2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2017 - 02 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

EC Number:

239-044-2

EC Name:

2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Cas Number:

14970-87-7

Molecular formula:

C6H14O2S2

IUPAC Name:

2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ordered from Envigo RMS srl, San Pietro al Natisone (UD), Italy and obtatined by Envigo Netherlands, Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: ca 200 g for males and ca. 160g for females
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum): tap water
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

The required amount of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) was suspended in the vehicle. The formulations were prepared daily (concentrations of 2, 6 and 18mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally, by gavage, at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in a separate study in the range from 1 to 20mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 90-110%; precision CV < 5%). A 28 hour stability at room temperature and an 8 day stability at +4°C were verified in the range from 1 to 20mg/mL.
The proposed formulation procedure for the test item was checked in the range from 1 to 20mg/mL by chemical analysis (concentration and homogeneity) in RTC Study no. A2370 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The acute toxicity expected (ATE) of DMDS is lower than 300mg/kg body weight, but higher than 50mg/kg body weight. Animals dosed at 300mg/kg showed tremors, muscular rigidity, ataxia and piloerection. No clinical signs were seen in animals dosed at 50mg/kg (RTC Study No. A2159).
Following daily oral administration at dose levels of 10, 30 and 100mg/kg/day for 2 weeks, DMDO did not show any signs of toxicity (RTC study no. E0103).
- Post-exposure recovery period in satellite groups: recovery period of 2 weeks for 5 males and 5 females from Groups 1 and 4.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (approximately 30 minutes -1 hour and 2 - 2.5 hours after dosing).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week from the start of treat- ment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, and unusual res- piratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter, on Day 28 and just prior to necropsy.

FOOD CONSUMPTION:
- The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4
- Anaesthetic used for blood collection: Yes (sofluorane)
- Animals fasted: No
- How many animals: 5 males and 5 females
- Parameters examined.
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
Large unstained cells
– Platelets
These parameters were analysed by Siemens Advia 120.
Coagulation
– Prothrombin time
This parameter was analysed by Instrumentation Laboratory ACL 3000 PLUS.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4
- Anaesthetic used for blood collection: Yes (sofluorane)
- Animals fasted: No
- How many animals: 5 males and 5 females
- Parameters examined.
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride
These parameters were analysed by Siemens Advia 1200.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.
– Appearance
– Volume
– Specific gravity
– pH
– Protein
– Glucose
- Ketones
– Bilirubin
– Urobilinogen
– Blood
These parameters were analysed byMenarini AUTION MAX 4280/AUTION ELEVEN AE 4020.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was
examined microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 of treatment
- Dose groups that were examined:
- Battery of functions tested: an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength

IMMUNOLOGY: No

OTHER:
-Vaginal smears and oestrous cycle
Prior to necropsy, vaginal smears were taken in the morning from all main phase animals. The vaginal smear data were examined to determine potential anomalies of the oestrous cycle.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).

ORGAN WEIGHTS: Yes
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate gland, Spleen, Testes, Thymus (where present), Thyroid gland.


HISTOPATHOLOGY: Yes
Bone marrow
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur. Smears prepared from these samples were air dried, fixed in methanol, stained using a May-Grunwald-Giemsa procedure and stored. In the first instance, the smears were examined for abnormalities and a differential count was performed including calculation of the myeloid/erythroid cell ratio from main phase control and high dose animals.

Histopathological examination
The tissues required for histopathological examination are listed below:
Abnormalities, Adrenal glands, Bone marrow (from sternum),, Brain (cerebrum, cerebellum, medulla/pons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes (note examined), Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes – mesenteric, Ovaries, Oviducts, Parathyroid glands, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal column (not examined), Spinal cord, Spleen, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus – cervix, Vagina.

In the first instance the examination was restricted as detailed below:
i Tissues specified above from all animals in the control and high dose groups and the kidneys from the low and mid-dose males killed after 4 weeks of treatment.
ii Tissues specified above from all animals dying during the treatment period.
iii All abnormalities in all main phase groups.
The examination was extended to include the spleen and the thymus from all other animals killed after 4 weeks of treatment and 2 weeks of recovery, since possible treatment-related changes were observed.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous, a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily clinical signs
Semi-closed eyes was the only sign observed in one male animal of Group 4 (180mg/kg/day) from Day 25 to Day 28 of the treatment period.
Hunched posture, decreased activity and dyspnea were observed in individual high dose females during Weeks 3 and 4 of the treatment period.
All the above mentioned signs were no longer observed during the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three male animals from Group 4 (nos. A2371042, A2371044 and A2371050) were found dead on Days 13, 25 and 26 of the treatment period. Decreased activity and dyspnoea or red staining of nose were observed in two of these animals prior to death.
Although some treatment-related findings were observed in these animals (mild congestion associated with yellow/brown pigmentation in red pulp of the spleen and/or mild extramedullary haematopoiesis, nephropathy in the kidneys and/or eosinophilic infiltration in the submucosa of the glandular region of the stomach), the pathological pictures observed at histopathological examination did not allow to establish the factor contributory to death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight statistically significant reductions in body weight and body weight gain were observed in males of Group 4 on Day 28 and in the females of the same group from Day 8 up to the end of treatment period. Very slight reductions in body weight were still observed in the high dose females during
recovery, with a complete recovery in the males.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant changes were observed in food consumption during treatment and recovery periods.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease of erythrocytes, haemoglobin and haematocrit was recorded in animals of both sexes dosed at 180mg/kg/day; reticulocytosis was recorded in the same animals. Platelets were increased in animals dosed at 180mg/kg/day. These changes were no longer observed at the end of the recovery period.

