12.01	p≤0.05	0.93	NS
CPA (10)*	12000	50.13	41.8	3.48	0.26			0.00	p≤0.001

* CPA slides coded from previously conducted positive control slide bank study (Covance Study Number 8360181)

† %MN PCE calculated from a total of 500 cells (PCE and NCE counted) per animal

Linear trend test P-value: 0.945, NS

NS        Not significant

MN        Micronucleated

SD        Standard deviation

Table 3: Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine: Summary and Statistical Analysis of Micronucleus Data – Bone Marrow - Females.

Treatment (mg/kg/day)	PCE Cell Total	% PCE	MN PCE	% MN PCE†	SD	Heterogeneity		Contingency X^2C
						X^2	S	P-value	Significance
0	20000	50.16	44	0.22	0.12	9.89	p≤0.05
30	20000	50.88	44	0.22	0.11	8.29	NS	0.46	NS
50	20000	47.72	44	0.22	0.11	8.06	NS	0.54	NS
100	20000	50.52	44	0.22	0.10	7.84	NS	054	NS
CPA (10)*	12000	45.40	427	3.56	0.29			0.00	p≤0.001

* CPA slides coded from previously conducted positive control slide bank study (Covance Study Number 8360181)

† %MN PCE calculated from a total of 500 cells (PCE and NCE counted) per animal

Linear trend test P-value: 0.500, NS

NS        Not significant

MN        Micronucleated

SD        Standard deviation

Conclusions:

The test item was considered non-clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP)

Executive summary:

The genotoxicity potential of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was investigated in an  Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422, adopted 29 July 2016). Covance study 8359422. Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg. The positive control slides were prepared under Covance Positive control Slide Bank Study (Covance study 8360181) from animals that had received two daily oral gavage doses of 10 mg/kg/day cyclophosphamide (dissolved in saline). Sampling of these animals was on Day 3 (approximately 24 hours post the last dose administration).

Animals treated with the test article at all doses exhibited group mean %PCE values that were either similar to or slightly higher than the value for the concurrent vehicle control group but which fell within the historical vehicle control range. As such, there was no evidence of bone marrow toxicity. Treatment with the test item exhibited group mean MN PCE frequencies that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle control group for all doses. Individual MN PCE values were consistent with historical vehicle control distribution ranges and similar to values observed in the concurrent vehicle control group.

Based on the conditions of the study, it can be stated that the test item did not induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of male and female Han-Wistar rats treated at doses of 30, 50 or 100 mg/kg/day. The test item was considered non clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

A reverse gene mutation assay (OECD 471) identified the test item to be a possible mutagen. A mammalian cell cytogenicity assay (OECD 473) identified that the test item to be a potential clastogen. In vivo testing (OECD 489) to further investigate this produced a positive result for genotoxicity in glandular stomach, without a clear linear dose-response. On this basis, a non-threshold mode of action is assumed for mutagenicity of Reaction mass of N,N,N’,N-tetrabutylmethylenediamine and dibutylamine in acetone.

In the absence of any information on species specific modes of action the available information is regarded as relevant for humans

Additional information

in vitro genotoxicity data

 

OECD 471 (2018) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Reaction mass of N,N,N’,N-tetrabutylmethylenediamine and dibutylamine in acetone at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate a screening experiment followed by concentrations of 31.25, 62.5, 125, 250, 500, 100 and 200 µg/plate (TA98 also tested at 400 and 750 µg/plate) in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix.

 

The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

 

It was concluded that the test article induced mutation in histidine-requiring strains TA98 and TA100, of Salmonella typhimurium when tested under the conditions of this study.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

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 f 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.

 

 

In vivo genotoxicity data

 

OECD 474 (incorporated into OECD 422 study design) - The genotoxicity potential of the Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was investigated in an  Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422, adopted 29 July 2016). Covance study 8359422. Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle: corn oil]), 30, 50, or 100 mg/kg/day test item once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg. The positive control slides were prepared under the Covance Positive control Slide Bank Study (Covance study 8360181) from animals that had received two daily oral gavage doses of 10 mg/kg/day cyclophosphamide (dissolved in saline). Sampling of these animals was on Day 3 (approximately 24 hours post the last dose administration).

 

Animals treated with the test item at all doses exhibited group mean %PCE values that were either similar to or slightly higher than the value for the concurrent vehicle control group but which fell within the historical vehicle control range. As such, there was no evidence of bone marrow toxicity. Treatment with the test item exhibited group mean MN PCE frequencies that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle control group for all doses. Individual MN PCE values were consistent with historical vehicle control distribution ranges and similar to values observed in the concurrent vehicle control group.

 

Based on the conditions of the study, it can be stated that the test item did not induce micronuclei in the polychromatic erythrocytes (PCE) of the bone marrow of male and female Han-Wistar rats treated at doses of 30, 50 or 100 mg/kg/day. The test item was considered non clastogenic and does not meet the criteria for classification in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).

 

OECD 489 Comet assay in the rat - Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was further investigated in an Alkaline in vivo Comet Assay performed according to the OECD TG 489 and in compliance with GLP. Males Wistar Han rats were treated twice (at 0 and 21 hours) by gavage administration with Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine  at doses of 0, 87.5, 175 and 350 mg/kg bw.  The vehicle was corn oil. Blood for bioanalysis of Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine in plasma was collected from satellite animals for the 350 mg/kg group (highest dose group) and from satellite animals for the vehicle control group. Blood was sampled 1, 2, 4, 6, and 24 h after the first dose of satellite animals dosed with the vehicle and the highest concentration of the test item. Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

 

No statistically significant increase in the mean Tail Intensity (%) was observed in liver and duodenum cells of test item treated male animals compared to the vehicle treated animals.

 

A statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals in all dose-groups and a significant trend was present. The values for the high and intermediate dose-groups were outside the historical control database of the negative controls.

 

The mean Tail Intensity in liver, glandular stomach and duodenum cells of vehicle-treated rats was 4.13 ± 0.95% (mean ± SD), 5.21 ± 1.02% (mean ± SD) and 11.96 ± 3.01% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control, except in the duodenum cells. Since the mean Tail Intensity was just above the upper limit of the acceptability criteria, this has no effect on the validity of the results. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 81.74 ± 2.35% (mean ± SD), 60.74 ± 2.91% (mean ± SD) and 56.65 ± 4.67% (mean ± SD) in male animals in liver, glandular stomach and duodenum cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

 

Histopathology of the stomach was performed. There were no morphologic alterations in the stomach following two administrations of the test material by oral gavage to male Wistar (Han) rats up to 350 mg/kg.

 

In conclusion, the test is valid, and Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine is genotoxic in glandular stomach cells and not genotoxic in liver and duodenum cells in the Comet assay when sampled approximately 3-4hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 350 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report.

Justification for classification or non-classification

Harmonised classification: 

The substance has no harmonised classification for genetic toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification: 

The substance meets the criteria for classification for germ cell mutagenicity Category 2 – H341: Suspected of causing genetic defects – in accordance with Regulation (EC) 1272/2008 (CLP).