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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other: Body responsible for the test
Title:
Unnamed
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Guidelines for Chemical Substances Control Law of Japan (1986)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-bis((2-mercaptoethyl)thio)-1-propanethiol
EC Number:
411-290-7
EC Name:
2,3-bis((2-mercaptoethyl)thio)-1-propanethiol
Cas Number:
131538-00-6
Molecular formula:
C7 H16 S5
IUPAC Name:
2,3-bis((2-mercaptoethyl)thio)-1-propanethiol
Details on test material:

- Analytical purity: no data

Test animals

Species:
rat
Strain:
other: Charles River CD(R)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 40 to 47 days
- Weight at study initiation: 107 to 133 g
- Housing: five of one sex per cage (Type TR18 (Modular Systems Development Ltd., London, England))
- Diet (e.g. ad libitum): expanded rodent diet, Laboratory Animal Diet No. 1 (Biosure, Manea, Cambridgeshire, England), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 12 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
All formulations were prepared freshly each day.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days, + 14 d recovery
Frequency of treatment:
daily, 7d/week
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control: 5
10 mg/kg bw/day: 5
50 mg/kg bw/day: 5
200 mg/kg bw/day: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dosages were selected an the basis of toxicity data provided by two preliminary investigations.
Initially five groups of three male and three female CD rats received the test item daily, by oral gavage, at dosages of 10, 50, 100, 500 or 1000 mg/kg/day for seven days.
An additional group of three males and three females received the vehicle only. Serial examinations were confined to observation of clinical signs, bodyweight, haematology, blood chemistry and organ weights.
All animals receiving 500 or 1000 mg/kg/day were either found dead or were killed in extremis within two days of the commencement of treatment. No indication of a treatment-related effect was found at any other dosage level.
Accordingly, a second preliminary investigation was performed using two groups of three male and three female CD rats which received the test item daily, by oral gavage, at dosages of 200 or 350 mg/kg/day. Serial examinations were confined to observations of clinical signs and bodyweight.
There were no deaths. Signs of reaction to treatment included reduced bodyweight gain, staining, hunched posture, piloerection, thin body conformation, lack of grooming, hair-loss and irregular respiration amongst animals receiving 350 mg/kg/day. No evidente of a treatment-related effect was observed in animals which received 200 mg/kg/day.
Dosages selected for the main study were 0, 10, 50 and 250 mg/kg/day. However, due to an administrative oversight the highest dosage administered to the animals throughout the study was 200 mg/kg/day. In view of the clear effects of treatment seen at the dosage of 200 mg/kg/day this error was not considered to have adversely affected the validity of the study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: Skin and fur, Eyes and mucous membranes, Respiratory system, Circulatory system, Autonomic and central nervous system, Somatomotor activity, Behaviour pattern

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily check for deaths and morbidity. Detailed weekly examination including palpation.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- mean weekly consumption per rat was calculated for each cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes (cagewise)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of main study, end of recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters: Packed cell volume (PCV), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Total and differentialt leucocyte count (WBC), Platelet count, Mean cell haemoglobin (MCH), Mean cell volume (MCV), Mean cell haemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (PTTK)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of main study, end of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters: AP, ALT, AST, GGT, Urea, Creatinine, Glucose, Total bilirubin concentration, Total cholesterin concentration, Total triglyceride concentration, Total protein concentration, Electrophoretic protein fractions, Sodium (Na), potassium (K), chloride (Cl), calcium (Ca) , inorganic phosphorus (P)Erythrocyte (cell) cholinesterase activity, Plasma cholinesterase activity

URINALYSIS: Yes
- Time schedule for collection of urine: prior to start of study, after 25 d; only main study animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: Appearance, Volume, pH by pH meter, Specific gravity, Protein, Total reducing substances, Glucose, Ketones, Bilirubin, Urobilin, Nitrite, Blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
weights: Adrenals, Brain, Kidneys, Liver, Ovaries, Testes
preserved, but not examined histopathologically: Brain, Eyes and optic nerves, Femoral bone and marrow, Lungs (with mainstem bronchi), Ovaries, Pituitary, Testes, Stomach, Thyroids (with parathyroids), Urinary bladder

