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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 January 2002 - 5 February 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Sponsor's Identification: p-Toluenesulfonamide
Lot No.: K017105ES
CAS No.: 70-55-3
purity: 99.3%; (by HPLC, taken from CoA supplied by Sigma-Aldrich)
Date Received: 02 Jan 2002
Physical Description: White, crystalline powder
Storage Conditions: Ambient temperature

Test animals

Species:
mouse
Strain:
other: Crl:CD-1 (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: young
- Weight at study initiation: mean of all groups; males ± 31.7 g, females ± 25.9
- Assigned to test groups randomly: yes, under following basis: by a computer program.
- Fasting period before study: no
- Housing: The animals were housed in sanitary, polycarbonate cages containing Sani-chips Hardwood Chip Laboratory bedding. The animals were housed up to five animals per cage during acclimation, and full dose groups after randomization.
- Diet (e.g. ad libitum): A commercial diet, PMI Feeds, Inc. Certified Rodent Diet # 5002 (pellets) was available ad libitum.
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 17-26
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 2 January 2002 - 5 February 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: A preliminary solubility test was conducted using deionized water and corn oil to examine which vehicle had the best solubility or produced a homogeneous suspension when mixed with the test article. The test article, p-Toluenesulfonarnide, was extremely hydrophobic in deionized water (heterogeneous with floating test article at 200 mg/mL). However, a homogenous suspension was formed in corn oil at the same concentration. Therefore, corn oil was selected as the vehicle for test article administration based on these results.
- Concentration of test material in vehicle: depending on the dose 18.75, 375, 75.0 or 150.0 mg/ml
- Amount of vehicle (if gavage or dermal): 10ml/kg
- Lot/batch no. (if required): Corn oil (Welsh, Nolme, and Clarke; Lot No. 12-394 [dose range-finding study] or 12-403 [micronucleus assay] CAS No. 8001-30-7).
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each concentration of the test article: p-Toluenesulfonamide, was prepared by adding the appropriate amounts of the corn oil and test article and mixing the components until in a homogeneous suspension.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
a single dose
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
187.5, 375, 750, 1500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
12 males and 12 females in the high dose group and the vehicle control and 6 males and 6 females in other dose groups.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (Sigma, Lot No. 108H0568; CAS No. 6055-19-2) was the positive control article for the micronucleus assay. The cyclophosphamide (CP) was dissolved in sterile deionized water (Covance, Batch No. 12-07-01) at approximately 8 mg/mL and administered by oral gavage at approximately 10 mL/kg to achieve a dose level of approximately 80 mg/kg. The positive control animals were dosed once with CP by oral and bone marrow was sampled approximately 24 hours after dosing.

Examinations

Tissues and cell types examined:
bone marrow, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose range-finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Samples were taken 24 hours after dosing and in the high dose group and the vehicle control also 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest timepoints, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones
(tibias) were removed for marrow extraction from five surviving animalddose level. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanized at the completion of the assay.
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanoI, stained in acridine orange, and protected by permanently mounted or control of bias, all slides were coded prior to analysis.

METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucieus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. Data from those experimental animals wit either fewer than 500 PCEs or with a PCE to NCE ratio less than 20% of the vehicle control alue were not used either in the individual animal data or for statistical evaluation.

The historical background frequency of micronucleated cells were expressed as percent micronucleated cells based on the number of PCEs analyzed. The historical background frequency of micronuclei in this mouse strain at this laboratory is 0.0 to 0.4%, which is within the published data (Salamone and Mavoumin, 1994). The frequency of PCEs versus mature erythrocytes (NCEs) were determined by scoring the number of PCEs and NCEs observed in optic fields while scoring at least the first 500 erythrocytes on the slide. The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occur. Micronuclei are sharp bordered and generally between onetwentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
OTHER: none
Evaluation criteria:
Acceptable Controls.
The vehicle control group had less than approximately 0.4% micronucleated PCEs. The positive control group had a statistically significantly higher
(p ≤0.01) number of micronucleated PCEs than the vehicle control group.
The criteria for a positive reponse was the detection of a statis ically significant increase in micronucleated PCEs for at least one dose level, and a statistically-significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

