3,3'-thiodi(propionic acid)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Only translated summary available, actual guideline not stated.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Only summary available, actual guideline not stated

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding center, Charles River Laboratories, Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: males 143-166 g, females 114-133 g
- Fasting period before study: no
- Housing: individually after separation into experimental groups
- Diet (e.g. ad libitum): solid feed for experimental animals (MF, Oriental Yeast Co., Ltd.), ad libitum
- Water (e.g. ad libitum): sterile-filtered and UV-radiated tap water, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was weighed and added to the solvent (0.5 % CMC) while being suspended with a mortar and pestle and being sufficiently stirred before being prepared into stocks with constant concentrations of 20, 40, and 100 mg/mL. The 4 mg/mL liquid required for administration to the low dose group was obtained by 10-times dilution of the 40 mg/ml stock liquid (mistake in materials and methods: they describe an 0.4 mg/ml administration liquid for the low dose group, but considering a dose volume of 10 ml/kg, a concentration of 4 mg/mL is required to obtain a dose of 40 mg/kg bw). The 20 and 100 mg/mL stocks were directly used for dosing of the mid and high dose group, respectively.

Fresh stocks were prepared at least once a week, and before being administered the preparations were stored refrigerated and dark for a maximum of 8 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 % CMC-Na aqueous solution (CMC-Na, Wako Pure Chemical Industries and Kanto Chemical), due to limited solubility in water (2.5 %, at 20°C)
- Concentration in vehicle: 20, 40, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw, calculated based on body weight measured just prior to start of the test

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The titration end-point method was used to analyze the concentration of the mid and high dose group administration liquids and the 40 mg/ml preparation fluid of the low dose group. Concentrations were confirmed to be in the set concentration ranges.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 (10 in control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on acute test and a 7-day repeat dose preliminary test (dosage: 1000, 500, 100, and 0 mg/kg with 5 males and females in each group), the latter demonstrating a high leukocyte count and triglyceride value thought to be by test material administration in the 1000 mg/kg bw group. Therefore, 1000 mg/kg bw was chosen as the highest dosage, the other dosages were set using the common ration of 5.
- Rationale for selecting satellite groups: Additional animals included in the control and the high dose group were chosen to investigate the reversibility of possible symptoms.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day prior to administration and 30 min after administration, once a day during the other periods.
- Cage side observations included: general conditions


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once on the day before the administration start date and once each week during the administration period, both during the afternoon; in home cage, during handling, and in the open field


BODY WEIGHT: Yes
- Time schedule for examinations: on days 1, 8, 15, 22, 28, 29 (1st day of recovery period), 36, 42, and 43


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes, during measurement periods of day 1 to 8, 8 to 15, 15 to 22, 24 to 28, 29 to 36, and 38 to 42.


HEMATOLOGY: Yes
- Time schedule for collection of blood: On the planned necropsy days at the end of the administration and the recovery period (days 29 and 43) from the postcava.
- Anaesthetic used for blood collection: Yes (sodium thiopental)
- Animals fasted: Yes, on the evening prior to necropsy
- How many animals: all
- Parameters examined: red blood cell count, hemoglobin concentration, hematocrit value, reticulocyte count, platelet count, leukocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the planned necropsy days at the end of the administration and the recovery period (days 29 and 43)
- Animals fasted: Yes, on the evening prior to necropsy
- How many animals: all
- Parameters examined: GOT, GPT, γGT, ALP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chlorine


URINALYSIS: Yes
- Time schedule for collection of urine: On day 23; since there were no findings the tests during recovery period were not conducted
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: pH, protein, glucose, keetones, bilirubin, occult blood, urobilinogen; as there were no findings, urinary sediment and urine quantitative analyses were not conducted.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once in the afternoon during th 4th week
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity to stimuli (approach response, touch response, auditory response, tail pinch response, aerial righting reaction) / grip strength (forelimb, hindlimb) / motor activity


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes [brain (cerebrum, cerebellum, pons), spinal cord, stomach, , duodenum, jejunum, ileum (incl. Peyer's patch), cecum colon, rectum, liver, kidneys, adrenals, spleen, heart, thymus, eyeballs, harderian glands, pituitary, thyroid (incl. parathyroid), trachea and lungs, testes, ovaries, epididymis, prostate, uterus, vagina, urinary bladder, mandibular lymph node, mesenteric lymph node, sciatic nerve (removed as attached to the thigh muscle), bone marrow (femur), visually abnormal areas]
HISTOPATHOLOGY: Yes, all organs and tissues included in gross pathology from all male and female cases of the control and high dose group after administration period and areas with visual abnormalities of all animals including those from the control group were stained with hematoxylin/eosin and microscopically examined.

