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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several studies are required to evaluate the genetic toxicity of a compound.
4,4'-dithiodimorpholine was not genotoxic in a bacterial reverse mutation assay (e.g. Ames test) and in a HGPRT cell gene mutation assay on CHO cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The mutagenic potential of test item was tested in cultured Chinese Hamster ovary (CHO) cells. Mutation at the hypowanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of test item was performed initially in a range of Aroclor 1254 -induced rat liver homogenate (S9) concentrations (0 -10%) followed by a confirmatory experiment at 0 and 5%S9. In mutagenicity assays, test item was tested up to concentrations which induced significant cytotoxicity (>50% cell killing).
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT (hypoxanthine guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The subclone K1BH4 of CHO cells was used. The cells were routinely maintained in our laboratory as log growing monolayer cultures in Ham's F12 medium supplemented with heat-inactivated 10% newborn calf serum in an incubator-controlled environment of a 95% humidified atmosphere of 5% +/-1% CO2 and 95% air at 37.5°C +/-2.
Metabolic activation:
with and without
Metabolic activation system:
S9 = Rat liver homogenate induced by Aroclor 1254
Test concentrations with justification for top dose:
Without S9 : 3.4-10 µg/ml
With S9 (1, 2, 5%): 10-34 µg/ml
With S9 (10%): 3.4-10 µg/ml
Vehicle / solvent:
Methanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
methanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene with S9, ethyl methane sulfonate without S9
Details on test system and experimental conditions:
Cytotoxicity determination :
CHO cells were seeded in 25cm² plastic culture flasks at 0.5 x 10^6 cells per flask in growth medium 18-24 hours before treatment. On the day of treatment, the medium was changed to Ham's F12 medium without serum (5 ml) with or without activation. Volumes of 25µl of different concentrations of the test chemical were added. After an incubation of 3 hours at 37.5°C+/-2, the treatment medium was discarded. The wells were washed with 5 ml Hank's balanced salt solution and the cells were removed from the flasks by trypsinization and counted. Three aliquots of approximately 200 cells were plated per sample for determination of cloning efficiency. The plates were returned to incubation for 6-9 days. The colonies developed were fiwed with 70% methanol, stained with 10% Giemsa, and counted by hand. Cytotoxicity was expressed as relative survival.

Mutagenesis :
CHO cells were plated on the day before treatment. The next day, they were treated with test chemicals, positive controls and negative solvent control. The cells were when processed as described above for cytotoxicity determination, except that in addition to plating 200 cells for cloning efficiency, 10^6 cells per sample were plated in 10mL of subculture medium. The cells were subcutured every 2-3 days as attached cultures for 7-9 days for the expression of the mutant phenotype. Mutant selection was performed using selective medium consisting of hypoxanthine-free ham's F12 medium supplemented with mM 6TG and 5%% dialyzed newborn calf serum. Approximatesly 10^6 cells per sample were plated in 100 mm plates (5 plates, 2x10^5 cells per plate) in 8 ml of selective medium plate for mutant selection. Three aliquotsof approximately 200 cells per sample were plated in 2 ml of selective medium without 6TG for the determination of cloning efficiency. The plates were returned to the incubator for 7-9 days. The colonies developed were fixed, stained and counted. Results were expressed as mutant frequency.
Evaluation criteria:
not precised
Statistics:
Mutagenicity data were analyzed according to the statistical method of Snee and Irr (1981) designed specifically for the CHO/HGPRT mutation assay. Mutant frequency values were transformed according to the equation Y=(X+1)^0.15, where Y= transformed lutant frequency and X= observed mutant frequency. Student's t-test was then used to compare treatment data to solvent data. The Snee and Irr analysis also allowed the determination of dose-response relationship as linear, quadratic, or higher-order.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 3.4 µg/ml and greater
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µg/ml (1%), and at 34 µg/ml (2-10%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increases in mutant frequency were observed in these experiments.
The positive controls yielded expected positive responses in mutagenicity indicating the adequacy of the experimental conditions.

