N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (Key, OECD 471), Ames: positive with and without metabolic activation

Mammalian Cytogenicity (Key, OECD 473), Chromosome Aberration: positive with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jan 2018 - 07 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon for S. typhimurium strains
trp operon for E. coli strains

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arocolor 1254.

Test concentrations with justification for top dose:

Pre-experiment (dose-range finding test, Direct Plate Assay): 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate
Main-experiment (Pre-incubation Assay): 17, 52, 164, 512, 1600, 5000 µg/plate (Based on the results of the dose-range finding test)

Vehicle / solvent:

- Vehicle/solvent used: water (Milli-Q-water, Millipore Corp., Bedford, MA., USA).
- Justification for choice of solvent/vehicle: The test item formed a clear colorless solution in Milli-Q water.

- Vehicle/solvent used for positive control items: Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, Direct Plate Assay, Pre-incubation assay

DURATION
- Preincubation period: 30 ± 2 min
- Exposure duration: at least 48 ± 4 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
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 negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

2.3-fold dose-related increase in the number of revertant colonies in the absence of S9-mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

1.9-fold dose-related increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the laboratory historical control data ranges and less than two-fold the concurrent solvent control

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Remarks:

Cytotoxicity was observed at 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Direct Plate Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains. Except for tester strain WP2uvrA, where no toxicity was observed in the absence or presence of S9-mix.

Pre-Incubation Assay
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 μg/plate onwards without metabolic activation and at 5000 μg/plate with metabolic activation. Except for tester strain WP2uvrA in the presence of S9-mix, where no toxicity was observed

  Table 3: Dose-Range Finding Test (Direct-Plate assay)

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 100	WP2uvrA
Vehicle Control		113 ± 11	33 ± 11
Test Substance	1.7	121 ± 15	25 ± 5
	5.4	123 ± 19	37 ± 2
	17	115 ± 12	21 ± 1
	52	134 ± 12	33 ± 6
	164	141 ± 13	37 ± 6
	512	166 ± 16	35 ± 0
	1600	292 ± 22^n	41 ± 9
	5000	0 ± 0^aNP	51 ± 4^nNP
Positive Control		744 ± 65	1602 ± 77
	S9-Mix	With
	Test item (µg/plate)	TA 100	WP2uvrA
Vehicle Control	-	118 ± 6	43 ± 7
Test Substance	1.7	100 ± 8	50 ± 6
	5.4	106 ± 13	40 ± 3
	17	116 ± 13	35 ± 9
	52	122 ± 6	38 ± 8
	164	135 ± 18	43 ± 4
	512	169 ± 21	58 ± 5
	1600	310 ± 19	75 ± 4
	5000	106 ± 94^1NP	52 ± 5^nNP
Positive Control		2156 ± 227	558 ± 54
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Table 4: Direct-Plate assay

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 1535	TA 1537	TA 98
Vehicle Control		12 ± 2	3 ± 2	11 ± 1
Test Substance	17	12 ± 2	3 ± 0	17 ± 5
	52	13 ± 5	4 ± 3	14 ± 3
	164	14 ± 4	2 ± 1	11 ± 3
	512	13 ± 2	2 ± 1	10 ± 6
	1600	11 ± 3^n	2 ± 1^n	12 ± 6^n
	5000	0 ± 0^aNP	2 ± 2^eNP	0 ± 0^aNP
Positive Control		1091 ± 65	834 ± 49	1141± 30
	S9-Mix	With
	Test item (µg/plate)	TA 1535	TA 1537	TA 98
Vehicle Control	-	11 ± 6	3 ± 4	16 ± 2
Test Substance	17	12 ± 5	4 ± 3	16 ± 3
	52	10 ± 2	7 ± 4	22 ± 6
	164	13 ± 5	4 ± 1	16 ± 4
	512	17 ± 8	3 ± 2	17 ± 4
	1600	18 ± 3	3 ± 2	19 ± 7
	5000	5 ± 8^1NP	1 ± 2^1NP	3 ± 2^1NP
Positive Control		408 ± 32	374± 108	1298± 195
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Table 5: Plate-Incubation Test

	EXPERIMENT 2 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100 (fold)	WP2uvrA (fold)
Vehicle Control		10 ± 2	3 ± 4	18 ± 3	106 ± 14	19 ± 5
Test Substance	17	13 ± 5	3 ± 1	17 ± 6	122 ± 15	28 ± 2
	52	12 ± 2	3 ± 2	13 ± 5	130 ± 5	29 ± 15 (1.5)
	164	19 ± 1	3 ± 2	14 ± 3	134 ± 16 (1.3)	35 ± 7 (1.8)
	512	13± 3^n	8 ± 2^n	17 ± 3^n	195 ± 5^n(1.8)	44 ± 8^n(2.3)
	1600	0 ± 0^a	0± 0^a	0± 0^a	0± 0^a	1± 0^1n
	5000	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP
Positive Control		1036 ± 33	138 ± 14	1146 ± 68	781 ± 11	152 ± 85
	S9-Mix	With
	Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Vehicle Control	-	11 ± 2	5 ± 3	19 ± 6	102 ± 2	30 ± 7
Test Substance	17	13 ± 2	4 ± 2	15 ± 6	166 ± 18	36 ± 6
	52	10 ± 5	7 ± 2	13 ± 2	180 ± 20	42 ± 6
	164	14 ± 3	7 ± 2	19 ± 4	139 ± 7	39 ± 8
	512	10 ± 0	5 ± 3^n	13 ± 2	190 ± 26 (1.9)	42 ± 4 (1.4)
	1600	14 ± 6	2 ± 1^s	15 ± 3^n	304 ± 22^n(3.0)	56 ± 13 (1.9)
	5000	0 ± 1^1nNP	0 ± 0^aNP	0 ± 0^aNP	0 ± 0^aNP	57 ± 11^nNP(1.9)
Positive Control		247 ± 22	230 ± 32	762 ± 14	1586 ± 89	587 ± 30
	1=Revertants were only observed on a small part of the plate with normal background, no bacterial growth or background was observed on a large part of the plate NP= No precipitate a = Bacterial background lawn absent n = Normal bacterial background lawn s = Bacterial background lawn slightly reduced

 

Conclusions:

Under the conditions of the conducted test the substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA100 and WP2uvrA both in the absence and presence of S9-mix. In the latter, only in the absence of S-9 mix in experiment 2, these increases exceeded the two-fold threshold (2.3-fold). Based on these results, the test substance is regarded to be mutagenic in bacteria.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 1986 - 01 July 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood was obtained by venepuncture from a healthy, non-smoking, male, human volunteer not currently taking any medication, and collected in heparinised vessels
- Sex, age and number of blood donors if applicable: One male volunteer, age not reported
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
Complete culture medium: 418.0 mL RPMI 1640 medium with HEPES, sodium bicarbonate and L-glutamine; 75.0 mL fetal calf serum; 5.0 mL Heparin (100 i.u./mlL); and 2.0 mL Penicillin/streptomycin (5000 i.u./mL; 5000 ug/mL)
Treatment medium: 480.5 mL RPMI 1640 medium with HEPES, sodium bicarbonate and L-glutamine; 12.5 mL fetal calf serum; 5.0 mL Heparin (100 i.u./mlL); and 2.0 mL Penicillin/streptomycin (5000 i.u./mL; 5000 ug/mL)

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction of Arcolor 1254 induced rats

Test concentrations with justification for top dose:

Preliminary experiment: 1.4, 7.2, 36, 180, 900 µg/mL, ± S9

Main experiment: 7.5, 15.0 and 30.0 ug/mL, -S9; 3.75, 7.5 and 15.0 µg/mL, +S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The compound was found to be soluble in dimethyl sulphoxide (DMSO) up to a maximum concentration of approximately 184 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 24 h with and without metabolic activation during the first two hours of exposure
- Expression time (cells in growth medium): 24 h (including a 3 hour Colcemid treatment)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After cells were fixed, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were
transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic test respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer pH 6.8), washed in buffer and left to air-dry. Permanent mounts were made using DPX mountant after clearing in xylene.

NUMBER OF CELLS EVALUATED: For the preliminary experiment, approximately 1000 lymphocytes per culture were examined using a light microscope, and the mitotic index was calculated as the percentage. For the main experiment, approximately 1000 cells were scored and the mitotic index calculated.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphases (with 46 centromeres)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Methods: Scoring followed the recommendations of the Ad Hoc committee of the Environmental Mutagen Society and the Institute for Medical Research, 1972

Evaluation criteria:

According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of the results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both, biological and if evaluated, statistical significance should be considered together. A test item is considered to be negative if there is no biologically relevant increase in the percentage of aberrant cells above concurrent control levels, at any dose group.

Statistics:

The Fisher Exact Probability test is a useful nonparametric technique for analysing data when comparing two small independent samples. It is used when the scores for the samples all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications.

Data from replicate cultures with or without S-9 mix were compared. If significant differences were found, each treatment was then compared with the respective negative control - with or without activation. Where no significant differences were found, data from activated and non-activated cultures were pooled for subsequent analysis.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

15 and 30 µg/mL

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

7.5 and 15 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

26% reduction at 15 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The preliminary test showed marked depression of cell division at an test item concentration of 36.0 µg/mL (65% reduction of mitotic index over control values without S-9 mix, 93% reduction with S-9 mix).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the absence of S-9 mix mean mitotic indices were 3.3, 2.7 and 2.4 for treatment levels of 7.5, 15.0 and 30.0 µg/mL respectively, compared with the solvent control value of 2.4 showing no reduction in mitotic activity at any level tested. In the presence of S-9 mix mean mitotic indices were 7.6, 7.3 and 6.1 for treatment levels of 3.75, 7.5 and 15 µg/mL, showing an approximately 26% reduction in mitotic activity at the highest test item concentration tested compared to the control value of 8.2.

Table 1: Main cytogenetic test – number and types of chromosomal aberrations

 

Group	Concentration (µg/mL)	S-9 Mix	Numbers of cells scored	Mean mytotic index	Cells with aberrations			Cells with aberrations
					Total	Range %	Mean %*	Total	Range %	Mean %*
DMSO	/	-	300	2.4	9	1-5	3.0	3	0-2	1.0
		+	300	8.2	6	1-4	2.0	0	0-0	0.0
Test substance	3.75	-	/	/	/	/	/	/	/	/
		+	300	7.6	14	3-7	4.7	4	0-2	1.3
	7.5	-	300	3.3	14	3-6	4.7	5	1-2	1.7
		+	300	7.3	14	2-7	4.7	6	1-3	2.0
	15.0	-	212	2.7	31	14-15.4	14.6	17	7-10	8.0
		+	300	6.1	23	5-10	7.7	9	2-4	3.0
	30.0	-	60	2.4	9	0-26.1	15.0	9	0-26.1	15.0
		+	/	/	/	/	/	/	/	/
Cyclophosphamide	6	-	300	5.3	4	0-4	1.3	0	0-0	0.0
		+	300	2.9	188	56-68	62.7	149	43-58	49.7
Chlorambucil	2.5	-	300	2.9	123	32-48	41.0	80	23-33	26.7
		+	/	/	/	/	/	/	/	/

*Mean% = (Number aberrant metaphases/ Total cells scored) x 100

Conclusions:

Under the conditions of the conducted test, the test substance induced damage to chromosomal structure in peripheral human lymphocytes. In the main experiment statistically significant increase in % aberrant cells was observed at test substance concentration of 15 and 30 µg/mL without metabolic activation and at 7.5 and 15 µg/mL with metabolic activation (cytotoxicity detected at 15 µg/mL). Therefore, the test substance is regarded to be clastogenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Combined Micronucleus and Alkaline Comet Test in the Rat (Key, OECD 474/489): negative

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

a combined micronucleus and alkaline comet test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Sept to 03 Nov 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 474. Mammalian Erythrocyte Micronucleus Test and OECD Guideline 489. In Vivo Mammalian Alkaline Comet Assay.

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian comet assay and mammalian erythrocyte micronucleus assay

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: 6-7 weeks
- Weight at study initiation: 152.0-160.8g
- Assigned to test groups randomly: Yes. Animals in poor health or at extremes of body weight range were not assigned to groups. Females (used in the dose ranging finding experiment only) were nulliparous and non-pregnant.
- Housing: Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
Up to 5 animals of the same sex and same dosing group were housed together.