Coagulation
No changes were recorded.

Bone marrow evaluation

Dosing phase
An increase of erythroid cells was observed in all female animals and one male dosed at 180mg/kg/day. This condition, often due to the increase of the immature and/or intermediate forms, was the result of increased normoblastic erythropoiesis, due to the anaemic conditions observed.
In addition, a slight reduction of myeloid mature forms, sometimes associated with a slight increase of immature/intermediate forms (left shift), was recorded in all treated females. This condition has no pathological relevance and is commonly seen in reactive bone marrow.
Recovery phase
A slight increase of erythroid cells was still recorded in one male and two females dosed at 180mg/kg/day (male no. 58 and female nos. 55 and 57). Animal nos. 58 and 57 also showed a left shift of the myeloid cells.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Fluctuations of some biochemical parameters were recorded in a number of treated animals. The severity of the findings observed was not considered to be suggestive of tissue/organ injury and could be related to metabolic changes. These changes were no longer observed at the end of the recovery period.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced diuresis was recorded in males dosed at 180mg/kg/day. In the absence of other related findings, this change was considered of no toxicological significance. No changes were recorded during the recovery period.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods. No differences between treated and control groups were evident at the functional tests and at the motor activity measurements at the end of treatment and recovery periods.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice
A statistically significant slight reduction of terminal body weight was mainly noted in high dose females (180mg/kg/day), when compared with their controls. An increase of the absolute and relative weight of the spleen and a reduction of the absolute and relative weight of the thymus were observed, with statistical significance, in males and females dosed at 180mg/kg/day. Such organ weight variations were considered treatment-related also on the basis of histopathological results.
Recovery sacrifice
A slight reduction in terminal body weight was still noted in the high dose females (180mg/kg/day), when compared with their controls. A slight increase in the absolute and relative weight of the spleen was again observed in high dose animals, with statistical significance in females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice
At post mortem examination, swollen spleen in one high dose male and reduced size of the thymus in one high dose female were the only findings that could be considered treatment related.
Recovery sacrifice
At post mortem examination, no relevant changes were noted in treated animals, when compared with controls.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice
Treatment-related changes were seen in males and females receiving DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) at 180mg/kg/day. The effects mainly observed in the spleen consisted in mild to moderate increase of yellow/brown pigmentation, probably resulting from increased hemosiderin pigment associated with extramedullary haematopoiesis (EMH) and congestion in the red pulp of the spleen. In addition, the white pulp of the spleen showed minimal decreases in the size and cellularity of the periarteriolar lymphoid sheaths (PALS) with decreased numbers of primary lymphoid follicles. A minimal presence of yellow/brown pigment and extramedullary haematopoiesis (EMH) was noted in controls and in females dosed at 20 and 60mg/kg/day respectively and was considered a background finding commonly observed in rodents. Minimal increase of apoptotic lymphocytes and consequently phagocytosis of cell debris from the cortex, resulting in a “starry-sky” appearance or tingible macrophages, were mainly noted in the thymus of high dose females. No remarkable changes were noted in the thymus of the remaining treated animals, when compared with controls.
Recovery sacrifice
The treatment-related changes were considered toward a complete recovery in the spleen and fully recovered in the thymus.

Other effects:

no effects observed

Description (incidence and severity):

Oestrous cycle
Evaluation of the oestrous cycle at the end of treatment did not indicate particular differences between groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

180 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, signs of effects related to treatment with DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) were observed in male and female animals when administered by oral gavage for 4 consecutive weeks at the dosage of 180mg/kg/day. Clinical signs, reduction in body weight, treatment-related histopathological changes in spleen and thymus were the major changes observed in male and, mainly, in female animals dosed at 180mg/kg/day. These signs were partially reversible after 2 weeks of recovery. Although a reduction in body weight was observed also at 60mg/kg/day, this sign was not
considered to be adverse, due to the low magnitude and complete reversibility.
Therefore, it can be concluded that the mid-dose of 60 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL) for this study.

