Butyl toluene-4-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacterial cells (Gijsbrechts, 2019)

Based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2019 to 19 August 2019

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- No correction was made for the purity/composition of the test material.
- A solubility test was performed based on visual assessment. The test material formed a clear (colourless) solution in dimethyl sulfoxide.
- To protect the test material from light, amber-coloured glassware or tubes wrapped in tinfoil were used for test material preparations.
- Test material concentrations were used within 2 hours after preparation.

Target gene:

Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The characteristics of the different Salmonella typhimurium strains were as follows: TA1537: mutation hisC3076 (frameshift); TA98: mutation his D3052/R-factor* (frameshift); TA1535: mutation hisG46 (base-pair substitutions); TA100: mutation hisG46/R-factor* (base-pair substitutions)
*R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa: deep rough (defective lipopolysaccharide cellcoat)
gal: mutation in the galactose metabolism
chl: mutation in nitrate reductase
bio: defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilisation of the strain using Tris-EDTA treatment. The strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in the freezer (-150 °C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- Source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 and 2.5 μg/plate, respectively.
- Method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter (0.22 μm)-sterilised. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5 % (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Dose-Range Finding Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate (TA100 and WP2uvrA - with and without metabolic activation)
Mutation Experiment: 0, 52, 164, 512, 1600, 2500, 5000 µg/plate (TA1535, TA1537, TA98 - with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (without metabolic activation - TA1537; 2.5 μg/ plate). 2-aminoanthracene (with metabolic activation - TA1535 and TA1537: 2.5 μg/ plate; TA98 and TA100: 1 μg/plate; WP2uvrA: 15 µg/plate)

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: plate incorporation

At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain. The dose-range finding study with two tester strains was reported as a part of the mutation assay. In the second part of the experiment, the test material was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.
After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines.

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 the Test Laboratory.
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.

INTERPRETATION OF RESULTS
A test material 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 times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three 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:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitate: Precipitation of the test material on the plates was not observed at the end of the incubation period in any tester strain
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA, where no toxicity was observed at any of the dose levels tested.
- Mutagenicity: In tester strain TA100, the test material induced up to 1.8- and 1.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were not two-fold the concurrent solvent control but were above the laboratory historical control data range in the presence of S9-mix.
In tester strain WP2uvrA, the test material induced up to 2.4- and 2.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than two-fold the concurrent solvent control but were within the laboratory historical control data range.
In tester strain TA1535, the test material induced up to 4.1- and 10-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than three-fold the concurrent solvent control and were above the laboratory historical control data range.
No increases were observed in tester strains TA1537 and TA98.

Mean number of revertant colonies/3 replicate plates (± S.D.)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 1.7 5.4 17 52 164 512 1600 2500 5000	97 ± 12 104 ± 21 110 ± 4 92 ± 11 115 ± 9 104 ± 9 130 ± 12 174 ± 24 - 141 ± 18s	9 ± 8 - - - 12 ± 3 12 ± 7 24 ± 5 37 ± 7 28 ± 3 26 ± 5s	14 ± 3 19 ± 1 13 ± 3 15 ± 6 11 ± 1 15 ± 1 16 ± 5 24 ± 6 - 34 ± 4	14 ± 5 - - - 15 ± 6 11 ± 3 9 ± 3 10 ± 0 9 ± 2 4 ± 4s	8 ± 0 - - - 5 ± 5 6 ± 6 8 ± 3 5± 0 6 ± 1 1 ± 1s
+	Solvent 1.7 5.4 17 52 164 512 1600 2500 5000	96 ± 15 85 ± 15 87 ± 17 96 ± 17 86 ± 11 102 ± 9 157 ± 8 182 ± 28 - 60 ± 4m	10 ± 6 - - - 14 ± 4 34 ± 8 64 ± 20 80 ± 13 102 ± 20 63 ± 17m	13 ± 3 19 ± 5 17 ± 5 21 ± 7 17 ± 6 22 ± 2 28 ± 1 32 ± 6 - 38 ± 4	18 ± 6 - - - 14 ± 5 15 ± 4 11 ± 4 12 ± 1 9 ± 2 9 ± 4m	4 ± 1 - - - 6 ± 2 4 ± 3 5 ± 3 4 ± 1 4 ± 1 3 ± 1m
Positive Controls
-	Name	MMS	SA	4-NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	725 ± 33	969 ± 46	1060 ± 100	1056 ± 25	876 ± 101
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	1598 ± 53	311 ± 3	423 ± 40	1396 ± 68	368 ± 38

m = Bacterial background lawn moderately reduced

s = Bacterial background lawn slightly reduced

MMS = methylmethanesulfonate

SA = Sodium azide

4-NQO = 4-nitroquinoline N-oxide

2NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

Conclusions:

Based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, and under GLP conditions.