HISTOPATHOLOGY: Yes
Adrenals, Heart, Kidneys, Liver, Spleen

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Throughout the treatment period animals receiving 200 mg/kg/day salivated immediately after dosing; from Week 2 the incidence was 95% or 100% of the maximum possible. Salivation was occasionally observed in animals receiving 50 mg/kg/day.
Signs observed among animals receiving 200 mg/kg/day included staining of the fur, hair-loss and lack of grooming. The staining of the fur was observed on the head, limbs and on the ventral and dorsal surfaces. Staining on the head was also evident during the first week of the reversibility period for females. Hair-loss from the head, limbs and from the ventral and dorsal surfaces was observed in males, and from the head and dorsal surface in females. The incidence tended to be higher in males. Hair-loss from the head and dorsal surface persisted throughout the reversibility period.
The staining of the head and hair-loss from the dorsal surface occasionally observed in animals receiving 10 or 50 mg/kg/day, were not considered to be related to treatment, as they were also observed among the controls.
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight gain of males and females receiving 200 mg/kg/day was inferior to that of the controls throughout the treatment period. Animals receiving 10 or 50 mg/kg/day were unaffected.
Over the first ten days of the reversibility period bodyweight gains of animals which had received 200 mg/kg/day were higher than those of the controls.
Weight losses recorded at the end of the treatment or reversibility periods were attributed to the collection of clinical pathology samples.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of males receiving 200 mg/kg/day was lower than that of the controls during the first two weeks of treatment, and similar to that of the controls during the third week. In contrast the food consumption of females receiving this dosage was similar to that of the controls during the first week and superior during the second and third weeks.
Food consumption of animals receiving 10 or 50 mg/kg/day was unaffected by treatment.
During the reversibility period food consumption of males and females which had received 200 mg/kg/day was superior to that of the controls, particularly during the first week.
The lower food consumption, during the fihal week of the treatment period for all groups was associated with the collection of urine samples. The reduction in food intake at this time in animals receiving 200 mg/kg/day was greater than in the controls.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food utilization was clearly less efficient in animals receiving 200 mg/kg/day when compared with control values. During the reversibility period lower food conversion ratios indicated improved food utilization efficiency for these rats.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption measurements in the final week of treatment confirmed that water intake among animals receiving 200 mg/kg/day, particularly females, was higher than that of the controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 29 days the following treatment-related changes, relative to the controls, were observed:
Slightly higher erythrocyte count, lower mean cell volume and mean cell haemoglobin in males receiving 200 mg/kg/day. High platelet count in males and females receiving 200 mg/kg/day.
Slightly longer prothrombin time in males and females receiving 200 mg/kg/day and in males receiving 50 mg/kg/day.
The effect was dosage-related in males.
Slightly langer activated partial thromboplastin time in males receiving 200 mg/kg/day.
After 14 days of the reversibility period only the high erythrocyte count, low mean cell volume and mean cell haemoglobin were still apparent for males which had received 200 mg/kg/day.
Examination of the blood films an Day 29 indicated slight anisocytosis and microcytosis in two males receiving 200 mg/kg/day; one of these animals had slight polychromasia in addition. On Day 15 of the reversibility period four of the five males which had received 200 mg/kg/day had abnormalities of the blood film which included anisocytosis, poikilocytes, microcytosis, target cells, spherocytes and polychromasia. The
blood films of controls were considered to be normal at both examinations.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After 29 days of treatment the following differences from the controls were noted:
Lower alkaline phosphatase activity in males-receiving 50 or 200 mg/kg/day and in two females at 200 mg/kg/day. Slightly higher alanine amino-transferase activity in animals receiving 200 mg/kg/day.
Slightly higher erythrocyte acetylcholinesterase activity in males receiving 200 mg/kg/day.
Slightly lower total cholesterol concentration in males receiving 200 mg/kg/day.
The lower plasma a-1 globulin level evident in all groups of treated females and the slightly higher chloride concentration evident in all groups of treated males were not considered to be related to treatment.
After 14 days of the reversibility period higher cell acetylcholinesterase activity was evident in males which had received 200 mg/kg/day. The alkaline phosphatase activities in three males previously treated at 200 mg/kg/day were slightly higher than in the controls. All other parameters examined were essentially the same for animals which had received 200 mg/kg/day and the controls.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary output was marginally greater for females receiving 200 mg/kg/day than for the controls after 25 days of treatment.
The number of crystals in the urine of females receiving 200 mg/kg/day was higher than for the controls. However, the number of crystals had also been higher prior to the commencement of treatment in this group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period absolute and bodyweight-relative liver weights were high for females receiving 200 mg/kg/day. The bodyweight-relative liver weights of males at this dosage were also higher than those of the controls after 29 days of treatment, although the differente was not statistically significant. At the end of the reversibility period the liver weights, both absolute and relative, of females which had received 200 mg/kg/day were low compared with the controls.
Slightly high bodyweight-relative testes weights were evident at the end of treatment and reversibility periods for males receiving 200 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal any internal changes which could be attributed to treatment. The higher incidences of hair-loss evident for animals which received 200 mg/kg/day reflected the changes seen in vivo.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Slight centriacinar hepatocytic fatty vacuolation was noted in three females which received 200 mg/kg/day, killed at the end of the treatment period. Single incidences of this finding were observed in females which received 10 or 50 mg/kg/day.
At the end of the reversibility period minimal centriacinar hepatocytic fatty vacuolation was observed in one female which had received 200 mg/kg/day.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
not specified
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
blood
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
It was concluded that treatment of CD rats with DMPT at 200 mg/kg/day was associated with non-specific toxicity and with minor changes in the blood and liver. The changes in the liver were reversed over a 14-day period without treatment; the haematological changes were not. It was considered that variations at 50 mg/kg/day were minimal and that this dosage was a no-toxic-effect level in this study.
Executive summary:

Two groups of five male and five female CD rats received DMPT by oral gavage, at dosages of 10 or 50 mg/kg/day for four weeks. A third group of ten male and ten female CD rats received the test item at a dosage of 200 mg/kg/day for four weeks; five males and five females of this group were held for a subsequent two week period without treatment, for reversibility studier. A control group, constituted similarly to the high dosage group, received the vehicle (maize oil) during the treatment period.

 

Administration of DMPT to CD rats at a dosage of 200 mg/kg/day resulted in a number of changes in the blood and liver and in bodyweight gain.

There was evidence that, with the exception of some haematological changes, these effects were reversible.

Salivation seen immediately after dosing is a common finding in studies with gavage administration and is not of itself of toxicological significance.

Evidence of toxicity included hair-loss and lack of grooming, indicating poor condition, and lower bodyweight gains in animals treated at 200 mg/kg/day. The effect upon bodyweight gain was not

clearly dependent an food consumption, as there was also a reduction in the efficiency of food utilization.

Changes in the haematological composition of the blood indicated an effect an haemopoiesis. The increase in erythrocyte count was not large and probably resulted from a response to the microcytosis. The aetiology of this effect is obscure, but it was considered to be of potential toxicological significance as these minor changes were still present at the end of the reversibility period, when the numbers of

animals with blood film abnormalities was greater than after 29 days of treatment. The variations in platelet count were small and were not toxicologically significant.

There was clear evidence for a minor effect of treatment an the liver in high dosage animals. This was indicated by increases in alanine amino-transferase activity and lower cholesterol concentration in the

plasma and by increased liver weight. The increases in prothrombin and activated partial thromboplastin times, although minor, were also probably associated with this change. The slight centriacinar hepatocytic fatty vacuolation noted in some females, although a common finding in CD rats, is further evidence that treatment with GST at 200 mg/kg/day resulted in a minor disturbance of hepatic function.

These changes were absent or greatly reduced after two weeks of the reversibility period.

Although an increase in alkaline phosphatase activity in the plasma is frequently a marker of liver toxicity, the decrease in high dosage animals after 29 days of treatment was probably associated with the lower bodyweight gain at this dosage. In young animals most of the alkaline phosphatase found in the plasma originates from osteoblastic activity, and the enzyme activity declines as bone growth decreases.

This hypothesis was supported by small increases in the activity of this enzyme at the end of the reversibility period, associated with a resumption of bodyweight gain.

The minor increases in water consumption and urinary volume in animals receiving 200 mg/kg/day were not associated with any change in plasma concentration, kidney weight or pathological appearance. This change is probably functional in origin and not of toxicological significance.

The high bodyweight-relative testes weight evident for males receiving 200 mg/kg/day compared with the controls was considered to reflect the disparity in bodyweight between these two groups as absolute testicular weight is usually dependent upon maturity rather than bodyweight.

In this study 50 mg/kg/day was considered to represent the no-toxic-effect-level as the variations observed in the cellular and chemical composition of the blood were minor and not of pathological

significance.

The changes observed among animals receiving 10 mg/kg/day were minimal and were considered to be unrelated to treatment.

 

It was concluded that treatment of CD rats with DMPT at 200 mg/kg bw/d was associated with non-specific toxicity and with minor changes in liver and haematology. The changes in the liver were reversed over a 14-day period without treatment; the haematological changes were not.

It was considered that variations at 50 mg/kg/day were minimal and that this dosage was a no-adverse-effect level in this study.