- Diet: Ad libitum, except during designated procedures. SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water: Freely available to each animal via water bottles. Municipal tap water.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22
- Humidity (%): 45 to 67
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 Sept 2022 To: 13 Oct 2022

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Water
- Justification for choice of solvent/vehicle: Stability analyses performed previously in conjunction with Study No. 20134629 demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the study.
- Concentration of test material in vehicle: 5, 10 and 20 mg/mL
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in Milli-Q water. This resulted in light pink solutions for all formulations. Test material concentrations were dosed within 5.5 hours after preparation.
Dose formulation analysis for concentration (all groups) and homogeneity (50 and 200 mg/kg bw/day groups) was performed on a single occasion.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

Daily

Post exposure period:

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

Dose / conc.:

50 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

200 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

Five male rats were used in each treatment group, with the exception of the highest dose group (8 animals). Three male rats in the vehicle control and highest dose group were used for bioanalysis.

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate (EMS)
Justification for choice of positive control(s): EMS is a known mutagen and is recommended in the OECD 489 test guideline for use with any target tissue.
- Route of administration: Oral
- Doses / concentrations: 200 mg/kg bw dissolved in physiological saline.
Positive control slides taken from male animals previously dosed with positive control cyclophosphamide (CP) (19 mg/kg bw/day at 10 mL/kg bw in physiological saline dosed orally 48 hours prior to sampling), as part of Test Facility Study No. 20337827 were also used.

Tissues and cell types examined:

Liver, glandular stomach, duodenum and bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Selection of an adequate dose-range for the main test was based on a dose-range finding (DRF) study. The test procedure and conditions were similar to those applied in the main test. In the dose-range finding study, 3 dose-groups were used to define the maximum tolerated dose (MTD) based on the toxic signs observed after dosing with different doses of the test material. One dose group, comprising of 3 males and 3 females, was dosed for three consecutive days (once daily) with the highest concentration of test material that was used for the main study. The other 2 groups consisted of 1 male and 1 female animal. The observation period after dosing was one to three days. During this period mortality and physical condition were recorded at least once a day.

The clinical signs of the DRF for the current OECD 489/474, did not indicate that the MTD had been reached. However, 200 mg/kg bw/day was considered to be a suitable top dose level for the main study. Based on the information provided by the Sponsor, in a previous study (OECD 422), all females were killed in extremis at 150 mg/kg bw/day and at 75 mg/kg bw/day showed clinical signs and adverse effects at necropsy. In an acute oral study, the LD50 was > 75 mg/kg bw, but < 300 mg/kg bw. Additionally, the OECD 422 DRF and acute oral study showed that the systemic toxicity is similar between males and females and therefore it was acceptable to run the main study with males only.

TREATMENT AND SAMPLING TIMES
The animals were dosed with vehicle or the test material for three consecutive days and twice with EMS. Bioanalysis blood samples were taken from 3 additional control and top dose males on Day 2 at 0, 1, 2, 4, 6, and 24 hrs post dose.
Approximately 3-4 hours after the third treatment with the test material bone marrow was isolated for the micronucleus test. In addition duodenum, glandular stomach and liver were collected/isolated and examined for DNA damage with the alkaline comet assay.

IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
Mortality: All animals were assessed twice daily from arrival to termination
Clinical observations: All animals were assessed daily from start of treatment to termination
Bodyweights: All animals were weighed prior to dosing

BIOANALYTICAL SAMPLE COLLECTION
Blood samples were taken from satelite animals control and top dose males on Day 2 at 0 hr (pre-dose) and 1, 2, 4, 6, 24 hrs post-dose via jugular vein puncture or if not feasible the retro-orbital sinus (under isoflurane anaesthesia). Samples were collected on ice. Resultant plasma was directly transferred into the stabilizer solution. Plasma/stabilizer solution must be in 1/1 (v/v) ratio.
Bioanalytical Sample Processing:
Immediately following the blood collection, samples were centrifuged within 15 minutes at approximately 2000 g for 10 minutes at 4-8 °C and the resultant plasma was separated, immediately transferred to uniquely labelled clear Micronic V-bottomed 1.4 mL tubes containing enough stabilizer solution to achieve 1:1 dilution (e.g. 50 µL of plasma was transferred to a tube containing 50 µL of stabilizer solution). Samples were mixed by gently inverting the tube few times and frozen immediately over dry ice or in a freezer set to maintain -80 °C.
Bioanalytical Sample Analysis:
Plasma samples were analyzed for concentration of Test Material using a LC-MS/MS method (liquid chromatography with tandem mass spectrometry) (Charles River project number 20365406).
Statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy and precision were performed.

DETAILS OF SLIDE PREPARATION
Liver cells
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.

Glandular stomach cells
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca2+, Mg2+ 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 consisted 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.

Duodenum cells
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 were collected in the mincing buffer present in the petri-dish. The mincing buffer consisted of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca2+, Mg2+ 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.

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, together with the part of the tail containing the identification number.

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 to 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 16 to 18 hrs) 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 minutes (glandular stomach and duodenum) or 30 minutes (liver) 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 4.0 °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 5 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.

Isolation of Bone Marrow:
Bone marrow were sampled 48 hrs after the first dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum. The cell suspensions were collected and centrifuged at 216 g for 5 min.

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 the 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 was based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
Positive control slides taken from male animals previously dosed with positive control (cyclophosphamide, 19 mg/kg bw/day at 10 mL/kg bw dosed orally 48 hours prior to sampling), as part of Test Facility Study No. 20337827, were added to the study slides for evaluation as scoring controls. Data from these animals was entered in the study as a positive control group for use in statistical evaluation. Arbitrary animal numbers were given based on the standard animal identification scheme. Slides from the positive control will be archived together with the study.

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 (Instem, Staffordshire, 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.

Analysis of the bone marrow smears for micronuclei:
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 proportion of immature erythrocytes was determined by counting and differentiating at least the first 500 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:
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.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg bw/day

Micronucleus Test:
A micronucleus test 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 concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.

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.

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.


 

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE FORMULATION ANALYSIS
Accuracy:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e., mean accuracies between 90% and 110%).
No test item was detected in the vehicle control group formulations.
Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

RESULTS OF RANGE-FINDING STUDY
The results of this dose-range finding study are presented in Table 1 (Any other information on results incl. tables).
No abnormalities were observed, except one female dosed at 200 mg/kg bw/day, which showed hunched posture, rough coat and eyes partly closed on Day 2 post-dose and on Day 3 pre- and post-dose.

