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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-11-16 - 1993-12-14
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:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
No data on sensory reactivity to stimuli of different types, assessment of grip strength, and motor activity; blood clinical chemistry did not include urea; urinalysis did not include osmolality or specific gravity.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Didodecyl 3,3'-thiodipropionate
EC Number:
204-614-1
EC Name:
Didodecyl 3,3'-thiodipropionate
Cas Number:
123-28-4
Molecular formula:
C30H58O4S
IUPAC Name:
didodecyl 3,3'-sulfanediyldipropanoate
Details on test material:
- Substance type: Organic
- Physical state: Solid, fine white powder
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Sprague Dawley Ico:OFA-SD (lOPS Caw)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO, L'Arbresle, FRANCE.
- Age at study initiation: 6 weeks
- Weight at study initiation: 162 - 193 g (males) and 142 - 180 g (females)
- Housing: Groups of 5 of the same sex and dose group in stainless steel mesh cages (555 x 350 x 200 mm)
- Diet (e.g. ad libitum): Ad libitum sterile Rat and Mouse pelleted complete diet (Diet A04C-10, Usine d'Alimentation Rationnelle, Epinay S/Orge, France)
- Water (e.g. ad libitum): Ad libitum filtered (0.2 µm) mains water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 35 - 75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Carboxymethylcellulose in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the test article was prepared as a suspension in the vehicle

VEHICLE
- Concentration in vehicle: 12.5, 35, and 100 mg/mL
- Amount of vehicle (if gavage): 10 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For day 1, it was used a Varian 9010 high-performance liquid chromatograph equipped with a 9095 autosampler and a Kontron 432 UV detector.
For weeks 4, 8, and 13, it was used a Shimadzu LC 6A high-performance liquid chromatograph equipped with a SIL 6A autosampler and a UV detector SPD 6A.
Column Nucleosil C18, 120 x 4.6 mm I.D., particle size 3 μm; flow rate at 1 mL/min.
Detection at 209 nm.
Injection volume: 15 μL of each sample.
Mobile phase: solvent A: acetonitrile; solvent B: methanol isocratic mode : 60 % (A) and 40 % (B).
Samples (2 x 2 g) from each preparation (including the control group) were taken at weeks 1, 4, 8, and 13 and were stored at approximately -20 °C.
Duration of treatment / exposure:
Males: 92 days; females: 93 days; recovery animals: 91 days (plus treatment-free recovery period of 4 weeks)
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 in each dose group of main study and 5 in recovery group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a dose range-finding study in the rat (study number 380/572). The high dose of 1000 mg/kg/day is the maximum required by the relevant Regulatory Guidelines, 125 mg/kg/day was the low dose level in the dose range-finding study and was expected to be a no-effect or no observable adverse effect level. The intermediate dose is an approximation of the geometric mean between the low. and high dose levels.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, at the beginning and at the end of each working day
- Cage side observations: Morbidity/mortality, clinical signs, and reaction to the treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and week 13
- Dose groups that were examined: all animals pretest, oil animals during week 13 in control and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 10 animals/sex/group. All recovery animals after week 17
- Parameters checked: Haemoglobin; Mean corpuscular haemoglobin (MCH); Mean corpuscular haemoglobin concentration (MCHC); Packed cell volume; Red blood cell count; Mean corpuscular volume (MCV); Platelet count; Prothrombin time; Activated partial thromboplastin time (APTT); Total white blood cell count; Differential white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Animals fasted: Yes
- How many animals: 10 animals/sex/group. All recovery animals after week 17
- Parameters checked: Sodium; Potassium; Chloride; Calcium; Inorganic phosphorus; Glucose; Blood urea nitrogen; Total cholesterol*; Total bilirubin; Total protein; Albumin; Globulin (calculated); Albumin/Globulin ratio; Creatinine; Alkaline phosphatase (AP)*; Aspartate aminotransferase (ASAT)*; Alanine aminotransferase (ALAT)*.
*Only these parameters were examined after week 17 ; total cholesterol was evaluated in females only.

URINALYSIS: Yes
- Time schedule for collection of urine: control and high-dose group after week 4, all groups after week 13, and recovery animals after week 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Animals deprived of food and, water received 20 mL/kg of mains water by gavage before being placed in the metabolism cages
- Parameters checked: Volume; Specific gravity; Appearance. Semi-quantitative estimation: pH*; Protein; Glucose; Ketones; Urobilinogen; Bilirubin; Blood; Reducing substances; Microscopic examination of spun deposit
*Only this parameter was examined after week 17.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were submitted to full necropsy procedures including examination of: the external surface,all orifices, the cranial cavity, the carcass, the external surface of the brain and of samples of the spinal cord (spinal cord was examined at the time of tissue processing for animals examined histopothologically), the thoracic, abdominal and pelvic cavities and viscera, the cervical tissues and organs.

