Butyl toluene-4-sulphonate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

Butyl toluene-4-sulphonate

EC Number:

212-295-5

EC Name:

Butyl toluene-4-sulphonate

Cas Number:

778-28-9

Molecular formula:

C11H16O3S

IUPAC Name:

butyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Details on test material:

- Appearance: Colourless to slightly yellow liquid
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

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

Test animals

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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group A (Main study) Vehicle control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group B (Main study)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group C (Main study)

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).