RESULTS OF DEFINITIVE STUDY
Based on information provided by the sponsor and the results of the dose-range finding study dose levels of 50, 100 and 200 mg/kg bw/day were selected as appropriate doses for the main test. The clinical signs of the DRF for the current OECD 489/474, did not indicate that the MTD had been reached. However, 200 mg/kg bw/day was considered to be a suitable top dose level for the main study. Based on the information provided by the Sponsor, in a previous study (OECD 422), all females were killed in extremis at 150 mg/kg bw/day and at 75 mg/kg bw/day showed clinical signs and adverse effects at necropsy. In an acute oral study, the LD50 was >75 mg/kg bw, but <300 mg/kg bw. Additionally, the OECD 422 DRF and acute oral study showed that the systemic toxicity is similar between males and females and therefore it was acceptable to run the main study with males only.
Five male animals were used in each treatment group, with the exception of the positive control and TK animals and the highest dose group (3 and 8 animals per group, respectively). The mean body weights per group were recorded immediately prior to dosing and are presented in Table 2 (Any other information on results incl. tables).

Mortality and Toxic Signs:
The animals of the groups treated with 50, 100 and 200 mg/kg bw/day and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

Micronucleated Polychromatic Erythrocytes:
The mean number of micronucleated polychromatic erythrocytes per group and the mean proportion of immature erythrocytes are presented in Table 3 (Any other information on results incl. tables). The individual data are described in Table 7 (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. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals 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 (Table 28, 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 29, Any other information on results incl. tables). Hence, all criteria for an acceptable assay were met.

Proportion of Immature Erythrocytes:
The animals of the groups which were treated with test material showed no decrease in the proportion of immature erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the proportion of immature 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 4 - 6 (Any other information on results incl. tables). The detailed data of the treatment groups is presented in Table 8-10 (Any other information on results incl. tables). The detailed data of the individual rats is presented in Table 11 - 25 (Any other information on results incl. tables). No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum, glandular stomach and liver cells of test material treated male treated animals compared to the vehicle treated animals. In addition, there were no Hedgehogs observed in vehicle and test material treated groups. The mean Tail Intensity in duodenum, glandular stomach and liver cells of vehicle-treated rats was 4.01 ± 1.19% (mean ± SD), 7.44 ± 1.76% (mean ± SD) and 3.35 ± 0.89% (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 26, Any other information on results incl. tables). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 44.08 ± 2.50% (mean ± SD, p<0.001 Students t test), 58.37 ± 4.46% (mean ± SD, p<0.001 Students t test) and 79.49 ± 0.82% (mean ± SD, p<0.001 Students t test) in male animals in duodenum, glandular stomach and liver cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database (Table 27, Any other information on results incl. tables). Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Bioanalysis:
Blood was sampled 1, 2, 4, 6, and 24 h after the second dose of toxicokinetic animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed no measurable amount of test material in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure. The bioanalytical phase report (Test Facility Study No. 20365399) tables are presented in the field "Overall remarks, attachments section".
 

Table 1: Mortality and Toxic Signs in the Dose-range Finding Study

Group	Sex	Animal Number	Dose mg/kg bw/day	Toxic signs*
				Day 1	Day 2		Day 3
				Post-dose	Pre-dose	Post-dose	Pre-dose	Post-dose
1	Male	101	75	B	B	B	B	B
1	Female	102	75	B	B	B	B	B
2	Male	103	100	B	B	B	B	B
2	Female	104	100	B	B	B	B	B
3	Male	105	200	B	B	B	B	B
3	Female	106	200	B	B	J, N, X	J, N, X	J, N, X
3	Male	107	200	B	B	B	B	B
3	Male	108	200	B	B	B	B	B
3	Female	109	200	B	B	B	B	N
3	Female	110	200	B	B	B	B	N

* Legend 'Mortality and toxic signs':

B = showed no abnormalities; J = hunched posture; N = rough coat; X= eyes partly closed.

 

Table 2: Mean Body Weight Immediately Prior to Dosing

Group code *	Dose (mg/kg bw/day)	Day 1 Body weight gram (Mean ± S.D.)			Day 2 Body weight gram (Mean ± S.D.)			Day 3 Body weight gram (Mean ± S.D.)
1	0	160.8	±	8.6	167.2	±	9.2	162.2	±	9.4
2	50	157.4	±	5.1	163.0	±	4.5	159.2	±	5.9
3	100	152.0	±	11.9	157.6	±	12.9	153.4	±	12.5
4	200	159.5	±	9.9	161.8	±	12.9	159.3	±	14.2
5	200 (EMS)	^#			160.0	±	9.2	155.0	±	10.0
TK 1	0	156.3	±	12.2	162.7	±	12.7	^$
TK 4	200	156.7	±	8.0	160.0	±	12.5	^$

^# Not dosed. was started on Day 2.

^$ Not weighed.

^* Positive control slides taken from male animals previously dosed with positive control CP (cyclophosphamide, 19 mg/kg bw/day at 10 mL/kg bw as part of Test Facility Study No. 20337827).           

 

Table 3: Mean Number of Micronucleated Polychromatic Erythrocytes and Proportion of Immature Erythrocytes

Group	Treatment	Number of Animals	Dose (mg/kg bw/day)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.) ^(*)			Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.) ^(#)
	MALES
1	Vehicle Control	5	0	4.0	±	1.6	0.56	±	0.05
2	Test Material	5	50	3.8	±	2.0	0.54	±	0.04
3	Test Material	5	100	5.4	±	2.1	0.55	±	0.05
4	Test Material	5	200	5.4	±	1.5	0.59	±	0.01
5	CP	3	19	66.7	±	28.7^($)	0.49	±	0.04^($)

Vehicle control = Milli-Q water.

CP = Cyclophosphamide.

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

(#)   The proportion was determined from at least the first 500 erythrocytes counted.