HISTOPATHOLOGY: Yes
The following organs/tissues were sampled for all animals: adrenals; aorta; bone (femur)* and articulation*; bone (sternum) with bone marrow; bone marrow smears; bronchi (mainstem); brain; cecum; colon; duodenum; epididymides; eyes; heart; ileum; jejunum; kidneys; lachrymal gland*; liver; lungs; lymph node (submaxillary); lymph node (mesenteric); mammary gland; esophagus; optic nerves; ovaries; pancreas; parathyroids; pituitary; prostate; rectum; salivary gland (submaxillary); sciatic nerve; seminal vesicles; skeletal muscle * (quadriceps femoris); skin; spinal cord (cervical, thoracic, lumbar); spleen; stomach (fundus, pylorus); testes; thymus (where identified); thyroids; trachea; urinary bladder; uterus (horn + cervix); all gross lesions;
* fixed but not processed initially
Other examinations:
The following organs were weighed at scheduled necropsy for all animals: Adrenals; Brain; Epididymides; Heart; Kidneys; Liver; Ovaries; Pituitary; Prostate; Spleen; Testes; Thyroids
Statistics:
For food consumption: arithmetic mean and standard deviation.

For body weights, body weight gains, haematology, blood clinical chemistry parameters, quantitative urinary parameters and organ weights, the tables show arithmetic mean and standard deviation calculated for each group and sex. The following parameters were analysed statistically for males and females separately: mean body weight on days 1, 29, and 92 and mean body weight gains over days 1 to 29, 1 to 64, 1 to 92, and 92 to 120; haematology (where appropriate), blood clinical chemistry, and urinary volume and specific gravity of weeks 4, 13 and 17; absolute and relative organ weights at weeks 13 and 17.

For each parameter Levene's test was used to test the equality of variance across groups.