RANGE-FINDING/SCREENING STUDIES:
An initial experiment was performed to test the cytotoxicity of the test material. A wide range of test material. A wide range of test concentrations (0.034, 0.065, 0.10, 0.34, 0.65, 1.0, 3.4, 10, 34 and 100 µg/ml) were used. Because of limited solubility of Sulfasan R in aqueous medium, 100 µg/ml was the higher dosage tested. The results of this experiment were used for a dose-selection purposes for the subsequent mutagenicity experiments.
The initial cytotoxicity determination of Sulfasan R in CHO cells at different S9 concentrations showed that the cytotoxicity of Sulfasan R decreased with increased S9 concentration. The concentrations for the cytotoxicity rangefinding experiment that induced significant cytotoxicitywere 10, 34, 34, 34 and 10 µg/ml in absence of S9, and in presence of 1%, 2%, 5% and 10% S9 respectively.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9 activation the cytotoxicity of the test sample decreased with increasing S9 concentration. Significant cytotoxicity was demonstrated for treatment levels of 10 µg/ml in the presence of 1% of S9 and at 34 µg/ml in the presence of 2, 5 and % S9.
Conclusions:
No test chemical related mutagenicity was observed in the initial mutagenicity test or in two confirmation experiments conducted in the absence of S9 or in the presence of S9. The test item is therefore concluded not to be a mutagen in CHO cells.
Executive summary:

The mutagenic potential of 4,4'-dithiodimorpholine was tested in cultured Chinese Hamster ovary (CHO) cells. Mutation at the hypowanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of Sulfasan R was performed initially in a range of Aroclor 1254 -induced rat liver homogenate (S9) concentrations (0 -10%) followed by a confirmatory experiment at 0 and 5%S9. In mutagenicity assays, Sulfasan R was tested up to concentrations which induced significant cytotoxicity (>50% cell killing). The ranges of concentrations for all experiments, including the cytotoxicity were 3.4 -10 µg/ml, 10 -34µg/ml, 10 -34 µg/ml, 10 -34 µg/ml and 3.4 µg/ml in the absence of S9 and in the presence of 1%, 2%, 5% and 10% of S9 respectively. No test chemical related mutagenicity was observed in the initial mutagenicity test or in two confirmation experiments conducted in the absence of S9 or in the presence of S9. 4,4'-dithiodimorpholine is therefore concluded not to be a mutagen in CHO cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine operon.
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
- 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 102 strain in both experiments,
- 19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1535 and TA 1537 strains in both experiments,
- 4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 µg/plate for the TA 98 and TA 100 strains in both experiments.

Experiments with S9 mix
- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for all tested strains in the first experiment,
- 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 1535 and TA 102 strains in the second experiment,
- 19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1537 and TA 98 strains in the second experiment,
- 9.77, 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate for the TA 100 strain in the second experiment,
- 156.3, 208.3, 312.5, 416.7, 625 and 1250 µg/plate for the TA 98 strain in the third experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item was freely soluble in DMSO at 50 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: sodium azide; 9-Aminoacridine; 2-Nitrofluorene; Mitomycin C; 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix was performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours, 37°C in growth medium

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
Not applicable.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

TOXICITY:
No S9:
moderate to strong toxicity at 156.3 µg/plate in TA 98 and TA 100, at = 312.5 µg/plate in TA 1535 and TA 1537 and at 1250 µg/plate in TA 102.
With S9:
Direct plate incorporation (first and third experiments): moderate to strong toxicity at = 625 µg/plate in TA 1537 and = 1250 µg/plate in TA 1535, TA 98, TA 100 and TA 102.
Preincubation (second experiment): moderate to strong toxicity at = 156.3 µg/plate in TA 1537 and TA 100 and = 312.5 µg/plate in TA 1535, TA 98 and TA 102.

