2-(2-thienyl)ethyl toluene-p-sulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

in vivo COMET assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - 16 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was initiated after final ECHA decision (decision number: TPE-D-2114473791-41-01/F). In this decision ECHA requested to carry out an in vivo mammalian alkaline comet assay (test method: OECD TG 489) in rats, oral route, on the following tissues: liver, glandular stomach and duodenum using the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-thienyl)ethyl toluene-p-sulphonate

EC Number:

254-911-5

EC Name:

2-(2-thienyl)ethyl toluene-p-sulphonate

Cas Number:

40412-06-4

Molecular formula:

C13H14O3S2

IUPAC Name:

2-(thiophen-2-yl)ethyl 4-methylbenzene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

Almost white powder

Details on test material:

Storage Conditions: At room temperature container flushed with nitrogen

Specific details on test material used for the study:

Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items.

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 weeks
- Weight at study initiation: 140 – 174g (males, main study)
- Assigned to test groups randomly: yes
- Fasting period before study: No, but only a limited quantity of food was supplied during the night before last dosing / necropsy (approximately 7 g/rat)
- Housing: Polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group together
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-20.4
- Humidity (%): 48-74
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 27 May 2020 - 05 Aug 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: PEG400 (adjustments were made to correct for specific gravity of the vehicle (1.13 g/mL))
- Amount of vehicle: 5 mL/kg
- Concentration in the dosing formulations: 0, 100, 200 and 400 mg/mL for the control group and the low, mid and high dose group, respectively.
- The vehicle was choosen since stability analyses performed previously in conjunction with Charles River Study 20189734 demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared daily and dosed within 2.5 hours after adding the vehicle to the test item. Test item concentrations were treated with ultra-sonic waves until the test item had completely dissolved.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Rats were dosed twice, on t=0 and t=21 hours.

Post exposure period:

Rats were sacrificed 3-4 hours after the second treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

3 (dose range finder study);
5 males (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS; RS344) at 200 mg/kg body weight dissolved in physiological saline. EMS was dosed orally within 3 hours after preparation at a dosing volume of 10 mL/kg body weight.

Examinations

Tissues and cell types examined:

Liver, stomach, duodenum

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dosing concentrations were determined based on the results of a dose range finder study. Three males and three females received the same treatment regimen as in the main study at 2000 mg/kg bw/day. No mortality or severe toxicity was observed, no substantial differences in toxicity between sexes was found.

DETAILS OF SLIDE PREPARATION:
Three up to four hours after the second treatment liver, duodenum and stomach will be collected. The isolation method for liver cells is based on Hu et al (2002). Cells from glandular stomach and duodenum will be collected based on the JACVAM Comet validation study. To the cell suspension melted low melting point agarose (LMAgarose) was added. The cells were mixed with the LMAgarose and 50-60 μL was layered on a precoated comet slides in duplicate. Three slides per tissue per animal were prepared (in total 6 agarose circles; 3 for scoring and 3 for backup). The slides were incubated for 14 to 20 minutes in the refrigerator in the dark until a clear ring appears at the edge of the comet slide area.
The cells on the slides were immersed in prechilled lysis solution for at least 60 minutes up to overnight in the refrigerator. After this incubation period, the slides were immersed/rinsed in neutralization buffer (0.4M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for30 minutes for liver and 20 minutes for duodenum and stomach 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 0.9-1 Volt/cm for liver and at 0.7 V/cm for stomach and duodenum. The electrophoresis was performed for 20 min for stomach and duodenum and for 30 minutes for liver under constant cooling. After completion of electrophoresis, the slides were immersed/rinsed in the neutralization buffer for 6 to 7 minutes. The slides were subsequently immersed for 5 to 6 minutes in Absolut ethanol (99.6%) and allowed to dry at room temperature. The slides were stained for approximately 5 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.

