Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1, 1987 - June 9, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
50 metaphase cells were observed per animal (instead of 100).
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Di(morpholin-4-yl) disulphide
EC Number:
203-103-0
EC Name:
Di(morpholin-4-yl) disulphide
Cas Number:
103-34-4
Molecular formula:
C8H16N2O2S2
IUPAC Name:
4-(morpholin-4-yldisulfanyl)morpholine

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories, Kingston, New York
- Age at study initiation: 61 days
- Weight at study initiation: males = 139-158g, females = 104-122g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: 18 hours prior dosing
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Wayne Rodent Blox, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Volume of administration = 10 ml/kg
Duration of treatment / exposure:
a single exposure
Frequency of treatment:
a single exposure
Post exposure period:
6, 18 and 30 hrs
Doses / concentrations
Remarks:
Doses / Concentrations:
2800 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 animals / sexe / group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclosphosphamide (20 ml/kg)

Examinations

Tissues and cell types examined:
Animals were sacrified at 6, 18 and 30 hours after dosing. 2/3h prior sacrifice, each animal was given a single peritoneal dose of colchicine at 4 mg/kg bw to arrest dividing cells in metaphase. A total of 500 well spred, intact metaphase cells were scored for the presence of chromosome aberration per experiment treatment point (50/animal) by 2 invertigators (25 each/animal).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the preliminay study and in discussion with the Sponsor, a dose of 2800 mg/kg was selected as an estimate of the maximum tolerated dose for evaluation in the metaphase analysis assay.

DETAILS OF SLIDE PREPARATION:
Slides were coded randomly by study number and number and letter designation by a person not involved in the actual scoring of the slides. The code was kept in the QAU's file until ail slides were evaluated. Ail animais were coded as a single set with no indication of dose or time of harvest. Following scoring of ail slides, data were decoded and placed into groups for statistical analysis.

METHOD OF ANALYSIS:
A total of 500 well spread, intact metaphase cells (if possible) were scored for the presence of chromosome aberration per experimental treatment point (50 per animal) by two investigators (25 each per animal). Cells were located by systematic searching of the slide under low power (20X-40X) magnification. Cells judged acceptable for analysis based on cell morphology and total centromere number were then further analyzed with a 100X oil immersion objective where abnormalities were detected and classified. Vernier coordinates were recorded for the first and last metaphase scored, as well as for any abnormal metaphases (including gaps) observed. The centromere number was recorded for all cells analyzed. Ail slides were scored for chromosome damage using a Nikon Optiphot microscope.
Evaluation criteria:
not precised
Statistics:
Mean number of aberrations per cell per rat (50 cells per rat) were analyzed for statistically significant increases in chromosome aberration by a one-way analysis of variance (ANOVA). Each sampling Lime was analyzed separately as compared to its concurrent vehicle control group. The CP group was not included in the ANOVA. Data from this group were analyzed separately by a one-tailed t test comparing CP with the 18 heur vehicle contrai. The mean and standard deviation of aberrations/tell were also determined for each group of rats (500 tells; 50 tells per rat). The number of aberrant metaphases was analyzed by Chi-square analysis for statistically significant increases. Statistical significance was determined at the p < or =0.05 probability level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY : 2 adult male and 2 adult female rats (55 days old)
- Dose range: 2800 mg/kg bw
Due to the pharmacotoxic signs observed at this dose, 2800 mg/kg was selected as an estimation of the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
Pharmacotoxic Effects of Treatment:
No pharmactoxic signs of treatment were observed in animals administered Sulfasan R immediately after dosing. Pharmacotoxic signs observed prior to colchicine administration in all groups treated with Sulfasan R were as follows :
-6 Hour Croup - (observed approximately 4 hours after dosing):
All rats had exhibited diarrhea, piloerection, decreased body tone, abnormal gait and a brownish discoloration on forepaws and around oral-nasal region. Four males and two females had an abnormal stance and arched back. Five males and two females had decreased activity and three females had body drop. One male died just prior to harvest. Gross necropsy of this animal revealed mottled lungs. This male was replaced by an other rat which also exhibited signs common to those in this group. Males in this group lost an average of 1.2 grams while the females gained 0.6 grams average from the original fasted weights.
-18 Hour Group - (observed approximately 16 hours after dosing):
All-rats exhibited diarrhea, decreased body tone, abnormal gait, abnormal stance, piloerection, arched back, and a brownish discoloration on forepaws and around nasal-oral region. Three females and four males exhibited chromodacryorrhea and one male and two females had lacrimation. Males and females bath lest an average of 5.2 grams from the initial fasted weights.
-30 Hour Group (observed approximately 28 hours after dosing):
Al rats had an abnormal gait, abnormal stance, decreased body tone, piloerection, arched back and a brownish discoloration on forepaws and around nasal-oral region. Additional signs observed in this group in one or more animais include diarrhea, decreased activity, chromodacryorrhea and pour grooming. Males lost an average of 3.6 grams by this time while females gained 2.2 grams average as compared to the original fasted weights.
-No pharmacotoxic signs were observed in rats administered the vehicle or positive controls with the exception of two males and one female in the 6 hour corn oil group which exhibited diarrhea prior to colchicine administration.
Males in the 6 hour corn oil group had an average weight loss of 1.4 grams white the females gained 0.6 grams average. In the 18 hour corn oil group, by sacrifice time, the males had an average weight gain of 6.4 grams while the females gained an average of 10 grams from the initial fasted weights. In the 30 hour corn oil group, males gained 9 grains average and the females gained 6 grams average.
Positive control males had an average gain of 6.4 grains and females gained an average of 7.6 grams compared to the initial fasted weights.

