N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α'-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo, other

Remarks:

Combined gene mutation (comet assay) and micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2022 - 26 September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: combined mammalian comet assay and mammalian micronucleus assay

Test material

Test material information

1

Reference substance name:

N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α'-diamine

EC Number:

264-438-6

EC Name:

N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α'-diamine

Cas Number:

63738-22-7

Molecular formula:

C20H28N2O4

IUPAC Name:

N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α'-diamine

Specific details on test material used for the study:

Physical description: light yellow liquid
Storage conditions: In refrigerator (2-8°C)
Test material handling: Handle in glove box or AtmosBag (nitrogen environment)
Specific gravity/density: 1.14 – 1.16 (25°C)
Solubility in vehicle:
- Water: insoluble
- Acetone: soluble
- Toluene: soluble
- Xylene: soluble
- N-Hexane: soluble
- Stability in vehicle: Stability analyses performed previously in conjunction with (Test Facility Study No. 20341993) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
- Water: not stable

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Females included in the study: only in the dose-range finding study. Females were nulliparous and non-pregnant
- Assigned to test groups randomly: yes, males and females were randomized separately.
- Fasting period before study: Not reported
- Housing: Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) containing sterilized sawdust as bedding material equipped with water bottles. During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water: ad libitum, municipal tap water.
- Contaminants: It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS (target ranges)
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 25 July 2022 To: 08 September 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: In the dose-range finding study, 5 dose-groups were used to define the MTD based on the toxic signs observed after dosing with different doses of the test material in the three different vehicles 0.1% Methylcellulose (1% MC), Propylene Glycol (PG) and Corn Oil. The suited vehicle was Corn Oil due to preliminary stability data obtained, in agreement with the Sponsor.
- Concentration of test material in vehicle: not reported
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: not reported
- Purity: not reported

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was suspended in corn oil. The specific gravity of corn oil is 1.0 g/mL. Test material concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. This resulted in yellow suspensions for all formulations. Test material concentrations were dosed within 2.5 hours after preparation.

Duration of treatment / exposure:

Three days

Frequency of treatment:

Once daily

Post exposure period:

3-4 hours after the third treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

187.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (only 3 animals per sex per dose)
- Justification for choice of positive control(s): generally used positive control for micronucleus test
- Route of administration: oral gavage
- Doses / concentrations: 19 mg/kg bw dissolved in physiological saline, dosed once

Ethylmethanesulphonate (only 3 animals per sex per dose)
- Justification for choice of positive control(s): generally used positive control for comet assay
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw/day dissolved in physiological saline, dosed twice

Examinations

Tissues and cell types examined:

Bone marrow was isolated for the micronucleus test.
In addition liver, duodenum and glandular stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a dose-range finding study 10 animals (5 dose groups: 1 male and 1 female per group) were dosed via oral gavage received a repeated (with 24 h interval) with 500, 750 and 1172 mg/kg body weight. In dose group 1 and 2, 1% Methylcellulose (1% MC) was used as vehicle for dose levels of 500 and 750 mg/kg body weight, respectively. In dose group 3, Propylene Glycol (PG) was used as vehicle for the dose level of 750 mg/kg body weight. In dose group 4 and 5, Corn Oil was used as vehicle for dose levels of 750 and 1172 mg/kg body weight, respectively. In the dose-range finding test, three male and three female animals dosed with 750 mg test material per kilogram body weight showed treatment related clinical signs.

TREATMENT AND SAMPLING TIMES: See table one under 'Any other information on materials and methods, incl. tables'

COMET assay
DETAILS OF SLIDE PREPARATION:
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.

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 duodeunum 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.


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.

Preparation of comet slides
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 were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 10-60 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 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 or 30 (liver) 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 – 1 Volt/cm. The electrophoresis was performed for 20 to 30 minutes under constant cooling (actual temperature 4 - 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 9-10 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.

METHOD OF ANALYSIS:
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.
In addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control.

MICRONUCLEUS assay
Preparation of bone marrow smears
The supernatant was removed with a Pasteur pipette. Approximately 500 µl serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.