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

 

Table 4: Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.01	1.19
Test Material 50 mg/kg bw/day	4.34	1.54
Test Material 100 mg/kg bw/day	3.34	1.30
Test Material 200 mg/kg bw/day	4.12	1.00
EMS 200 mg/kg bw/day	44.08	2.50

 

Table 5: Overview Tail Intensity in Glandular Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	7.44	1.76
Test Material 50 mg/kg bw/day	5.19	2.13
Test Material 100 mg/kg bw/day	4.43	1.36
Test Material 200 mg/kg bw/day	6.30	1.89
EMS 200 mg/kg bw/day	58.37	4.46

 

Table 6: Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	3.35	0.89
Test Material 50 mg/kg bw/day	3.22	1.06
Test Material 100 mg/kg bw/day	3.16	0.81
Test Material 200 mg/kg bw/day	2.60	1.42
EMS 200 mg/kg bw/day	79.49	0.82

 

Table 7: Individual Data Micronucleus Assay

Individual data (males) (Group 1: oral intubation of the vehicle) (Group 2: oral intubation at 50 mg/kg bw/day) (Group 3: oral intubation at CD3 at 100 mg/kg bw/day) (Group 4: oral intubation at 200 mg/kg bw/day) (Group 6: oral intubation of cyclophosphamide at 19 mg/kg bw/day)
	Group	Animal number	Number of polychromatic erythrocytes^(*)	Number of normochromatic erythrocytes^(*)	Proportion of immature erythrocytes ^(*)	Number of micronucleated polychromatic erythrocytes	Number of polychromatic erythrocytes scored for micronuclei
	1	1	307	213	0.59	4	4001
	1	2	326	223	0.59	6	4005
	1	3	319	208	0.61	3	4002
	1	4	283	282	0.50	5	4000
	1	5	269	246	0.52	2	4000
	2	6	281	253	0.53	4	4001
	2	7	275	287	0.49	2	4000
	2	8	353	299	0.54	7	4000
	2	9	292	263	0.53	2	4000
	2	10	302	200	0.60	4	4000
	3	11	308	198	0.61	5	4000
	3	12	259	253	0.51	7	4000
	3	13	325	218	0.60	4	4004
	3	14	269	256	0.51	8	4006
	3	15	278	262	0.51	3	4001
	4	16	312	239	0.57	6	4000
	4	17	315	218	0.59	6	4000
	4	18	299	214	0.58	7	4000
	4	19	338	234	0.59	3	4003
	4	20	324	214	0.60	5	4002
	6	117	251	254	0.50	93	4000
	6	118	271	250	0.52	71	4000
	6	119	246	311	0.44	36	4000

(*)   The proportion was determined from the first 500 erythrocytes counted.

 

Table 8: Mean Tail Intensity in Duodenum Cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	4.16
	Rat 2	5.76
	Rat 3	4.07
	Rat 4	2.43
	Rat 5	3.64
	Mean	4.01
	S.D.	1.19
Test Material 50 mg/kg bw/day		Tail Intensity (%)
Group 2	Rat 6	4.69
	Rat 7	2.80
	Rat 8	4.76
	Rat 9	6.54
	Rat 10	2.92
	Mean	4.34
	S.D.	1.54
Test Material 100 mg/kg bw/day		Tail Intensity (%)
Group 3	Rat 11	3.20
	Rat 12	2.37
	Rat 13	5.53
	Rat 14	3.27
	Rat 15	2.34
	Mean	3.34
	S.D.	1.30
Test Material 200 mg/kg bw/day		Tail Intensity (%)
Group 4	Rat 16	4.06
	Rat 17	5.82
	Rat 18	3.62
	Rat 19	3.25
	Rat 20	3.86
	Mean	4.12
	S.D.	1.00
EMS 200 mg/kg bw/day		Tail Intensity (%)
Group 5	Rat 24	43.81
	Rat 25	46.70
	Rat 26	41.73
	Mean	44.08
	S.D.	2.50

 

Table 9: Mean Tail Intensity in Glandular Stomach Cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	8.83
	Rat 2	9.79
	Rat 3	6.20
	Rat 4	5.79
	Rat 5	6.60
	Mean	7.44
	S.D.	1.76
Test Material 50 mg/kg bw/day		Tail Intensity (%)
Group 2	Rat 6	4.98
	Rat 7	7.04
	Rat 8	5.69
	Rat 9	6.61
	Rat 10	1.66
	Mean	5.19
	S.D.	2.13
Test Material 100 mg/kg bw/day		Tail Intensity (%)
Group 3	Rat 11	5.69
	Rat 12	5.64
	Rat 13	2.39
	Rat 14	4.42
	Rat 15	3.99
	Mean	4.43
	S.D.	1.36
Test Material 200 mg/kg bw/day		Tail Intensity (%)
Group 4	Rat 16	8.01
	Rat 17	8.52
	Rat 18	4.08
	Rat 19	5.19
	Rat 20	5.73
	Mean	6.30
	S.D.	1.89
EMS 200 mg/kg bw/day		Tail Intensity (%)
Group 5	Rat 24	53.80
	Rat 25	62.71
	Rat 26	58.62
	Mean	58.37
	S.D.	4.46

 

Table 10: Mean Tail Intensity in Liver Cells

Vehicle Controls		Tail Intensity (%)
Group 1	Rat 1	3.23
	Rat 2	2.57
	Rat 3	4.16
	Rat 4	2.43
	Rat 5	4.36
	Mean	3.35
	S.D.	0.89
Test Material 50 mg/kg bw/day		Tail Intensity (%)
Group 2	Rat 6	2.98
	Rat 7	2.95
	Rat 8	4.97
	Rat 9	3.11
	Rat 10	2.08
	Mean	3.22
	S.D.	1.06
Test Material 100 mg/kg bw/day		Tail Intensity (%)
Group 3	Rat 11	3.68
	Rat 12	3.54
	Rat 13	3.99
	Rat 14	2.28
	Rat 15	2.30
	Mean	3.16
	S.D.	0.81
Test Material 200 mg/kg bw/day		Tail Intensity (%)
Group 4	Rat 16	3.80
	Rat 17	4.06
	Rat 18	2.79
	Rat 19	1.58
	Rat 20	0.74
	Mean	2.60
	S.D.	1.42
EMS 200 mg/kg bw/day		Tail Intensity (%)
Group 5	Rat 24	80.11
	Rat 25	79.79
	Rat 26	78.56
	Mean	79.49
	S.D.	0.82

 

 Table 11: Individual Rat Data in Duodenum cells (Group 1: 0 mg/kg bw/day)