As a suspected effect of the test material was found in the stomachs of males and females in the high dose group, additional examinations were performed on the stomachs of males and females in the low and mid dose groups necropsied after administration period and the stomachs of males and females necropsied after recovery period.

Other examinations:

Organ weights, relative organ weight: liver, kidneys, adrenal, testes, epididymis, ovaries, thymus, spleen, brain, heart

Statistics:

Measured data was subjected to Bartlett's test for equality of variance; if distribution was uniform, one-way ANOVA was performed, if distribution was not uniform, Kruskal-Wallis test was performed. If a significant difference was found among the groups, the Dunnett method or Dunnett type multiple comparison test was performed. The measurement data was subjected to a*b chi² test, and if a significant difference was found, a comparison between the control gorup and all dosage groups was made using Armitage chi² test. The significance level was 5 % in all cases.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred. Rales were heard in 3/10 males and 1/10 females in the high dose group which disappeared by day 2. Slight salivation was found in 3/10 males and 2/10 females in the high dose group on days 27 and 28, but these observation were rather contributed to irritation than to neurotoxicity or systemic effects of the test material. The observation of bulging eye balls in 1/10 females on day 2 in the high dose group disappeared after 1 day and was not considered to be treatment-related. A walking abnormality observed in 1/5 males from day 28 to day 39 of the recovery period was confirmed by necropsy to be related to a fall (broken femur).

BODY WEIGHT AND WEIGHT GAIN
No significant differences observed compared to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences observed compared to controls.

HAEMATOLOGY
No changes considered to be treatment-related were observed.

At the end of the administration period a high red blood cell count was observed in high dose females. Due to the lack of other changes accompanying red bood cell related parameters this finding was considerd not to be toxicologically significant. At the end of the recovery period a high red blood cell count, low mean corpuscular volume (MCV) and low mean corpuscular hemoglobin (MCH) were found in the high dose males, but as these changes were only minor and had not been found at the end of the administration period they were considered incidental.

CLINICAL CHEMISTRY
No changes considered to be treatment-related were observed.

At the end of the administration period a high value for albumin was found in the low dose males, due to lack of dose-correlation this finding was considered to be incidental. Tests at the end of the recovery period found a low GOT, and high glucose and A/G ratio in the high dose males, and low inorganic phosphorus in the high dose females. However, these observations were not observed at the end of the administration period and thererfore considered incidental.

URINALYSIS
No changes considered to be treatment-related were observed.

During the final week of administration a high pH for low dose males and a low pH for high dose males was found. However, since this was not found in females and the pH of males in the control group was concentrated around pH 8.5, these changes were considered to be incidental.

NEUROBEHAVIOUR
No changes considered to be treatment-related were observed, neither in the detailed symptom observation, the functional tests, nor the motor activity tests.

ORGAN WEIGHTS
No changes considered to be treatment-related were observed.

At the end of the administration period low absolute testes weights were found in low dose males. However, due to the lack of dose-correlation this finding was considered to be incidental. At the end of the recovery period a low relative liver weight was found in the high dose males, however, as this change was not found at the end of the administration period it was also considered incidental.

GROSS PATHOLOGY
No changes considered to be treatment-related were observed.

Several changes observed in necropsies at the end of administration and recovery period did not occur frequently and were determined to be incidental. A broken femur was identified as reason for walking abnormalities in a high dose male.

HISTOPATHOLOGY: NON-NEOPLASTIC
A change considered to be test material-related was found in the stomachs of males and females. Animals necropsied at the end of the administration period showed an increase in globule leukocytes in the glandular stomach mucous membrane for all males and females of the high dose group and eosinophil infiltration in the glandular stomach mucous membrane and the mucous membrane submucosa for 2/5 males and all females of the high dose group. Animals necropsied at the end of the recovery period did not show this change.