Executive summary:

The oral toxicity of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) in rats following daily oral administration for 4 consecutive weeks and recovery from any treatment-related effect during a treatment-free period of 2 weeks, were investigated in this study. Three groups, each of 5 male and 5 female Sprague Dawley rats, received the test item by gavage at dosages of 20, 60 and 180 mg/kg/day for 4 consecutive weeks. A fourth similarly constituted group received the vehicle alone (0.5 % aqueous solution of carboxymethylcellu- lose) and acted as a control. Five additional animals for each sex were included in the high dose and control groups for recovery  assessment. The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, oestrous cycle, clinical pathological investigations (including bone marrow smears), terminal body weight, organ weights, macroscopic observations and histopathological examinations.

Three male animals from Group 4 (dosed at 180mg/kg/day) were found dead during Weeks 3 and 4 of the treatment period. Decreased activity and dyspnoea or red staining of nose were the clinical signs observed in two of these animals on the day of death. Although some treatment-related changes were present at macroscopic and microscopic observations, these data did not allow to clearly establish the factor contributory to death. Decreased activity, dyspnoea, staining of nose and/or semi-closed eyes were observed during the treatment period at 180mg/kg/day, on daily occasions. These clinical signs were reversible at the end of the recovery period. No other significant signs of toxic or neurotoxic effects were seen during the in vivo phase of the study. No particular differences in oestrous cycle were noted between groups. Statistically significant reduction in body weight and body weight gain was observed in male and female animals of the high dose group, with higher severity in females, starting from Day 8 of the treatment period. These reductions were completely reversible only in male animals. Food consumption was not affected by treatment. Slight decrease of erythrocytes, haemoglobin and haematocrit was recorded in animals of both sexes dosed at 180mg/kg/day; reticulocytosis was recorded in the same animals. Platelets were increased in animals dosed at 180mg/kg/day. Fluctuations of some biochemical parameters were recorded in a number of treated animals. The severity of the findings observed was not considered to be suggestive of tissue/organ injury and could be related to metabolic changes. An increase in total bilirubin was also observed in the high dose animals This increase, since is not associated with other hepatic markers finding, was not considered indicative of liver/cholestatic change. These changes were no longer observed at the end of the recovery period. No changes of toxicological relevance were observed in urinalysis at the end of treatment and recovery periods. An increase of erythroid cells was observed in the majority of male and female animals dosed at 180 mg/kg/day at the end of treatment period. This condition was regarded as the result of the anaemic condition observed in animals dosed at 180 mg/kg/day. This increase was still present in 1 male and 2 females dosed at 180 mg/kg/day at the end of recovery.

Post mortem observations showed a statistically significant slight reduction of terminal body weight mainly in high dose females. Moreover, an increase of the absolute and relative weight of the spleen and a reduction of the absolute and relative weight of the thymus were observed, with statistical significance, in males and females dosed at 180mg/kg/day. Such organ weight variations were considered treatment-related also on the basis of histopathological results. A slight increase in the absolute and relative weight of the spleen was again observed in high dose animals. Macroscopic examination reported a swollen spleen in one high dose male and reduced size of the thymus in one high dose female, that could be considered treatment-related. These changes were no longer present at the end of recovery. At microscopic examination, treatment-related changes were seen in males and females receiving the test item at 180mg/kg/day. The effects mainly observed in the spleen consisted in mild to moderate increase of yellow/brown pigmentation, probably resulting from increased hemosiderin pigment associated with extramedullary haematopoiesis (EMH) and congestion in the red pulp of the spleen. The observed increases in EMH were characterized by a predominance of erythroid precursors in all treated animals. Such pathological patterns were associated with the increase in spleen weights and reflected a physiological effect or a consequence of the haematological alterations. Furthermore, minimal increases of apoptotic lymphocytes were mainly noted in the thymus of high dose females. The treatment-related changes were considered toward a complete recovery in the spleen and fully recovered in the thymus.

In conclusion, signs of effects related to treatment with DIMERCAPTO-1,8-DIOXA-3,6 -OCTANE (DMDO) were observed in male and female animals when administered by oral gavage for 4 consecutive weeks at the dosage of 180mg/kg/day. Clinical signs, reduction in body weight, treatment-related histopathological changes in spleen and thymus were the major changes observed in male and, mainly, in female animals dosed at 180mg/kg/day. These signs were partially reversible after 2 weeks of recovery. Even if a reduction in body weight was observed also at 60mg/kg/day, this sign was not considered to be adverse, due to the low magnitude and complete reversibility. Therefore, it can be concluded that the mid-dose of 60 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL) for this study.