Staining of bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

METHOD OF ANALYSIS
To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.

Evaluation criteria:

Comet assay
A test material 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 material 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.


Micronucleus assay
A test material is considered positive in the micronucleus test 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 the frequency of micronucleated polychromatic erythrocytes 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 material is considered negative in the micronucleus test if:

a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes 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.

Statistics:

Comet assay: ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.
As the Step-down Jonckheere-Terpstra Test Procedure shows that there are statistically significant differences between one or more of the test material 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.

Micronucleus assay: ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. As the William’s Multiple t test shows that there are statistically significant differences between one or more of the test material groups and the vehicle control group, a Cochran Armitage Trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Micronucleus assay: not clastogenic or aneugenic

Toxicity:

yes

Remarks:

Clinical observations were made in the groups treated with 750 mg test material/kg bw/day

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

Comet assay: equivocal results obtained in the liver cells; positive results obtained in the duodenum cells; negative results obtained in the glandular stomach cells

Toxicity:

yes

Remarks:

Clinical observations were made in the groups treated with 750 mg test material /kg bw/day

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose formulation analysis
Accuracy
In the vehicle, no test material was detected.
The concentrations analyzed in the dose formulation samples were in agreement with target concentrations ((i.e., mean sample concentration results were within or equal to 85%-115%).

Homogeneity
The dose formulation samples were homogeneous (i.e., coefficient of variation ≤ 10%).

Mortality and Toxic Signs
The animals of the group treated with 187.5 and 350 mg test material/kg body weight/day and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Clinical observations were made in the groups treated with 750 mg test material/kg body weight/day.

Micronucleated Polychromatic Erythrocytes
The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in table 2 under 'Any other information on results incl. tables'. The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.
An increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the highest concentration of the test material, compared to the vehicle treated animals. However, the data was within the 95% control limits of the distribution of the historical negative control database and no trend was observed (p=0.1356) and therefore the increase was considered as not biologically relevant.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database (Table 3 under 'Any other information on results incl. tables').
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database (Table 4 under 'Any other information on results incl. tables'). Hence, all criteria for an acceptable assay were met.

The animals of the groups, which were treated with test material and the positive control showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Comet Slide Analysis
Comet slides were prepared and analyzed. An overview of the mean Tail Intensity is presented in Table 5 -7 under 'Any other information on results incl. tables'.
The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.74 ± 1.97% (mean ± SD), 8.28 ± 3.37% (mean ± SD), and 6.52 ± 1.51% (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 (Table 8). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 89.51 ± 0.88% (mean ± SD), 56.01 ± 3.47% (mean ± SD), and 66.06 ± 3.66% (mean ± SD) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database (Table 9).
Adequate numbers of cells (150 cells per animal) and doses were analysed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.
A statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male animals compared to the vehicle treated animals.
In liver cells, the two highest test item-treated groups showed statistically significant increases in tail intensity and in addition a significant trend analysis was observed. However, the tail intensity increases were within the 95% control limits of the distribution of the historical control data for the vehicle control and were therefore considered as equivocal.
In duodenum cells, the two highest test item-treated groups showed statistically significant increases in tail intensity and in addition a significant trend analysis was observed. In addition, the tail intensity increases were outside the 95% control limits of the distribution of the historical control data for the vehicle control and were therefore considered as biologically relevant.
In glandular stomach cells, the highest test item-treated group showed a statistically significant increase in tail intensity. However, no significant trend was observed and the tail intensity increases were within the 95% control limits of the distribution of the historical control data for the vehicle control and was therefore considered as not biologically relevant.

Histopathology
Test material-related microscopic findings in the duodenum were present at all doses and consisted of
• Eosinophilic inflammatory infiltrates in the lamina propria at all doses (up to moderate degree)
• Apoptosis/necrosis of single mucosal cells at all doses (up to moderate degree)
• Slight regeneration of the mucosa at all doses
• Villous atrophy (in few animals with additional necrosis of lamina propria and hemorrhages) at 750 mg/kg bw/day (up to moderate degree)
Following three administrations of N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α’-diamine by oral gavage to male Wistar (Han) rats up to 750 mg/kg bw, treatment related morphologic alterations were present in the duodenum starting at 187.5 mg/kg bw/day (inflammatory infiltrates, apoptosis/necrosis single cells, mucosal regeneration and/or villous atrophy).

