2,2-bis(methylthio)propane

Endpoint summary

Description of key information

The potential repeated dose toxicity of bismethylthiopropane (BMTP) was evaluated following daily oral administration (gavage) to rats in studies conducted according to OECD guidelines no. 408, 407 and 421. The No Observed Adverse Effect Level (NOAEL) was established at the dose-level of 300 mg/kg/day in the oral subchronic repeated dose toxicity (OECD 408) study.

 

Key study

The potential repeated dose toxicity of bismethylthiopropane (BMTP) was evaluated following daily oral administration (gavage) to rats in a study conducted according to OECD guidelines no. 408 (Bentz, 2015). Three groups of 10 (low- and mid-dose groups) or 16 (high-dose group) male and female Sprague-Dawley rats each received BMTP by daily oral gavage administration for at least 13 weeks. BMTP was administered at 10, 100 and 300 mg/kg/day as a solution in the vehicle (corn oil) under a constant dosage-volume of 5 mL/kg/day. A control group of 16 animals per sex received the vehicle alone under the same experimental conditions. On completion of the treatment period, the animals in each group were sacrificed, except for the first six animals per sex in the control and high-dose groups which were kept for a 6 week treatment-free period. The concentration of the dose formulations was checked at each preparation (thus 5 times during the study) before the administration to the animals. The animals were checked at least twice daily during the treatment period or once daily after the treatment period for mortality and morbidity, and once daily for clinical signs. In addition, detailed clinical examinations were performed weekly. The body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as well as food consumption. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals at the end of the treatment period. Estrous cycle stage was determined daily for the last 4 consecutive days of the treatment period. Hematology and blood biochemistry were performed towards the end of the treatment and treatment-free periods. Just before sacrifice, under deep anesthesia, left epididymides and testes were sampled from all males for sperm analyses (motility, morphology and counts). Animals were euthanized and submitted to a full macroscopic post mortem examination. Designated organs were weighed and several tissues were preserved. A microscopic examination was performed on selected tissues from animals of the control and high-dose groups sacrificed at the end of the treatment period, on liver and spleen (both sexes) from animals of the low-and mid-dose groups sacrificed at the end of the treatment period, on liver and spleen (both sexes) and on kidneys (males) from recovery animals of the control and high-dose groups, and on all macroscopic lesions. Males kidney slides immunostained with an antibody for Alpha 2µ-globulin protein were also microscopically examined at the end of treatment and treatment-free periods.

BMTP concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%) and no test item was detected in control formulations. There were no test item treatment-related deaths or clinical signs. Mean body weight and mean body weight gain were not affected by the test item treatment. In males treated at 300 mg/kg/day, there was higher mean food consumption during the treatment period from week 5, considered to be of limited toxicologically significance. In females, there were no test item treated effects on mean food consumption. There were no toxicologically significant effects on mean food consumption during the treatment-free period. There were no ophthalmological findings and no test item-treated effects on estrous cycle data at the end of the treatment period. At hematology at the end of the treatment period, when compared with controls, mean red blood cell count and hemoglobin concentration were statistically significantly decreased along with higher mean cell volume and reticulocyte count in females given 300 mg/kg/day. These variations were no longer observed at the end of the treatment-free period and there were no test item-related effects in males. These findings were not considered to be adverse. At blood biochemistry in females treated at 300 mg/kg/day, mean glucose concentration was lower than in controls. Higher mean phosphorus levels were recorded at 100 and 300 mg/kg/day. In males and when compared with controls, there was higher mean cholesterol concentration at 300 mg/kg/day. All these findings were not considered to be adverse and recovery was complete at the end of the treatment-free period. At sperm analyses, there were no test item treatment-related effects on mean percent of motile epididymal sperm cells and their morphology. The relationship of the test item treatment with the variations observed in mean epididymal sperm cells count (+14/15% from controls) and mean testicular sperm head count and daily sperm production rate (-15%) at 300 mg/kg/day was questionable but considered to be of no toxicological significance in rats. At necropsy at the end of the treatment period, there were higher mean kidney and liver (absolute and/or relative-to body weight) weights in both sexes at 300 mg/kg/day and higher mean relative kidney weights in females at 100 mg/kg/day. There were also higher mean spleen weights in females at 100 and 300 mg/kg/day. At microscopic examination, presence of hyaline droplets (Alpha 2µ-globulin) in kidneys of males from all test item-treated groups correlated with increases in mean kidney weights at necropsy; it was observed with a dose-related trend and associated at 100 and 300 mg/kg/day with a higher incidence and/or severity of tubular basophilia and mononuclear cell infiltrate (300 mg/kg/day). These findings were considered to be adverse at these dose-levels but non-relevant for human considering the male rat-specific mechanism. Centrilobular hepatocellular hypertrophy at 100 (males) and 300 (both sexes but mainly males) mg/kg/day correlated with the higher mean liver weight, and the accentuated lobular pattern in males; it is usually seen as an adaptative response and in the absence of associated degenerative changes considered not to be adverse. Midzonal vacuolation was seen at a higher incidence in all test item-treated group males than in controls, without dose-related trend, correlating with tan discoloration and/or accentuated lobular pattern noted at necropsy. At 100 and 300 mg/kg/day, there was a trend towards a minimally increased extramedullary hematopoiesis in the spleen of females considered to correlate with the decrease in red blood cell mass noted clinically and considered to be non-adverse. This was accompanied with a slightly higher severity of pigment deposit (suggestive of hemosiderin) which was also considered to be secondary to the lower red blood cell mass noted clinically. Following a 6-week treatment-free period signs of recovery were seen in the liver, kidneys (males) and spleen.