Rat 1	Slide	Tail Intensity (%)
	52a2	3.25
	52b1	5.37
	52a1	3.87
	Mean	4.16
	S.D.	1.09
Rat 2	Slide	Tail Intensity (%)
	64a2	6.78
	64b1	5.38
	64a1	5.10
	mean	5.76
	S.D.	0.90
Rat 3	Slide	Tail Intensity (%)
	67a2	4.76
	67b1	4.55
	67a1	2.89
	Mean	4.07
	S.D.	1.03
Rat 4	Slide	Tail Intensity (%)
	60a2	3.24
	60b1	1.95
	60a1	2.12
	Mean	2.43
	S.D.	0.70
Rat 5	Slide	Tail Intensity (%)
	47a2	2.33
	47b1	3.35
	47a1	5.23
	Mean	3.64
	S.D.	1.47

 

 Table 12: Individual Rat Data in Duodenum Cells (Group 2: Test Material 50 mg/kg bw/day)

Rat 6	Slide	Tail Intensity (%)
	62a2	4.14
	62b1	4.18
	62a1	5.74
	Mean	4.69
	S.D.	0.91
Rat 7	Slide	Tail Intensity (%)
	50a2	3.64
	50b1	2.91
	50a1	1.85
	mean	2.80
	S.D.	0.90
Rat 8	Slide	Tail Intensity (%)
	65a2	5.09
	65b1	3.83
	65a1	5.35
	Mean	4.76
	S.D.	0.81
Rat 9	Slide	Tail Intensity (%)
	55a2	6.71
	55b1	5.51
	55a1	7.39
	Mean	6.54
	S.D.	0.95
Rat 10	Slide	Tail Intensity (%)
	59a1	2.39
	59b1	2.54
	59a2	3.82
	Mean	2.92
	S.D.	0.78

 

Table 13: Individual Rat Data in Duodenum Cells (Group 3: Test Material 100 mg/kg bw/day)

Rat 11	Slide	Tail Intensity (%)
	61b1	2.46
	61a1	2.89
	61a2	4.27
	Mean	3.20
	S.D.	0.95
Rat 12	Slide	Tail Intensity (%)
	69a2	1.49
	69b1	2.51
	69a1	3.11
	mean	2.37
	S.D.	0.82
Rat 13	Slide	Tail Intensity (%)
	66b1	5.90
	66a1	5.24
	66a2	5.46
	Mean	5.53
	S.D.	0.33
Rat 14	Slide	Tail Intensity (%)
	53a2	2.47
	53b1	3.13
	53a1	4.22
	Mean	3.27
	S.D.	0.88
Rat 15	Slide	Tail Intensity (%)
	49a2	3.13
	49b1	1.46
	49a1	2.43
	Mean	2.34
	S.D.	0.84

 

Table 14: Individual Rat Data in Duodenum Cells (Group 4: Test Material 200 mg/kg bw/day)

Rat 16	Slide	Tail Intensity (%)
	56a2	4.25
	56b1	3.33
	56a1	4.61
	Mean	4.06
	S.D.	0.66
Rat 17	Slide	Tail Intensity (%)
	63a2	6.19
	63b1	5.12
	63a1	6.14
	mean	5.82
	S.D.	0.60
Rat 18	Slide	Tail Intensity (%)
	68a2	3.42
	68b1	2.40
	68a1	5.04
	Mean	3.62
	S.D.	1.33
Rat 19	Slide	Tail Intensity (%)
	58a2	2.75
	58b1	1.30
	58a1	5.69
	Mean	3.25
	S.D.	2.24
Rat 20	Slide	Tail Intensity (%)
	51a2	4.26
	51b1	4.05
	51a1	3.27
	Mean	3.86
	S.D.	0.52

 

Table 15: Individual Rat Data in Duodenum Cells (Group 5: EMS 200 mg/kg bw/day)

Rat 24	Slide	Tail Intensity (%)
	48a2	41.03
	48b1	45.93
	48a1	44.47
	Mean	43.81
	S.D.	2.52
Rat 25	Slide	Tail Intensity (%)
	57a2	48.00
	57b1	41.69
	57a1	50.41
	Mean	46.70
	S.D.	4.50
Rat 26	Slide	Tail Intensity (%)
	54a2	44.57
	54b1	46.78
	54a1	33.83
	Mean	41.73
	S.D.	6.93

 

Table 16: Individual Rat Data in Glandular Stomach Cells (Group 1: 0 mg/kg bw/day)

Rat 1	Slide	Tail Intensity (%)
	31a2	12.57
	31b1	6.45
	31a1	7.46
	Mean	8.83
	S.D.	3.29
Rat 2	Slide	Tail Intensity (%)
	44a2	12.35
	44b1	7.45
	44a1	9.56
	mean	9.79
	S.D.	2.46
Rat 3	Slide	Tail Intensity (%)
	46a2	3.36
	46b1	7.50
	46a1	7.74
	Mean	6.20
	S.D.	2.46
Rat 4	Slide	Tail Intensity (%)
	39a2	7.57
	39b1	5.72
	39a1	4.09
	Mean	5.79
	S.D.	1.74
Rat 5	Slide	Tail Intensity (%)
	27a2	3.97
	27b1	8.86
	27a1	6.97
	Mean	6.60
	S.D.	2.46

 

Table 17: Individual Rat Data in Glandular Stomach Cells (Group 2: Test Material 50 mg/kg bw/day)

Rat 6	Slide	Tail Intensity (%)
	32a2	5.40
	32b1	6.45
	32a1	3.07
	Mean	4.98
	S.D.	1.73
Rat 7	Slide	Tail Intensity (%)
	37a2	8.26
	37b1	5.43
	37a1	7.44
	mean	7.04
	S.D.	1.46
Rat 8	Slide	Tail Intensity (%)
	43a2	7.92
	43b1	5.00
	43a1	4.15
	Mean	5.69
	S.D.	1.98
Rat 9	Slide	Tail Intensity (%)
	25a2	8.86
	25b1	7.77
	25a1	3.19
	Mean	6.61
	S.D.	3.01
Rat 10	Slide	Tail Intensity (%)
	33a2	3.57
	33b1	1.17
	33a1	0.24
	Mean	1.66
	S.D.	1.72

 

Table 18: Individual Rat Data in Glandular Stomach Cells (Group 3: Test Material 100 mg/kg bw/day)