Any other information on results incl. tables

Table 2. Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group	Treatment	Number of Animals	Dose	Number of micronucleated polychromatic erythrocytes			Ratio polychromatic/ normochromatic erythrocytes
				(mean ± S.D.) ^(1)
			(mg/kg body weight)				(mean ± S.D.) ^(2)
	MALES
1	Vehicle Control	5	0	2.4	±	1.1	1.44	±	0.28
2	Test Material	5	187.5	4.0	±	1.4	1.07	±	0.08 ^(5)
3	Test Material	5	375	3.6	±	1.5	1.04	±	0.08 ^(5)
4	Test Material	5	750	5.0	±	2.3 ^(4)	0.83	±	0.10 ^(5)
5	CP	3	19	27.7	±	6.5 ^(3)	0.26	±	0.03 ^(3)

Vehicle control = Corn Oil

CP = Cyclophosphamide.

(1)   At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(2)   The ratio was determined from at least the first 500 erythrocytes counted.

(3)   Significantly different from corresponding control group (Students t test, P < 0.001).

(4)   Significantly different from corresponding control group (Williams’ t test, P ≤ 0.05).

(5)   Significantly different from corresponding control group (Step-down Jonckheere-Terpstra Test Procedure, P ≤ 0.05).

 

Table 3. Historical Negative Control Data for Micronucleus Studies

 

	Male
Mean Number of Micronucleated cells per 4000 cells	3.8
SD	1.4
N	59
Lower Control Limit (95% Control Limits)	1
Upper Control Limit (95% Control Limits)	7

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between May 2019 and May 2022.

 

Table 4. Historical Positive Control Data for Micronucleus Studies

	Male
Mean Number of Micronucleated cells per 4000 cells	34.6
SD	23.6
N	55
Lower Control Limit (95% Control Limits)	-12
Upper Control Limit (95% Control Limits)	81

 

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between May 2019 and May 2022.

 

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

	Tail Intensity (%)	S.D.
Vehicle Control	2.74	1.97
Test Material 187.5 mg/kg	3.36	0.96
Test Material 375 mg/kg	3.60	0.70
Test Material 750 mg/kg	4.48	1.47
EMS 200 mg/kg	89.51	0.88

 

Table 6. Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	8.28	3.37
Test Material 187.5 mg/kg	12.05	2.30
Test Material 375 mg/kg	17.11	5.41
Test Material 750 mg/kg	19.57	3.20
EMS 200 mg/kg	56.01	3.47

 

 Table 7. Overview Tail Intensity in Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	6.52	1.51
Test Material 187.5 mg/kg	7.92	0.40
Test Material 375 mg/kg	8.76	3.54
Test Material 750 mg/kg	8.36	0.78
EMS 200 mg/kg	66.06	3.66

 

Table 8.

Historical Negative Control Data for Comet Assay

 

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	2.7	6.5	6.4
SD	1.4	3.0	3.1
n	49	34	33
Lower control limit (95% control limits)	-0.1	0.6	0.3
Upper control limit (95% control limits)	5.5	12.3	12.5

SD = Standard deviation

n = Number of observations

 

Liver, Duodenum, Stomach:

Historical control data from experiments performed in May 2019 – May 2022

 

Table 9. Historical Positive Control Data for Comet Assay 

	Liver Tail Intensity (%) Males and Females	Duodenum Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	81.6	50.7	56.5
SD	7.6	9.6	8.7
n	44	34	33
Lower control limit (95% control limits)	66.6	31.9	39.5
Upper control limit (95% control limits)	96.6	69.5	73.5

SD = Standard deviation

n = Number of observations

 

Liver, Duodenum, Stomach:

Historical control data from experiments performed in May 2019 – May 2022

Applicant's summary and conclusion

Conclusions:

An in vivo combined comet/micronucleus assay has been performed according to OECD TG 489 and 474 and in accordance with GLP principles. Based on the results, the test substance is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 750 mg/kg bw/day (the maximum tolerated dose). The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 750 mg/kg bw/day with the test item (the maximum tolerated dose) under the experimental conditions described as found to be valid. The test material is equivocal in the Comet assay in liver cells as there was a significant dose-response shown however within historical control range of the vehicle control. The test material is positive in the Comet assay in duodenum cells as there was a significant dose-response shown and outside historical control range of the vehicle control. The morphological alterations observed in the duodenum could possibly be the cause of the positive outcome in the Comet assay. The test material is not genotoxic in the Comet assay in glandular stomach cells.

Executive summary:

An in vivo combined comet/micronucleus assay has been performed according to OECD TG 489 and 474 and in accordance with GLP principles.

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 materials. Moreover, historical control background data has been generated with this strain.

In the vehicle, no test material was detected. The concentrations analyzed in the dose formulation samples were in agreement with target concentrations ((i.e., mean sample concentration results were within or equal to 85%-115%). The dose formulation samples were homogeneous (i.e., coefficient of variation ≤ 10%).

Based on the results of the dose-range finding study test concentrations of 750 mg/kg bw/day for male animals was selected as maximum dose for the main test (the maximum tolerated dose). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

In the main study male animals were dosed three times by oral gavage with vehicle or with 187.5, 375 and 750 mg test material per kg body weight for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group for the micronucleus assay was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control (3 animals per group).

Clinical signs of toxicity were limited to the high dose group and included rough coat and hunched posture.

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. Bone marrow smears were prepared for micronucleus analysis.

Micronucleus

No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

The groups that were treated with the test material and with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, demonstrating toxic effects on erythropoiesis.

Comet

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.74 ± 1.97% (mean ± SD), 8.28 ± 3.37% (mean ± SD), and 6.52 ± 1.51% (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. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 89.51 ± 0.88% (mean ± SD), 56.01 ± 3.47% (mean ± SD), and 66.06 ± 3.66% (mean ± SD) in male animals in liver, duodenum and glandular stomach 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.

A statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male animals compared to the vehicle treated animals.

In liver cells, the two highest test item-treated groups showed statistically significant increases in tail intensity and in addition a significant trend analysis was observed. However, the tail intensity increases were within the 95% control limits of the distribution of the historical control data for the vehicle control and were therefore considered as equivocal.

In duodenum cells, the two highest test item-treated groups showed statistically significant increases in tail intensity and in addition a significant trend analysis was observed. Furthermore, the tail intensity increases were outside the 95% control limits of the distribution of the historical control data for the vehicle control and were therefore considered as biologically relevant.

Duodenum tissues collected from group 1-4 were evaluated histopathologically. There were morphologic alterations in the duodenum following three administrations of N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α’-diamine by oral gavage to male Wistar (Han) rats up to 750 mg/kg bw, treatment related morphologic alterations were present in the duodenum starting at 187.5 mg/kg bw/day (inflammatory infiltrates, apoptosis/necrosis single cells, mucosal regeneration and/or villous atrophy).

In glandular stomach cells, the highest test item-treated group showed a statistically significant increase in tail intensity. However, no significant trend was observed, and the tail intensity increases were within the 95% control limits of the distribution of the historical control data for the vehicle control and was therefore considered as not biologically relevant.

In conclusion, N,N,N’,N’-tetrakis(2,3-epoxypropyl)-m-xylene-α,α’-diamine is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 750 mg/kg bw (the maximum tolerated dose) under the experimental conditions described in this report.

The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 750 mg/kg bw with the test item (the maximum tolerated dose) under the experimental conditions described in this report as found to be valid. The test material is equivocal in the Comet assay in liver cells as there was a significant dose-response shown however within historical control range of the vehicle control. The test material is positive in the Comet assay in duodenum cells as there was a significant dose-response shown and outside historical control range of the vehicle control. The morphological alterations observed in the duodenum could possibly be the cause of the positive outcome in the Comet assay. The test material is not genotoxic in the Comet assay in glandular stomach cells.