The objective of the study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test was performed in the direct plate assay with DMSO as the solvent.

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In tester strain TA100, the test material induced up to 1.8- and 1.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were not two-fold the concurrent solvent control but were above the laboratory historical control data range in the presence of S9-mix.

In tester strain WP2uvrA, the test material induced up to 2.4- and 2.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than two-fold the concurrent solvent control but were within the laboratory historical control data range.

In the first mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix.

In tester strain TA1535, the test material induced up to 4.1- and 10-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than three-fold the concurrent solvent control and were above the laboratory historical control data range.

No increases were observed in tester strains TA1537 and TA98.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material induced a significant dose-related increase in the number of revertant (His+) colonies in tester strain TA1535. The increases were more than three-fold the concurrent solvent control, were above the laboratory historical control data range and observed in the absence and presence of S9-mix.

In tester strains TA100 and WP2uvrA, dose-related increases in the number of revertant colonies both in the absence and presence of S9-mix were observed. However the increases observed were either below the laboratory historical control data ranges or less than two-fold the concurrent solvent control.

In conclusion, based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay (Eurlings, 2020)

Under the conditions of the study, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). However, the test material is genotoxic in the Comet assay in liver cells of male rats when sampled approximately 3-4 hours post oral gavage dosing, for three consecutive days up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 December 2019 to 30 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

The test method is a protocol based on the OECD 474 and 489 guidelines combined.

GLP compliance:

yes

Type of assay:

other: in vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay

Specific details on test material used for the study:

No correction was made for the purity/composition of the test material.

Species:

rat

Strain:

Wistar

Remarks:

(Wistar-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 non-clinical toxicity test by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 9 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes (the body weights of the rats at the start of the treatment were within 20% of the mean. The mean body weights were 272 ± 8.5 g and the range 260 – 294 g. The animals were allocated at random to treatment groups. Male animals were randomized. Animals in poor health or at extremes of body weight range were not assigned to groups).
- Fasting period before study: A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat) until maximum 4 hours after administration of the test material.
- Housing: Animals were housed in groups up to 5 (by sex) in polycarbonate cages containing sterilised sawdust as bedding material equipped with water bottles. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: pellets, ad libitum (except during designated procedures)
- Water: municipal tap water, ad libitum (via water bottles)
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 21°C
- Humidity: 48 to 53%
- Air changes: Ten or more air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light (except during designated procedures)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
A solubility test was performed based on visual assessment. The test material was dissolved (clear yellow solution) in corn oil.

Details on exposure:

Test material concentrations were dosed within 3 hours after preparation. To protect the test material from light, amber-colored glassware or tubes wrapped in tin-foil were used for test material preparations.
Any residual volumes were discarded.

Duration of treatment / exposure:

The rats were dosed for three consecutive days (vehicle or test material).
The rats were dosed twice with EMS or once with CP.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group A (Main study) Vehicle control

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group B (Main study)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group C (Main study)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group D (Main study)

No. of animals per sex per dose:

5 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control for the micronucleus test was cyclophosphamide (CP) at 19 mg/kg bw dissolved in physiological saline. The route of administration was oral and the dosing volume was 10 mL/kg body weight.
The positive control for the Alkaline Comet test was ethyl methanesulfonate (EMS) at 200 mg/kg bw dissolved in physiological saline. EMS was used within 2 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight.

Tissues and cell types examined:

TERMINAL PROCEDURES
- Scheduled Euthanasia
The main animals were euthanised by abdominal aorta bleeding under isoflurane anesthesia and subject to gross necropsy.

COMET ASSAY
> Isolation Cells
Approximately 3-4 hours after the third treatment with the test material, bone marrow was isolated for the micronucleus test. In addition, liver were collected/isolated and examined for DNA damage with the alkaline Comet assay.
- Isolation of bone marrow for the micronucleus test
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 foetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.
- Liver
A portion of 0.6-0.7 g 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) 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.