Where Levene's test showed no significant difference in the group variances data were analysed using parametric procedures. Such analysis consisted of a one way analysis of variance (ANOVA) allowing for a group effect, followed by pairwise comparisons using a protected t-test (pre-treatment body weight data) or Dunnett's t-test to assess the significance of any intergroup differences. A dose-response test was also performed.
Where Levene's test showed a significant difference in the variance across groups the data were analysed using non-parametric methods. Such analysis consisted of the Kruskal-Wallis ANOVA followed by the protected Wilcoxon rank sum test for pairwise comparisons to assess the significance of intergroup differences and the Terpstra - Jonckheere test for a dose-response.
The parameters analysed using non parametric procedures were ASAT, ALAT (females) and inorganic phosphorus (males) in week 13.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related eye lesions.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 4 weeks of treatment, the mean platelet count of high dose males was statistically significantly higher than the control mean (p < 0.05). However, in the absence of any statistically significant difference in females at the some stage of the study, or in any other treatment group at the end of the treatment period, the toxicological significance of this small change was considered doubtful.
The mean activated partial thromboplastin time (APTT) of high dose males was also higher than the control mean at the week 4 sampling, but this was almost entirely due to animal n° 39. The reason for this high value is unclear, but because of the isolated nature of the change, it is considered to be unrelated to treatment.
At the end of the treatment period (week 13), high dose males had higher haemoglobin concentrations (Hb), packed cell volumes (PCV), and red blood cell counts (RBC) than respective controls. These differences were statistically significant (p < 0.05 and p < 0.001).
It should be borne in mind, however, that all the individual values in the high dose group for these parameters were within the extreme normal background range of the testing facility (with the exception of the PCV for animal n° 44) and that the control means for Hb and PCV were lower than the normal background mean.
In females at the end of the treatment period, all treated groups tended to have higher Hb concentrations than controls, although the difference was very small in intermediate dose females. In addition, high dose females had higher mean cell haemoglobin values than controls. These differences were statistically significant. In the absence of any dose-relationship and since the individual values fell consistently within the normal background range, the changes were considered unlikely to be a direct toxic effect of the test article.
A small number of high dose animals had slightly higher absolute or percentage neutrophil counts than controls at the end of treatment resulting in the difference between the means being statistically significant (p < 0.05). Since the majority of the individual values were within the normal background range, and based on the low incidence of this change, its toxicological relevance was considered to be doubtful.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In general, high dose animals tended to have higher serum potassium, calcium, and phosphorus concentrations than controls at the end of the treatment period. In addition, the calcium concentrations of intermediate dose males were also slightly elevated compared with controls. However, the differences were small, often not dose-related and generally well within the normal background range. They were therefore considered to be of doubtful toxicological significance.
At the end of the treatment period, the mean cholesterol concentration of high dose females was higher than the control mean (p < 0.01). However, only three individual values fell at or outside two standard deviations of the background mean. When this parameter was measured at the end of 4 weeks without treatment there was no difference between the controls and those females previously treated at the high dose level, thus indicating that this small change was not a direct toxic effect of the test article.
High dose animals had higher serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities than controls at the end of 13 weeks of treatment. There was also some evidence for a similar but much smaller effect on ALAT in intermediate dose females (p < 0.05). However, the magnitude of the difference was not large and the individual values were within the normal background range. In addition, a change in this parameter is not indicative of an effect on the heart. Consequently, the change was considered to be incidental to treatment. There were no differences between control animals and those previously treated at the high dose level when the parameters were measured in week 17.
None of the other small differences between treated and control groups, although occasionally reaching the level of statistical significance, was considered to represent a direct toxic effect of the test article.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Although the urine of high dose animals was slightly more acidic than that of the controls after 13 weeks of treatment when the pH of the urine was measured at the end of the treatment-free period there was no meaningful difference in the acidity of the urine collected from control animals and that collected from animals previously treated at the high dose level. There were no other differences in the urine parameters to indicate any effect of the test article.
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):
When compared with the control group, the mean liver weight was higher for high dose group males when expressed relative to body weight and for females when expressed in absolute terms and relative to body weight. In addition, when compared with controls, the mean testis weight was lower for high dose group (absolute and relative to brain weight).
High dose group females also had a lower mean ovarian weight when expressed relative to brain weight. When compared with the control group, the mean liver weight was higher for high dose group males when expressed relative to body weight and for females when expressed in absolute terms and relative to body weight. In addition, when compared with controls, the mean testis weight was lower for high dose group (absolute and relative to brain weight). High dose group females also had a lower mean ovarian weight when expressed relative to brain weight. In the absence of any microscopic lesions in these organs the weight changes were considered to be of doubtful toxicological significance.
At the end of the treatment-free period, the mean absolute brain weight for high dose group males was higher than the control mean and this group also had a statistically significantly higher mean (absolute and relative to brain weight) prostate and epididymidis weight than the control. In the absence of any microscopic lesions in these organs the weight changes were considered to be of doubtful toxicological significance. In the absence of any microscopic lesions in these organs, the weight changes were considered to be of doubtful toxicological significance.
Historical control data for organ weights were not given in the report.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Most tissues were unremarkable at necropsy. The most common finding was pale area(s) on the stomach mucosa. The incidence of this finding was higher for high dose group males and females, but was also seen in the control group. Alopecia seen in high dose group males female was also seen in control females killed at the end of the treatment-free period and it was considered to be of no toxicological significance. Other macroscopic changes were considered to be either agonal or incidental in origin.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were seen in the heart. The lesion which was seen in intermediate dose group males and in high dose group males and females, was described as small foci of degenerated or necrotic fibers associated with minimal to moderate mononuclear cell infiltration. This association suggested early or on-going myocarditis. In high dose group males and females the lesions involved the right and/or the left ventricular wall and occasionally the interventricular septum. Such findings were multifocal and slightly more severe in males than in females. In intermediate dose group males, the lesions were minimal or slight focal and most often located in the subendocardial zones, in the interventricular septum, or in the papillary muscles.
In one female (N° 78), the mononuclear cell infiltration was in the right and left ventricular wall and multifocal, but it was not associated with small foci of degenerate or necrotic fibers. No ventricular infiltration was seen in the other females nor in the males.
Inflammatory changes in control and low dose group males and females were few and they were also in the subendocardial zones, in the septum, or in the papillary muscles. No change was seen in the ventricular walls.
Special staining by phosphotungstic acid haematoxylin (PTAH) did not indicate any modification of the striation of the myocardial fibers in the areas next to the small foci of necrosis.
The above findings were clearly related to the test article in high dose group males and females. In addition, this finding was correlated in males with an increase in serum aspartate aminotransferase (ASAT) activity. The activity of this enzyme is known to increase in cases of slight myocardial necrosis. However, in intermediate dose group the focal distribution of the histological changes (very focal and most often in the septum) and the lower grade of severity of the lesions (minimal), suggested that such findings had a doubtful toxicological significance, particularly in the absence of elevated ASAT levels, and were most probably unrelated to the administration of the test article. It is known that small foci of necrosis and focal inflammation and fibrosis occur spontaneously in the septum or in the papillary muscles in young rats, probably as a result of focal ischemia, and that this change becomes more common with increasing age. No abnormality was seen in the heart at the end of the recovery period.
In the stomach, mucous adherence on the glandular mucosa was correlated with the macroscopic observation of "pale areas". In addition, two superficial erosions of the mucosa were seen, one in male N° 31 from the intermediate dose group and one in female N° 95 from the high dose group. This finding was most probably related to the administration procedure (gavage).
Other findings were few or those normally observed in rats and they were considered to be agonal or incidental in origin and unrelated to the administration of the test article. No change was seen in the organs for which the weight was modified.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Signs of early or ongoing myocarditis observed at 1000 mg/kg bw. Non-adverse changes in haematology and biochemistry at 350 mg/kg bw. All findindings reversible within the 4-week recovery period.
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see above

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
heart
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
The NOAEL in the rat after 13 weeks oral administration was 350 mg/kg bw/day.
The NOEL was 125 mg/kg bw/day.