Rat 11	Slide	Tail Intensity (%)
	28a2	6.86
	28b1	3.27
	28a1	6.94
	Mean	5.69
	S.D.	2.10
Rat 12	Slide	Tail Intensity (%)
	38a2	3.99
	38b1	8.62
	38a1	4.30
	mean	5.64
	S.D.	2.59
Rat 13	Slide	Tail Intensity (%)
	41a2	2.30
	41b1	1.21
	41a1	3.66
	Mean	2.39
	S.D.	1.22
Rat 14	Slide	Tail Intensity (%)
	45a2	3.85
	45b1	5.30
	45a1	4.11
	Mean	4.42
	S.D.	0.77
Rat 15	Slide	Tail Intensity (%)
	34a2	4.62
	34b1	4.14
	34a1	3.21
	Mean	3.99
	S.D.	0.72

 

Table 19: Individual Rat Data in Glandular Stomach Cells (Group 4: Test Material 200 mg/kg bw/day)

Rat 16	Slide	Tail Intensity (%)
	26a2	11.26
	26b1	7.55
	26a1	5.22
	Mean	8.01
	S.D.	3.05
Rat 17	Slide	Tail Intensity (%)
	36a2	8.56
	36b1	7.83
	36a1	9.15
	mean	8.52
	S.D.	0.66
Rat 18	Slide	Tail Intensity (%)
	42a2	3.52
	42b1	3.39
	42a1	5.33
	Mean	4.08
	S.D.	1.09
Rat 19	Slide	Tail Intensity (%)
	29a2	8.77
	29b1	3.00
	29a1	3.80
	Mean	5.19
	S.D.	3.12
Rat 20	Slide	Tail Intensity (%)
	40a2	4.79
	40b1	7.04
	40a1	5.35
	Mean	5.73
	S.D.	1.17

 

Table 20: Individual Rat Data in Glandular Stomach Cells (Group 5: EMS 200 mg/kg bw/day)

Rat 24	Slide	Tail Intensity (%)
	35a2	54.07
	35b1	55.68
	35a1	51.64
	Mean	53.80
	S.D.	2.04
Rat 25	Slide	Tail Intensity (%)
	24a1	60.34
	24b1	66.72
	24a2	61.07
	Mean	62.71
	S.D.	3.49
Rat 26	Slide	Tail Intensity (%)
	30a2	59.16
	30b1	53.23
	30a1	63.47
	Mean	58.62
	S.D.	5.14

 

Table 21: Individual Rat Data in Liver Cells (Group 1: 0 mg/kg bw/day)

Rat 1	Slide	Tail Intensity (%)
	16a2	3.14
	16b1	2.36
	16a1	4.20
	Mean	3.23
	S.D.	0.93
Rat 2	Slide	Tail Intensity (%)
	11a2	2.01
	11b1	3.31
	11a1	2.40
	mean	2.57
	S.D.	0.67
Rat 3	Slide	Tail Intensity (%)
	7a2	4.55
	7b1	4.91
	7a1	3.01
	Mean	4.16
	S.D.	1.01
Rat 4	Slide	Tail Intensity (%)
	20a2	2.27
	20a1	3.18
	20b	1.85
	Mean	2.43
	S.D.	0.68
Rat 5	Slide	Tail Intensity (%)
	1a2	3.18
	1b1	4.41
	1a1	5.48
	Mean	4.36
	S.D.	1.15

 

Table 22: Individual Rat Data in Liver Cells (Group 2: Test Material 50 mg/kg bw/day)

Rat 6	Slide	Tail Intensity (%)
	8b1	2.76
	8a1	1.92
	8a2	4.27
	Mean	2.98
	S.D.	1.19
Rat 7	Slide	Tail Intensity (%)
	17a2	2.99
	17b1	3.51
	17a1	2.34
	mean	2.95
	S.D.	0.59
Rat 8	Slide	Tail Intensity (%)
	22a2	6.23
	22b1	5.85
	22a1	2.84
	Mean	4.97
	S.D.	1.86
Rat 9	Slide	Tail Intensity (%)
	2a2	3.06
	2b1	2.15
	2a1	4.14
	Mean	3.11
	S.D.	1.00
Rat 10	Slide	Tail Intensity (%)
	13a2	1.36
	13b1	2.72
	13a1	2.17
	Mean	2.08
	S.D.	0.69

 

Table 23: Individual Rat Data in Liver Cells (Group 3: Test Material 100 mg/kg bw/day)

Rat 11	Slide	Tail Intensity (%)
	10a2	3.89
	10b1	3.05
	10a1	4.10
	Mean	3.68
	S.D.	0.56
Rat 12	Slide	Tail Intensity (%)
	18a2	3.73
	18b1	3.38
	18a1	3.51
	mean	3.54
	S.D.	0.17
Rat 13	Slide	Tail Intensity (%)
	21a2	4.14
	21b1	2.62
	21a1	5.21
	Mean	3.99
	S.D.	1.30
Rat 14	Slide	Tail Intensity (%)
	5a2	2.51
	5b1	3.04
	5a1	1.28
	Mean	2.28
	S.D.	0.91
Rat 15	Slide	Tail Intensity (%)
	14a2	2.25
	14b1	2.13
	14a1	2.53
	Mean	2.30
	S.D.	0.21

 

Table 24: Individual Rat Data in Liver Cells (Group 4: Test Material 200 mg/kg bw/day)

Rat 16	Slide	Tail Intensity (%)
	19a2	4.14
	19b1	3.44
	19a1	3.83
	Mean	3.80
	S.D.	0.35
Rat 17	Slide	Tail Intensity (%)
	15a1	5.48
	15b1	3.45
	15a2	3.24
	mean	4.06
	S.D.	1.24
Rat 18	Slide	Tail Intensity (%)
	23a2	2.10
	23b1	2.38
	23a1	3.89
	Mean	2.79
	S.D.	0.96
Rat 19	Slide	Tail Intensity (%)
	9a2	1.15
	9b1	2.55
	9a1	1.05
	Mean	1.58
	S.D.	0.84
Rat 20	Slide	Tail Intensity (%)
	3a1	1.14
	3b1	0.52
	3a2	0.57
	Mean	0.74
	S.D.	0.34

 

Table 25: Individual Rat Data in Liver Cells (Group 5: EMS 200 mg/kg bw/day)

Rat 24	Slide	Tail Intensity (%)
	4a2	80.18
	4b1	81.13
	4a1	79.01
	Mean	80.11
	S.D.	1.06
Rat 25	Slide	Tail Intensity (%)
	12b1	84.05
	12a1	76.19
	12a2	79.14
	Mean	79.79
	S.D.	3.97
Rat 26	Slide	Tail Intensity (%)
	6a2	77.18
	6b1	77.68
	6a1	80.82
	Mean	78.56
	S.D.	1.98