Details of tissue and slide preparation:

COMET ASSAY
- Preparation of Comet Slides
To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide in duplicate. Three slides per tissue were prepared. The slides were incubated for 11 - 15 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
- Lysis, Electrophoresis and Staining of the Slides
The cells on the slides were overnight (approximately 16 h) immersed in pre-chilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralisation buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 30 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 – 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralisation buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolute ethanol and allowed to dry at room temperature. The slides were stained for approximately 5 - 6 minutes with the fluorescent dye SYBR® Gold 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.
- Comet Scoring
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides, one of the agarose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.
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 (Comets without a head which may be an indication of cytotoxicity) 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. There were no hedgehogs present at the vehicle control and all test item concentrations tested.

HISTOPATHOLOGY
Part of the liver from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) were collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The piece of the tail containing the identification number of the animal was added for correct identification. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin fot histopathological examination.

MICRONUCLEUS
- 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/ether 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 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. This staining is based on Giemsa. The dry slides were automatically embedded and mounted with a coverslip with an automated coverslipper.
- Analysis of the bone marrow smears for micronuclei
To prevent bias, all slides were randomly coded before examination. 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:

- Micronucleus Test
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.

- 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.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
As there were statistically significant differences between one or more of the test material groups and the vehicle control group (in the Comet assay) a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

(micronucleus assay)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

(Comet assay)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

> Main Study
- Mortality and Toxic Signs
The animals of the groups treated with test material 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 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 within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, 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.
- Ratio Polychromatic to Normochromatic Erythrocytes
The animals of the groups, which were treated with test material showed no decrease in the ratio of polychromatic to normochromatic 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 ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Comet Slide Analysis
The mean Tail Intensity in liver cells of vehicle-treated rats was 2.41 ± 0.32% (mean ± SD) in male, 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 86 ± 2.05% (mean ± SD, 36-fold induction; p<0.001 Welch-t test) in male rats in liver cells. 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 (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 (% tail DNA) was observed in liver cells of test material-treated animals compared to the vehicle-treated animals. The two highest test material-treated groups showed tail intensity increases outside of the historical control interval of vehicle-treated animals and in addition a significant trend analysis was observed (p<0.001).

- Histopathology
Microscopic examination was performed on the liver all Group A, B, C and D animals.
Microscopic test item-related findings were present in:
Liver: Hepatocellular hypertrophy was present in 1/5 rats treated at 500 mg/kg at minimal degree and in 2/5 rats treated at 1000 mg/kg at minimal and mild degree. This finding does not represent cell death.
All other microscopic findings were within the range of background pathology encountered in rats of this age and strain.
Morphologic alterations following the administration of test material to male Wistar (Han) rats, were present in the liver at 500 and 1000 mg/kg/day.

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group	Treatment	Dose (mg/kg bw)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.)^(1,2)			Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.)^(1,3)
A	Vehicle Control	0	2.4	±	0.9	0.70	±	0.03
B	Test material	250	3.2	±	1.9	0.68	±	0.08
C	Test material	500	3.2	±	1.9	0.64	±	0.16
D	Test material	1000	1.6	±	0.5	0.51	±	0.07
E	CP	19	53.8	±	8.9^(4)	0.17	±	0.02

Vehicle control = Corn oil.

CP = Cyclophosphamide.

(1) Five animals per treatment group.

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

(3) The ratio was determined from at least the first 1000 erythrocytes counted.

(4) Significantly different from corresponding control group (Welch t test, P < 0.001).

Overview Tail Intensity in liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	2.41	0.32
Test material 250 mg/kg	3.59	1.93
Test material 500 mg/kg	4.04	1.38
Test material 1000 mg/kg	13.49	2.92
EMS 200 mg/kg	86.41	2.05

Conclusions:

Under the conditions of the study, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). However, the test material is genotoxic in the Comet assay in liver cells of male rats when sampled approximately 3-4 hours post oral gavage dosing, for three consecutive days up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Executive summary:

The potential genotoxicity of the test material, when administered to rats at the maximum recommended dose, was investigated in a study which was conducted according to a method based on the standardised guidelines OECD 474, OECD 489 and EU Method B.12, and under GLP conditions. During the study the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow, and the increase in DNA strand breaks in liver, were both measured.

