3-((3,4-dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 06 June 2017 to 28 July 2017. Report issued: 03 November 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(adopted 29 July 2016)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome damage or aneuploidy

Test material

Test material information

Constituent 1

Reference substance name:

3-((3,4-Dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Cas Number:

569316-81-0

Molecular formula:

C14H17N3O5S2

IUPAC Name:

3-((3,4-Dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

other: albino Hsd: ICR (CD-1)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Envigo, B.V., Horst, The Netherlands. At the start of the main test the mice weighed 20.1g to 29.9g and were approximately six to ten weeks old. The bodyweights of the individual mice were within 20% of the dose group mean. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of five in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Information as provided by the supplier. (W M Hodgson & Co)
Identification: Arachis oil BP
Test house Serial Number: V-6418
Expiry Date: 26 January 2018
Storage Conditions: Room temperature
Purity: Treated as 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

For the purpose of this study the positive control item (cyclophosphamide) was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Duration of treatment / exposure:

All animals were dosed once only at the appropriate dose level.

One group of mice was euthanised by cervical dislocation 24 hours following treatment and the second group was euthanised after 48 hours. The vehicle and positive controls were euthanised 24 hours following dosing.

Frequency of treatment:

Once

Post exposure period:

One group of mice was euthanised by cervical dislocation 24 hours following treatment and the second group was euthanised after 48 hours.

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Test substance

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow
erythrocytes

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.

A positive mutagenic response is demonstrated when a statistically significant and toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group and the number of micronucleated polychromatic erythrocytes in the animals exceed the historical control data range. If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Test
No evidence of toxicity was observed in animals dosed with test item via the oral route at either 1000mg/kg or the maximum recommended dose level of 2000 mg/kg. Therefore, the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test using the treatment schedule for non-toxic test items.


Micronucleus Test
Mortality Data and Clinical Observations
There were no premature deaths or clinical signs observed in either of the test item dose groups.

Evaluation of Bone Marrow Slides
There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test item groups when compared to the vehicle control group.

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group, and the group mean values were within the range of the group mean values of the historical vehicle control data.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all vehicle control animals were within the historical vehicle control data range.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 29 July 2016), Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods

A range-finding test was performed to find suitable dose levels of the test item and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in the toxicity of the test item between the sexes; therefore, the main test was performed using only male mice using the treatment schedule for non-toxic test items. The micronucleus test was conducted using the oral route in groups of five mice (males) at the maximum recommended dose (MRD) of 2000 mg/kg using arachis oil as the vehicle. Animals were euthanised 24 or 48 hours after dosing, the bone marrow was extracted and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity.

Two additional groups of mice were given a single oral dose of arachis oil, or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. Vehicle and positive control animals were euthanised 24 hours after dosing.

Before commencing the main test one of the stock animals was found dead. It was decided that one of the animals should be removed from the positive control group. Therefore only 4 animals were used in the positive control group.

Results

There were no premature deaths or clinical signs in either of the test item dose groups. There were also no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group.

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group, and the group mean values were within the range of the group mean values of the historical vehicle control data.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all vehicle control animals were within the historical vehicle control data range.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion

The test item was considered to be non-genotoxic under the conditions of the test.