METHOD OF ANALYSIS:
Slides were randomly coded (per tissue) before examination of the Comets to prevent bias. One hundred fifty comets were analyzed in total.
The following criteria for scoring of comets were used:
- The comets must be horizontal, with the head on the left and the tail on the right;
- Cells that overlap or are not sharp were not be scored.
In addition the frequency of hedgehogs were determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs were noted for all treatment groups and the control. Since there was no effect of the test item, hedgehogs data was not reported

Evaluation criteria:

ACCEPTABILITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05);
c) Adequate numbers of cells and doses have been analysed;
d) The highest test dose is the MTD or 2000 mg/kg/day.

Statistics:

STATISTICAL ANALYSES:
Equivocal results will be clarified by further testing using modification of experimental conditions. ToxRat Professional will be used for statistical analysis of the data. Appropriate statistical
tests will be selected for data evaluation. A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity compared with the concurrent negative control;
b) The increase is dose related when evaluated with a trend test;
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity 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.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality (1/5) and clinical signs (lethargy, hunched posture and rough coat) were noted at 2000 mg/kg bw/day

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The animals of the groups treated with 500 and 1000 mg test item/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. One animal treated with 2000 mg/kg showed lethargy, hunched posture and rough coat. Three animals showed lethargy and hunched posture and one animal was found dead before the second dosing.

The full study report is attached for reference.

The mean Tail Intensity in liver, duodenum and stomach cells of vehicle-treated rats was 1.82 ± 0.39% (mean ± SD), 4.30 ± 1.3% (mean ± SD) and 4.54 ± 0.91% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 78 ± 4.7% (mean ± SD, 43-fold induction; p<0.001 Welch t test;), 55 ± 4.2% (mean ± SD, 13-fold induction; p<0.001 Welch t test;) and 43 ± 6.1% (mean ± SD, 9.5-fold induction; p<0.001 Welch t test;) in male animals in liver, duodenum and stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

No biologically relevant statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of test item treated male animals compared to the vehicle treated animals. In liver and duodenum cells a slight, but statistically significant increase of the Tail Intensity (%) was found. This effect did not exceed the 95% confidence interval for the historical negative controls. Furthermore, the maximum increase never exceeded more than 2 times increase (maximum of 1.8 fold increase in liver and 1.4 in duodenum at the highest dose) and was thus concluded to be very limited. In comparison, the increase for the positive control was 43 fold in liver and 13 fold in duodenum. Based on these considerations, the observed increase in duodenum and liver were concluded not to be toxicologically relevant and not to be an indication of genotoxic properties.

Any other information on results incl. tables

Dose range finder study

No abnormalities were observed within 2 hours after first dosing. Within 21 hours after first dosing, all animals were found to be lethargic. In addition, all animals (except one female) showed hunched posture and rough coat. Within 2 hours of second dosing, all animals remained lethargic and were found in hunched posture. One male and one female rat showed ataxia. All rats, except one female, had a rough coat.

Formulation analyses

Accuracy

No test item was detected in the control group formulation. The concentrations analyzed in the formulations of the low, mid and high dose group were in agreement with target concentrations (i.e. mean accuracies between 97% and 104%).

Homogeneity

The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤ 2.3%).

 

Table: Overview Tail Intensity in Liver Cells of Male Rats   

	Tail Intensity (%)	S.D.
0 mg/kg	1.82	0.39
Test item 500 mg/kg	2.06	0.74
Test item 1000 mg/kg	2.80	0.52
Test item 2000 mg/kg	3.27	0.73
EMS 200 mg/kg	77.86	4.74

 

Table: Overview Tail Intensity in Duodenum Cells of Male Rats          

	Tail Intensity (%)	S.D.
0 mg/kg	4.30	1.34
Test item 500 mg/kg	4.72	1.45
Test item 1000 mg/kg	5.32	0.78
Test item 2000 mg/kg	6.16	0.93
EMS 200 mg/kg	55.19	4.20

 