Summary of Metaphases scored: A total of 500 metaphases/group (50/rat) were. analysed in each group, except for the Cl) group in which a total of only 460 metaphases were analysed.

Proportion of Cells with Aberrations: The proportion of cells with one or more aberrations (1 aberration or greater) was analyzed by Chi-square analysis by comparing the number of cells with aberrations versus the number of normal cells in each treatment group versus the vehicle control. Each time of sacrifice was analyzed separately and groups within each sacrifice time were compared individually to the vehicle control. Cells with gaps only were net considered aberrant for this analysis. A statistically significant increase (p < 0.01) was detected with the positive control CP group at the 18 hour sacrifice. No other significant differences were noted.

Analysis of Aberrations per Cell: The mean number of aberrations per cell per animal were analyzed for statistically significant increases by a one-way ANOVA for each time interval. Data reflect the total number of aberrations seen in all cells scored per group. No statistically significant differences (p < 0.05) from the vehicle controls were detected by this analysis in animals treated with Sulfasan RR.

Animals treated with CP gave a statistically significant (p < 0.01) increase in the number of aberrations. In this group 139 metaphases were severely damaged. These metaphases could not accurately be scored due to multiple aberrations with tangles (134 metaphases) and/or partially shattered chromosomes (5 metaphases). This metaphase population comprised 23.2% of ail metaphases scored in the CP group. These cells were noted on the scoring sheets but were not included in the total of 460 metaphases analyzed statistically.

Any other information on results incl. tables

Table : Results of in vivo test

Compound

dose

Harvest time

# rats

# metaphases analyzed

Aberrations/group

Corn oil

10 ml/kg

6 hr

10

500

2

DTDM

2800 mg/kg

6 hr

10

500

5

Corn oil

10 ml/kg

18 hr

10

500

2

DTDM

2800 mg/kg

18 hr

10

500

5

Positive control

20 mg/kg

18 hr

10

460

1719

Corn oil

10 ml/kg

30 hr

10

500

2

DTDM

2800 mg/kg

30 hr

10

500

6

Applicant's summary and conclusion

Conclusions:
DTDM was judged negative in its ability to induce structural chromosomal aberrations to the hemopoietic cells of the rat bone marrow under the experimental conditions of this assay.
Executive summary:

This study was designed to evaluate the potential of 4,4'-dithiodimorpholine (Sulfasan R) to induce structural chromosomal aberrations in the hemopoietic cells of the rat bonemarrow when administered by oral gavage. Sulfasan R was evaluated in a preliminary study at a dose of 2800 mg/kg of body weight. Due to the pharmacotoxic signs observed in this dose group, 2800 mg/kg was selected as anestimation of the maximum tolerated dose.

Sulfasan R (2800 mg/kg) and the vehicle control (corn oil) wereadministered in single oral doses to three groups of Fisher 344 rats per eachtreatment level and sacrificed 6, 18 and 30 hours after dosing. An extragroup of rats was dosed with the positive control, Cyclophosphamide (CP; 20mg/kg) and sacrificed 18 hours later. Approximately two hours prior to eachsacrifice, animals were administered colchicine at 4 mg/kg of body weight toarrest tells in metaphase. At the appropriate time, animals were sacrificedand both femurs were removed from each animal and metaphase slides prepared.Slides were stained, coded and scored for chromosomal aberrations.

Ail rats dosed with Sulfasan R {2800 mg/kg) exhibited from mild to severepharmacotoxic signs at all time intervals evaluated. These observations suggest that Sulfasan R was evaluated near the maximum tolerated dose.

A total of 50 metaphase cells were analyzed for each animal (when possible) for the presence of chromatid and chromosome type aberrations.Aberrations were classified according to type on a standard scoring sheet and the number of aberrations in each cell tabulated. The number of centromeresin each cell was counted and recorded.

Data were evaluated for statistically significant increases inaberrations per cell in treatment groups as compared tothevehicle controlgroups. The proportion of aberrant metaphases was also evaluated forstatistically significant increases over the vehicle control groups. Datawere evaluated separately for each harvest interval.

The positive control article, CP, resulted in significant increases inthe incidence of chromosome aberrations and in the proportion of metaphases with one or more aberrations.

No statistically significant increases in the incidence of aberrations orin the number of cells with one or more aberrations were observed in animals treated with Sulfasan R at a dose of 2800 mg/kg at any of the three sampling times evaluated. Therefore, under the conditions of thisassay, Sulfasan R was not clastogenic to the hemopoietic cells of the rat bone marrow.