Acceptable High Dose.
The high dose of 1500 mg/kg produced clinical toxicity and death.
Statistics:
Assay data analysis were performed using a one-way analysis of variance (Winer, 1971) on untransformed proportions of cells with rnicronuclei per animal when the variances are homogeneous. Ranked proportions were used for heterogeneous variances. The statistical analyses was conducted using the "ANOVA-Version 5.01,05108197" statistical software analysis program from the Covance Statistics Library entitled "One-way Analysis of Variance and Related Procedures7'. If the analysis of variance was significant (p ≤ 0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each sampling time and sex combination.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see "additional information of results"
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the dose range-finding study, the test article was suspended in corn oil and administered by oral gavage at dose levels of 0, 250, 500, 1000, 1500, and 2000 mgikg to five male and five female mice per dose level. The animals were observed for signs of clinical toxicity immediately and 1 hour postdose and at least once daily for up to 2 days after dosing for toxic signs and/or mortality.
Clinical signs in males were observed 1 hour post dosing in the 500, 1000 and 1500 mg/kg bw dose groups. These signs were: hypoactivity, ataxia, recumbency, flattened posture and squinted eyes. In the high dose group the animals were sacrificed for humane reasons approximately 20 hours postdose. No effects were observed in the vehicle control or 250 mg/kg bw dose groups.
Clinical signs in females were observed 1 hour post dosing in the 500 and 1000 mg/kg bw dose groups and up to 2 days postdoing in one animal of the 1500 and 2000 mg/kg bw dose groups. These signs were: labored breathing, chromodacryorrhea, hypoactivity, ataxia, recumbency, flattened posture and squinted eyes. In the high dose group 4 out of 5 animals were sacrificed for humane reasons approximately 20 hours postdose. No effects were observed in the vehicle control or 250 mg/kg bw dose groups. Treatment effects on bodyweight were not observed.

Based on the dose range-finding study doses of 0 (vehicle control), 187.5, 375, 750, and 1500 mg/kg were selected for the micronucleus assay.


RESULTS OF DEFINITIVE STUDY
Death occurred in p-Toluenesulfonamide-treated animals at the 750 mg/kg dose level (1 of 6 females) and 1500 mg/kg dose level (4 of 18 females and 1 of 18 males). There were no signs of toxicity at the 187.5 mg/kg dose level. At 375 mg/kg, hypoactivity and/or squinted eyes were noted in one animalhex. At 750 mg/kg, animals were noted with hypoactivity, flattened posture, ataxia, squinted eyes, andor recumbency. At 1500 mg/kg, findings included hypoactivity, cold to touch, flattened posture, recumbency, ataxia, squinted and/or sealed-shut eyes, labored breathing, and/or rolling over to the right. No significant or biologically relevant consistent body weight losses were observed in the surviving male or female mice in any test-article-treated group.
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase in micronucleus frequencies occurred in polychromatic erythrocytes (PCEs) in male or female mice treated with up to 1500 mg/kg of p-Toluenesulfonamide.
However, a statistically significant decrease was observed in the PCE:normochromatic erythrocytes (NCE) ratio in the 750 mg/kg females at the 24-hour harvest timepoint. When observed, a significant decrease in the PCE:NCE ratio is direct evidence of test article exposure to the bone marrow resulting in cytotoxicity.
The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs a compared to that of the vehicle controls, with a mean and standard error of 2.04 ± 0.30% for the males and 2.27 ± 0.49% for the females.
- Ratio of PCE/NCE (for Micronucleus assay): see "remarks on results...."
- Appropriateness of dose levels and route: "remarks on results...."
- Statistical evaluation: "remarks on results...."