In addition, a variety of histopathological findings were found. However, these chantges were nonspecifically expressed in the rats, and their state of expression showed no clear significant difference between groups, therefore, these changes were determined to be unrelated to test material administration.

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs (rales, slight salivation); histopathology (eosinophil inflammation)

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at 200 mg/kg bw/day

Critical effects observed:

not specified

Conclusion:

Changes to be thought test material-related were found in the general conditions and in the stomach. Rales were heard in males an a female of the high dose group, this change disappeared by day 2. In addition, slight salivation was found in males and females of the high dose group on days 27 and 28. When the test material was in a 20°C saturated aqueous solution it had a pH of 2.0, so it was supposed that the high dose administration liquid was strongly acidic. Therefore, the temporary rales and salivation are thought to have possibly been caused by the irritation of the test material. In addition, since salivation disappeared before administration on the next day, it differed from continuous salivation caused by neural toxicity.

The increase in globule leukocytes and eosinophilic infiltration in the glandular stomachs of the high dose group observed in this study is also known to occur in cases of administration of certain food additives and reflects an eosinophilic gastritis, but animals recovered once administration was stopped.

In view of the above, since changes considered to be test material-related were found in both males and females in the 1000 mg/kg group, the NOEL was determined to be 200 mg/kg bw/d for males and females under the conditions of this study.

Executive summary:

3,3'-Thiobispropanoic acid was administered to Crj: CD(SD) rats (5/sex/dose) at dose levels of 0 (control), 40, 200 and 1000 mg/kg bw/d in CMC (carboxymethyl cellulose) for 28 days. A satellite recovery group consisted of additional 5 animals/sex/dose in the control and high dose group.

Test item related changes were found in the general conditions and in the stomach. Rales were heard in males and a female of the high dose group, this change disappeared by day 2. In addition, slight salivation was found in males and females of the high dose group on days 27 and 28. When the test material was in a 20°C saturated aqueous solution it had a pH of 2.0, so it was supposed that the high dose administration liquid was strongly acidic. Therefore, the temporary rales and salivation are thought to have possibly been caused by the irritation of the test material. In addition, since salivation disappeared before administration on the next day, it differed from continuous salivation caused by neural toxicity.

The increase in globule leukocytes and eosinophilic infiltration in the glandular stomachs of the high dose group observed in this study is also known to occur in cases of administration of certain food additives and reflects an eosinophilic gastritis, but animals recovered once administration was stopped.

In view of the above, since changes considered to be test material-related were found in both males and females in the 1000 mg/kg group, the NOEL was determined to be 200 mg/kg bw/d for males and females under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

3,3'-Thiobispropanoic acid was administered to Crj: CD(SD) rats (5/sex/dose) at dose levels of 0 (control), 40, 200 and 1000 mg/kg bw/d in CMC (carboxymethyl cellulose) for 28 days. A satellite recovery group consisted of additional 5 animals/sex/dose in the control and high dose group.

Test item related changes were found in the general conditions and in the stomach. Rales were heard in males and a female of the high dose group, this change disappeared by day 2. In addition, slight salivation was found in males and females of the high dose group on days 27 and 28. When the test material was in a 20°C saturated aqueous solution it had a pH of 2.0, so it was supposed that the high dose administration liquid was strongly acidic. Therefore, the temporary rales and salivation are thought to have possibly been caused by the irritation of the test material. In addition, since salivation disappeared before administration on the next day, it differed from continuous salivation caused by neural toxicity.

The increase in globule leukocytes and eosinophilic infiltration in the glandular stomachs of the high dose group observed in this study is also known to occur in cases of administration of certain food additives and reflects an eosinophilic gastritis, but animals recovered once administration was stopped.

In view of the above, since changes considered to be test material-related were found in both males and females in the 1000 mg/kg group, the NOEL was determined to be 200 mg/kg bw/d for males and females under the conditions of this study.

Justification for classification or non-classification

Based on the available data, 3,3'-Thiobispropanoic acid does not need to be classified for repeated dose toxicity according to regulation (EC) 1272/2008. Thus, no labelling is required.