 

Table 26: Historical Negative Control Data for Comet Assay

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	7.0	2.9	6.7
SD	2.8	1.3	2.9
n	39	50	33
Lower control limit (95% control limits)	1.4	0.3	1.0
Upper control limit (95% control limits)	12.5	5.5	12.4

SD = Standard deviation

n = Number of observations

Liver, Stomach, Duodenum: Historical control data from experiments performed in November 2019 – November 2022

 

Table 27: Historical Positive Control Data for Comet Assay

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	50.5	80.6	57.7
SD	9.5	7.8	7.8
n	39	50	33
Lower control limit (95% control limits)	32.0	65.3	42.3
Upper control limit (95% control limits)	69.1	95.8	73.0

SD = Standard deviation

n = Number of observations

Liver, Stomach, Duodenum: Historical control data from experiments performed in November 2019 – November 2022

 

Table 28: Historical Negative Control Data for Micronucleus Studies

	Male
Mean Number of Micronucleated cells per 4000 cells	3.7
SD	1.6
N	70
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 November 2019 and November 2022.

 

Table 29: Historical Positive Control Data for Micronucleus Studies

	Male
Mean Number of Micronucleated cells per 4000 cells	33.7
SD	22.7
N	65
Lower Control Limit (95% Control Limits)	-11
Upper Control Limit (95% Control Limits)	78

SD = Standard deviation

n = Number of observations

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

Conclusions:

It was concluded that the test substance is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 200 mg/kg bw/day (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in the report. Additionally, the comet assay was found to be valid and the test substance is not genotoxic in the comet assay in duodenum, glandular stomach and liver cells when sampled approximately 3-4 hours post dosing, for male rats that were dosed via oral gavage for three consecutive days up to a dose of 200 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions described in the report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed according to OECD TG 471 under GLP conditions (Groot, 2018). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvr A were tested according to the direct plate and the pre-incubation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). A dose-range finding experiment (direct plate assay) at concentrations from 1.7 to 5000 µg/plate were performed. The main experiments (direct plate assay, pre-incubation assay) were conducted at concentrations from 17 to 5000 µg/plate (vehicle: water), including positive controls. The test substance induced significant dose-related increases in the number of revertant colonies in the tester strain TA 100 both in the absence and presence of S9-mix. The increases were observed in both experiments. In tester strain WP2 uvrA, dose-related increases in the number of revertant colonies both in the absence and presence of S9-mix were observed. However, these increases remained mostly below the two-fold threshold. Only in the absence of S-9 mix in experiment 2, the test item induced an up to 2.3-fold dose-related increase in the number of revertant colonies in tester strain WP2uvrA. However, the increase was more than two-fold the concurrent solvent control. Cytotoxicity, evident by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains from 1600 µg/plate onwards without metabolic activation and at 5000 µg/plate with metabolic activation, except for tester strain WP2uvrA, where no toxicity was observed. The included positive and negative controls showed the expected results. Hence, the test substance was mutagenic in the TA 100 strain tested with and without metabolic activation and in WP2uvrA without metabolic activation.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with the test substance in accordance with OECD TG 473 (Bootman, 1986). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of freshly heparinised human whole blood cultures, with and without a metabolic activation system (S9 -mix from rats treated with Arocolor 1254). Test substance concentrations of 1.4 - 900 µg/mL (preliminary experiment), 7.5 - 30 µg/mL (main experiment: without metabolic activation) and 3.75 -15 µg/mL (main experiment: with metabolic activation) were applied for 24 hours. Cell division was arrested by the addition of colcemid, three hours before the cells were harvested. In the preliminary study marked depression of cell division was observed at concentrations of 36 µg/mL (65% reduction of mitotic index over control values without S-9 mix, 93% reduction with S-9 mix). In the main experiment statistically significant increase in % aberrant cells was observed at test substance concentration of 15 and 30 µg/mL without metbaolic activation and at 7.5 and 15 µg/mL with metabolic activation. In the absence of S-9 mix no reduction in mitotic activity were observed. In the presence of S-9 mix mean mitotic indices were 7.6, 7.3 and 6.1 for treatment levels of 3.75, 7.5 and 15 µg/mL, showing an approximately 26% reduction in mitotic activity at the highest test substance concentration tested compared to the control value of 8.2. The negative as well as the positive controls showed the expected results. Therefore, under the conditions of the study, the test substance showed clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

 

Genetic toxicity (clastogenic or aneugenic and genotoxicity) in rat in vivo

An in vivo combined micronucleus and alkaline comet test in rat was conducted with the test substance in accordance with OECD TG 474 and 489 (Eurlings, 2023).

The potential genotoxicity of the test substance when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines was assessed by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in duodenum, glandular stomach and liver.

In the main study male animals were dosed three times by oral gavage with vehicle or with 50, 100 and 200 mg/kg bw/day of the test substance for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) /kg bw/day. Positive control slides were taken from male animals previously dosed with 19 mg cyclophosphamide (CP) /kg bw/day and dosed once by oral gavage were added to this study for evaluation as scoring controls in the micronucleus study. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals, and highest dose group (3 and 8 animals per group, respectively).

In addition, blood for bioanalysis of the test substance in plasma was collected from toxicokinetic animals for the 200 mg/kg bw/day group (highest dose group) and from toxicokinetic animals for the vehicle control group.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test substance 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 substance showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum, glandular stomach and liver cells of test substance treated animals compared to the vehicle treated animals.The mean Tail Intensity in duodenum, glandular stomach and liver cells of vehicle-treated rats was 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 which 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.

It was concluded that the test substance is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 200 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions of the study.

Moreover, the comet assay was considered valid and it was concluded that the test substance is not genotoxic in the comet assay in duodenum, glandular stomach and liver cells when sampled approximately 3-4 hours post dosing, in male rats that were dosed via oral gavage for three consecutive days up to a dose of 200 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions of the study.

 

Conclusion

Based on the findings from the combined in vivo micronucleus and alkaline comet test, the registered substance is concluded not to be a mutagen or clastogen/aneugen in vivo.

Justification for classification or non-classification

The available data on the registered substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and is therefore conclusive but not sufficient for classification of the registered substance.