Any other information on results incl. tables

% micronucleated PCEs            mean of 2000 per animal ± SD

Ratio PCE:NCE     mean ± SD

Treatment

Dose mg/kg

Harvest time hrs

Males

Females

Total

Males

Females

Vehicle control

Corn oil

24

0.02 ± 0.03

0.06 ± 0.07

0.04 ± 0.05

0.82 ± 0.09

0.96 ± 0.10

48

0.09 ± 0.08

0.10 ± 0.12

0.10 ± 0.10

0.79 ± 0.10

0.89 ± 0.11

Postitive control

80

24

2.04 ± 0.66*

2.27 ± 1.08*

2.16 ± 0.86*

0.84 ± 0.10

0.95 ± 0.11

p-TSA

187.5

24

0.07 ± 0.04

0.08 ± 0.03

0.08 ± 0.04

0.85 ± 0.09

0.92 ± 0.07

375

24

0.07 ± 0.04

0.06 ± 0.04

0.07 ± 0.04

0.88 ± 0.09

0.95 ± 0.26

750

24

0.03 ± 0.03

0.08 ± 0.06

0.06 ± 0.05

0.79 ± 0.07

0.63 ± 0.07**

1500

24

0.07 ± 0.08

0.10 ± 0.08

0.09 ± 0.08

0.74 ± 0.09

0.82 ± 0.18

48

0.09 ± 0.09

0.09 ± 0.07

0.09 ± 0.06

0.87 ± 0.17

0.81 ± 0.22

*Significantly greater than the corresponding vehicle control, p≤0.01

** Significantly less than the corresponding vehicle control, p≤0.05.

PCE = Polychromatic erythrocyte

NCE = Normochromatic erythrocyte

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based upon the statistical analysis, review by the Study Director, and the Study Evaluation Criteria, p-Toluenesulfonamide was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Executive summary:

The objective of this study was to evaluate the test article, p-Toluenesulfonamide, for in vivo clastogenic activity andlor disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1 (ICR) BR mouse bone marrow. The assay design was based on OECD Guideline 474, updated and adopted July 21, 1997 and OPPTS Guideline 870.5395. The study was performed under GLP.

In the dose range-finding study, the test article was suspended in corn oil and administered by oral gavage at dose levels of 0,250,500, 1000, 1500, and 2000 mgikg to five male and five female mice per dose level. The animals were observed for signs of clinical toxicity immediately and 1 hour postdose and at least once daily for up to 2 days after dosing for toxic signs and/or mortality. Based on the dose range-finding study and discussions with the Sponsor, doses of 0 (vehicle control), 187.5, 375,750, and 1500 mg/kg were selected. Since differences in toxicity between the sexes were observed in the dose range-finding assay, both male and female mice were tested in the micronucleus assay.

The animals were observed for signs of clinical toxicity immediately and 1 and 6 hours postdose and at least once daily and body weights were collected daily until the respective harvest timepoint. Five animals dosed with the test article at the 187.5, 375, and 750 mg/kg dose levels and five animals dosed with the positive control article were euthanized approximately 24 hours after dosing for extraction of the bone marrow. Five animals dosed with the test article at the1500 mg/kg bw dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.

Death occurred in p-Toluenesulfonamide-treated animals at the 750 mg/kg dose level (1 of 6 females) and 1500 mg/kg dose level (4 of 18 females and 1 of 18 males). There were no signs of toxicity at the 187.5 mg/kg dose level. At 375 mg/kg, hypoactivity and/or squinted eyes were noted in one animalhex. At 750 mg/kg, animals were noted with hypoactivity, flattened posture, ataxia, squinted eyes, andor recumbency. At 1500 mg/kg, findings included hypoactivity, cold to touch, flattened posture, recumbency, ataxia, squinted and/or sealed-shut eyes, labored breathing, and/or rolling over to the right. No significant or biologically relevant consistent body weight losses were observed in the surviving male or female mice in any test-article-treated group.

No statistically significant increase in micronucleus frequencies occurred in polychromatic erythrocytes (PCEs) in either male or female mice treated with up to 1500 mg/kg of p-Toluenesulfonamide. However, a statistically significant decrease was observed in the PCE:normochromatic erythrocytes (NCE) ratio in the 750 mglkg females at the 24-hour harvest timepoint. No other statistically significant decreases were observed in the PCE:NCE ratios at any other test article dose level examined at either bone marrow sampling time of 24 or 48 hours postdose. When observed, a significant decrease in the PCE:NCE ratio is direct evidence of test article exposure to the bone marrow resulting in cytotoxicity.

In conclusion, p-Toluenesulfonamide was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.