Based on the results of the dose-range finding study test material concentrations of 1000 mg/kg bw/day was selected as maximum dose for the main test (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 once daily, by oral gavage with vehicle or with 250, 500 and 1000 mg/kg bw, for three consecutive days. A positive control group was dosed twice by oral gavage with Ethyl Methane Sulfonate (EMS) at 200 mg/kg bw and a positive control group for the micronucleus assay was dosed once by oral gavage with cyclophosphamide (CP) at 19 mg/kg bw. In total 6 treatment groups were used, each consisting of 5 animals. Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and tissues were isolated.  Single cell suspensions from liver were made followed by Comet slide preparation. The slides were analysed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

In the micronucleus assay, no 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 item, 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 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.

In the Comet assay, the mean Tail Intensity in liver cells of vehicle-treated rats was 2.41 ± 0.32% (mean ± SD), 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 86 ± 2.05% (mean ± SD) in liver cells. 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 (% tail DNA) was observed in liver cells of test material-treated animals compared to the vehicle-treated animals. The two highest test material-treated groups showed tail intensity increases outside of the historical control interval of vehicle-treated animals and in addition a significant trend analysis was observed. Histopathology in the liver did not show any findings related to cell death which could explain the increased mean tail intensity.

In conclusion, under the conditions of the study, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). However, the test material is genotoxic in the Comet assay in liver cells of male rats when sampled approximately 3-4 hours post oral gavage dosing, for three consecutive days up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

In vitro gene mutation in bacterial cells (Gijsbrechts, 2019)

The mutagenic potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test was performed in the direct plate assay with DMSO as the solvent.

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In tester strain TA100, the test material induced up to 1.8- and 1.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were not two-fold the concurrent solvent control but were above the laboratory historical control data range in the presence of S9-mix.

In tester strain WP2uvrA, the test material induced up to 2.4- and 2.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than two-fold the concurrent solvent control but were within the laboratory historical control data range.

In the first mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix.

In tester strain TA1535, the test material induced up to 4.1- and 10-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than three-fold the concurrent solvent control and were above the laboratory historical control data range.

No increases were observed in tester strains TA1537 and TA98.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material induced a significant dose-related increase in the number of revertant (His+) colonies in tester strain TA1535. The increases were more than three-fold the concurrent solvent control, were above the laboratory historical control data range and observed in the absence and presence of S9-mix.

In tester strains TA100 and WP2uvrA, dose-related increases in the number of revertant colonies both in the absence and presence of S9-mix were observed. However the increases observed were either below the laboratory historical control data ranges or less than two-fold the concurrent solvent control.

In conclusion, based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.

In vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay (Eurlings, 2020)

The potential genotoxicity of the test material, when administered to rats at the maximum recommended dose, was investigated in a study which was conducted according to a combined method based on the standardised guidelines OECD 474, OECD 489 and EU Method B.12, and under GLP conditions. During the study the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow, and the increase in DNA strand breaks in liver, were both measured.

Based on the results of the dose-range finding study test material concentrations of 1000 mg/kg bw/day was selected as maximum dose for the main test (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 once daily, by oral gavage with vehicle or with 250, 500 and 1000 mg/kg bw, for three consecutive days. A positive control group was dosed twice by oral gavage with Ethyl Methane Sulfonate (EMS) at 200 mg/kg bw and a positive control group for the micronucleus assay was dosed once by oral gavage with cyclophosphamide (CP) at 19 mg/kg bw. In total 6 treatment groups were used, each consisting of 5 animals. Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and tissues were isolated.  Single cell suspensions from liver were made followed by Comet slide preparation. The slides were analysed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

In the micronucleus assay, no 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 item, 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 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.

In the Comet assay, the mean Tail Intensity in liver cells of vehicle-treated rats was 2.41 ± 0.32% (mean ± SD), 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 86 ± 2.05% (mean ± SD) in liver cells. 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 (% tail DNA) was observed in liver cells of test material-treated animals compared to the vehicle-treated animals. The two highest test material-treated groups showed tail intensity increases outside of the historical control interval of vehicle-treated animals and in addition a significant trend analysis was observed. Histopathology in the liver did not show any findings related to cell death which could explain the increased mean tail intensity.

In conclusion, under the conditions of the study, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines). However, the test material is genotoxic in the Comet assay in liver cells of male rats when sampled approximately 3-4 hours post oral gavage dosing, for three consecutive days up to a dose of 1000 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to genetic toxicity (Category 2) and is assigned the hazard statement H341 (Suspected of causing genetic defects).