BMTP was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats. At the dose-levels of 100 and 300 mg/kg/day, adverse test item treatment-related effects were observed in the kidneys of male rats: the hyaline droplets in the tubular cells were associated with Alpha 2µ-globulin accumulation as demonstrated by immunohistochemistry and with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. Alpha 2µ-globulin nephropathy of male rats is a common finding following the administration of hydrocarbons (Khan et al., 2002) and has already been associated with karyomegaly within the proximal tubules (Williams et al., 2001). It is considered to be a rat-specific effect with no relevance for human risk assessment (Swenberg et al., 1999). No other adverse effects were observed in the study. Therefore, excluding the male rat-specific kidney effect, a No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was established for human risk assessment and classification.

 

Supporting studies

4-Week oral repeated dose toxicity study in rats (OECD no. 407)

In the OECD guideline no. 407 study (Chevalier, 2008), three groups of six male and six female Sprague-Dawley rats were administered BMTP, daily by the oral route (gavage) at the dose-level of 80, 240 or 720 mg/kg/day for 4 weeks. An additional group of six males and six females received the vehicle alone (corn oil) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The animals were housed in two in Individually Ventilated Cages (IVC). Clinical signs and mortality were checked respectively once a day. A detailed clinical observation was performed prior to start the dosing and once a week throughout the study. At the end of the treatment period, effects on the central nervous system were evaluated by means of a Functional Observation Battery (FOB), including motor activity testing. Body weight was recorded once before group allocation, on the first day of treatment, then once a week during the treatment period. Food consumption was recorded once a week during the treatment period. Hematological and blood biochemical were performed on all animals prior to the necropsy. On completion of the treatment period, all surviving animals were sacrificed and subjected to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. Microscopic examination were performed on designated tissues from the control and high-dose group animals sacrificed at the end of the treatment period and on macroscopic lesions, the kidneys, the liver and the spleen from all animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period. Additional immunohistochemistry was carried out on liver and kidneys.

A satisfactory agreement was observed between the theoretical and measured concentrations of BMTP in the formulations since the deviations varied from -8% and +4%. The dosage forms prepared at 16, 48 and 144 mg/mL were stable over 4 weeks. No deaths occurred during the study. No signs of toxicological importance were observed, hypersalivation (recorded as ptyalism), not considered as adverse, was the only sign noted from day 4 among all animals given 720 mg/kg/day. No effects on the central nervous system were evidenced by the FOB tests. Neither mean body weight nor mean body weight change were affected. Food consumption was not affected. When compared to the control mean values, red blood cell parameters recorded in females given 720 or 240 mg/kg/day were decreased (red blood cell count, hemoglobin concentration, pack cell volume, mean cell hemoglobin concentration or increased (mean cell volume, mean cell hemoglobin). These differences were minimal, not always clearly dose-related and the values were still in the range of the historical controls. None of the blood biochemistry parameters were disturbed by BMTP treatment.