Table: Overview Tail Intensity in Stomach Cells of Male Rats         

	Tail Intensity (%)	S.D.
0 mg/kg	4.54	0.91
Test item 500 mg/kg	3.02	0.73
Test item 1000 mg/kg	3.55	0.66
Test item 2000 mg/kg	4.09	1.17
EMS 200 mg/kg	42.60	6.07

Statistical evaluations (nb: group 3 = mid dose group, group 4 = high dose group)

Comet assay in Liver

Group	Treatment	Dose (mg/kg body weight)	Sex	p-value (one sided)	Decision (95% CL)
3^1	Test Item	1000	Male	<0.001	Significant
4^1	Test Item	2000	Male	<0.001	Significant
All^2	Test Item	-	Male	<0.001	Significant

¹Comparison with the corresponding vehicle control group by using the Williams Multiple Sequentional t-test
²Analysis of a dose-response using a linear trend test

Positive control¹:

Group	Treatment	Dose (mg/kg body weight)	Sex	p-value (one sided)	Decision (95% CL)
5	EMS	200		<0.001	Significant

¹Comparison with the corresponding vehicle control group by using the Welch t-test

Comet assay in Duodenum

Group	Treatment	Dose (mg/kg body weight)	Sex	p-value (one sided)	Decision (95% CL)
4^1	Test Item	2000	Male	<0.001	Significant
All^2	Test Item	-	Male	0.012	Significant

¹Comparison with the corresponding vehicle control group by using the Williams Multiple Sequentional t-test
²Analysis of a dose-response using a linear trend test

Positive control¹:

Group	Treatment	Dose (mg/kg body weight)	Sex	p-value (one sided)	Decision (95% CL)
5	EMS	200	Male	<0.001	Significant

¹Comparison with the corresponding vehicle control group by using the Welch t-test

Comet assay in Stomach

Test Item:

Comparison with the corresponding vehicle control group by using the Dunnett’s test, no significant differences

 

Positive control¹:

Group	Treatment	Dose (mg/kg body weight)	Sex	p-value (one sided)	Decision (95% CL)
5	EMS	200	Male	<0.001	Significant

¹Comparison with the corresponding vehicle control group by using the Welch t-test

Historical control data:

Historical data Comet assay Negative control

 

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	4.3	2.4	3.5
SD	2.0	1.6	1.8
n	19	34	22
Lower control limit (95% control limits)	0.3	-0.8	0.0
Upper control limit (95% control limits)	8.2	5.6	7.0

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in July 2017 – June 2020

 

Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days)

 

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	45.4	87.7	55.3
SD	12.1	6.7	11.6
n	19	33	22
Lower control limit (95% control limits)	21.7	74.5	32.6
Upper control limit (95% control limits)	69.1	100.9	78.0

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in July 2017 – June 2020

Applicant's summary and conclusion

Conclusions:

Based on the results of an in vivo Comet assay performed with the registered substance according to OECD guideline 489 and following GLP principles, it was concluded that there are no indications of genotoxic properties of the registered substance.

Executive summary:

An in vivo Comet assay was performed with the registered substance according to OECD guideline 489 and following GLP principles. Based on the results of a dose range finder study, the test concentrations were established at 500, 1000 and 2000 mg/kg bw/day. The main study was done in males, as no difference in toxicity was observed between the sexes in the dose range finder study. Dose formulations analyses confirmed correct dosing of the animals.

Clinical signs of toxicity were observed in the high dose group only and included lethargy, rough coat and hunched posture. No biologically relevant statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of test item treated male animals compared to the vehicle treated animals. In liver and duodenum cells a slight, but statistically significant increase of the Tail Intensity (%) was found, however these did not exceed the 95% confidence interval for the historical negative controls and are therefore not biologically relevant. The results of the vehicle control and the positive control EMS demonstrated that the test system was reliable. Adequate numbers of cells and doses were analysed and the highest test dose was the maximum dose required by the guidelines. In conclusion, the test is valid and there are no indications the registered substance is genotoxic in vivo.