MUTATIONS:
Slight increases in the number of revertants were occasionally noted. In most cases they did not exceed the threshold of 2-fold the vehicle control value; in all cases they were neither dose-related nor reproducible and therefore were not considered as biologically relevant.
Conclusions:
The test item 4,4-Dithiodimorpholine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, with or without metabolic activation system.
Executive summary:

The potential of 4,4' -Dithiodimorpholine to induce reverse mutation was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.  

The selected treatment-levels witout S9 mix were 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 102 strain in both experiments, 19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1535 and TA 1537 strains in both experiments and  4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 µg/plate for the TA 98 and TA 100 strains in both experiments. A moderate to strong toxicity was noted at 156.3 µg/plate in the TA 98 and TA 100 strain, at dose-levels >=312.5 µg/plate in the TA 1535 and TA 1537 strains and at 1250 µg/plate in the TA 102 strain. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

The selected treatment-levels with S9 mix were: 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for all tester strains in the first experiment, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 1535 and TA 102 strains in the second experiment, 19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1537 and TA 98 strains in the second experiment, 9.77, 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate for the TA 100 strain in the second experiment, and 156.3, 208.3, 312.5, 416.7, 625 and 1250 µg/plate for the TA 98 strain in the third experiment. 

Using the direct plate incorporation method (first and third experiments), a moderate to strong toxicity was noted at dose-levels >= 625 µg/plate in the TA 1537 strain and >= 1250 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strain. Using the preincubation method (second experiment), a moderate to strong toxicity was noted at dose-levels >= 156.3 µg/plate in the TA 1537 and TA 100 strain and >= 312.5 µg/plate in the TA 1535, TA 98 and TA 102 strains. The test item did not induce any noteworthy increase in the number of revertants in the TA 1535, TA 1537, TA 100 and TA 102 strains in either experiment. A slight increase in the number of revertants was noted in the TA 98 strain in the first experiment using the direct incorporation method (up to 2.4-fold the vehicle control value). Since this increase was not dose-related, was not observed in the second experiment (using the preincubation method) and was not reproduced in the third experiment (despite the use of a narrower range of dose-levels), it was considered as non biologically relevant.

4,4' -Dithiodimorpholine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

4,4'-dithiodimorpholine was not genotoxic in an in vivo chromosome aberration assay in rats.

Link to relevant study records
Reference
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
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
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
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
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)
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.
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation assays

Bacterial reverse mutation assay (e. g. Ames test)

The potential of 4,4'-Dithiodimorpholine to induce reverse mutation was evaluated in Salmonella typhimurium (Sire, 2010). This key study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolvedin dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines. The selected treatment-levels without S9 mix were39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 102 strain in both experiments,19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1535 and TA 1537 strains in both experiments and  4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 µg/plate for the TA 98 and TA 100 strains in both experiments. A moderate to strong toxicity was noted at 156.3 µg/plate in the TA 98 and TA 100 strain, at dose-levels >=312.5 µg/plate in the TA 1535 and TA 1537 strains and at 1250 µg/plate in the TA 102 strain. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains. The selected treatment-levels with S9 mix were:78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for all tester strains in the first experiment,39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the TA 1535 and TA 102 strains in the second experiment,19.53, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate for the TA 1537 and TA 98 strains in the second experiment,9.77, 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate for the TA 100 strain in the second experiment, and 156.3, 208.3, 312.5, 416.7, 625 and 1250 µg/plate for the TA 98 strain in the third experiment. Using the direct plate incorporation method (first and third experiments), a moderate to strong toxicity was noted at dose-levels >=625 µg/plate in the TA 1537 strain and >=1250 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strain. Using the preincubation method (second experiment), a moderate to strong toxicity was noted at dose-levels >=156.3 µg/plate in the TA 1537 and TA 100 strain and >=312.5 µg/plate in the TA 1535, TA 98 and TA 102 strains. The test item did not induce any noteworthy increase in the number of revertants in the TA 1535, TA 1537, TA 100 and TA 102 strains in either experiment. A slight increase in the number of revertants was noted in the TA 98 strain in the first experiment using the direct incorporation method (up to 2.4-fold the vehicle control value). Since this increase was not dose-related, was not observed in the second experiment (using the preincubation method) and was not reproduced in the third experiment (despite the use of a narrower range of dose-levels), it was considered as non biologically relevant.

