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

Genetic toxicity: in vivo

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetrabutylmethylenediamine

EC Number:

243-678-5

EC Name:

N,N,N',N'-tetrabutylmethylenediamine

Cas Number:

20280-10-8

Molecular formula:

C17H38N2

IUPAC Name:

dibutyl[(dibutylamino)methyl]amine

Constituent 2

Reference substance name:

Dibutylamine

EC Number:

203-921-8

EC Name:

Dibutylamine

Cas Number:

111-92-2

Molecular formula:

C8H19N

IUPAC Name:

1-Butanamine, N-butyl-

Test material form:

liquid

Details on test material:

- Physical description: Clear light yellow liquid
- Storage conditions: In refrigerator (2-8°C) protected from light
- Expiration date of the lot / batch: 01 July 2022

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)

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

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.

Examinations

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 .

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.