4,4' –Dithiodimorpholine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

 

In a supportive study (Anonymous, 1996) 4,4'-Dithiodimorpholine was negative for reverse gene mutations in Salmonella typhimurium: TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and inEscherichia. coliWP2 uvr A.

Gene mutation assay in mammalian cells

The mutagenic potential of 4,4'-dithiodimorpholine was tested in cultured Chinese Hamster ovary (CHO) cells (Flowers, 1987). Mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of 4,4'-dithiodimorpholine was performed initially in a range of Aroclor 1254 -induced rat liver homogenate (S9) concentrations (0 -10%) followed by a confirmatory experiment at 0 and 5%S9. In mutagenicity assays, 4,4'-dithiodimorpholine was tested up to concentrations which induced significant cytotoxicity (>50% cell killing). The ranges of concentrations for all experiments, including the cytotoxicity were 3.4 -10 µg/ml, 10 -34µg/ml, 10 -34 µg/ml, 10 -34 µg/ml and 3.4µg/ml in the absence of S9 and in the presence of 1%, 2%, 5% and 10% of S9 respectively. No test chemical related mutagenicitywas observed in the initial mutagenicity test or in two confirmation experiments conducted in the absence of S9 or in the presence of S9. 4,4'-dithiodimorpholine is therefore concluded not to be a mutagen in CHO cells.

Chromosomal aberration assay

The potential of 4,4'-dithiodimorpholine to induce structural chromosomal aberrations was evaluated in the hemopoietic cells of the rat bone marrow when administered by oral gavage (San Sebastien, 1986). 4,4'-dithiodimorpholine 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 an estimation of the maximum tolerated dose. 4,4'-dithiodimorpholine (2800 mg/kg) and the vehicle control (corn oil) were administered in single oral doses to three groups of Fisher 344 rats per each treatment level and sacrificed 6, 18 and 30 hours after dosing. An extra group of rats was dosed with the positive control, Cyclophosphamide (CP; 20mg/kg) and sacrificed 18 hours later. Approximately two hours prior to each sacrifice, animals were administered colchicine at 4 mg/kg of body weight to arrest cells in metaphase. At the appropriate time, animals were sacrificed and both femurs were removed from each animal and metaphase slides prepared. Slides were stained, coded and scored for chromosomal aberrations. All rats dosed with4,4'-dithiodimorpholine (2800 mg/kg) exhibited from mild to severe pharmacotoxic signs at all time intervals evaluated. These observations suggest that 4,4'-dithiodimorpholine 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 centromeres in each cell was counted and recorded. Data were evaluated for statistically significant increases in aberrations per cell in treatment groups as compared to the vehicle control groups. The proportion of aberrant metaphases was also evaluated for statistically significant increases over the vehicle control groups. Data were evaluated separately for each harvest interval. The positive control article, CP, resulted in significant increases in the incidence of chromosome aberrations and in the proportion of metaphases with one or more aberrations. No statistically significant increases in the incidence of aberrations or in the number of cells with one or more aberrations were observed in animals treated with 4,4'-dithiodimorpholine. Therefore, under the conditions of thisassay,4,4'-dithiodimorpholinewas not clastogenic to the hemopoietic cells of the rat bone marrow.

Justification for classification or non-classification

Based on the available data, DTDM is considered to be not genotoxic according to the Regulation EC N°1272/2008.

Justification: DTDM did not induce gene mutation (Ames and HPRT tests are negative) or chromosomal aberrations (in vivo micronucleus assay is negative).