There were no relevant differences in organ weights in the 80 mg/kg/day group. High organ weights that were considered to be treatment-related were recorded at 240 and 720 mg/kg/day groups, in the male kidneys, the liver (males and females) and the female spleen, as shown (data expressed as percentage):

 

Treatment-related organ weight differences (expressed in %)

Sex	male			female
Dose-level (mg/kg/day)	80	240	720	80	240	720
- Kidneys
.absolute	+3	+15*	+24**	0	+11	+8
.relative	+3	+21**	+24**	0	+5	+11*
- Liver
.absolute	+3	+9	+39**	+5	+22**	+26**
.relative	+3	+15**	+39**	+6	+15	+29
- Spleen
.absolute	-9	-8	+1%	+16	+50**	+50**
.relative	-10	-3	0	+16	+41**	+52**

*: p<0.05, **: p<0.01.

 

In the liver of males and females given 720 and 240 mg/kg/day, hepatocellular hypertrophy was noted along with (males only) increase in extramedullary hematopoiesis. At immunohistochemistry, the hepatocellular hypertrophy was associated with increased in P450 enzyme proteins (CYP2B1/2 and CYP3A1/2) indicating an adaptive answer to the metabolism of BMTP. According to the study report, findings in the kidneys of males from 80 mg/kg/day (eosinophilic hyaline droplets in the cytoplasm of tubular cells of tubules, accompanied by tubular degeneration/necrosis of the cells) were considered to be rat-specific and with no relevance for human risk assessment [1][2]. Increased extramedullary hematopoiesis was noted in the spleen of rats treated from 240 mg/kg/day and an increased hemosiderosis was observed in females given the high dose. Both are an adaptive answer to the slight changes in red blood cell parameters.

Based on the experimental conditions of this study, and considering the minimal effects on the red blood cell parameters of the females and the associated adaptive effects on the spleen, it can be concluded that the dose-level of 240 mg/kg/day was the effect level and 80 mg/kg bw/d was the No Observed Adverse Effect Level (NOAEL).

 

Reproduction/developmental toxicity screening test by oral route in rats (OECD no. 421)

In the OECD guideline No. 421 study[4], three groups of 10 male and 10 female Sprague-Dawley rats received the test item, daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 5p. p. (5 weeks of treatment for the males and at least 6 weeks for the females). The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 40, 160 and 640 mg/kg/day. Another group of 10 males and 10 females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recorded once a week. The males were sacrificed after completion of the pairing period and the dams on day 6p. p. Final body weights and selected organs weights (epididymides, prostate, testes, kidneys, liver and spleen) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (epididymides, ovaries, testes, kidneys, liver, spleen) from the control- and high-dose groups, on kidneys (males), spleen (females) and liver (both sexes) from the low- and intermediate-dose groups and on all macroscopic lesions.

The test item concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%). There were no test item-related deaths in parents. At 640 mg/kg/day, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and abdominal breathing were observed at the beginning of the treatment period. They reappeared towards the end of gestation and disappeared in the lactation period. All animals of that group were affected by at least one of these clinical signs, females more than males. Although these clinical signs lasted the first 4 days and reappeared at the end of gestation only, they were particularly strong and likely induced in one female an abnormal maternal behavior leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day. At 160 mg/kg/day, there were also hypoactivity and mainly loss of balance in males and especially in females at the beginning of the treatment period only. These clinical signs were considered as non-adverse at this dose-level as less severe. Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered not to be adverse. There were no test item-related effects on mean body weight and on mean food consumption at any dose-level. At necropsy, there were higher mean kidney and liver weights in both sexes at 640 mg/kg/day and higher mean kidney weights in males at 160 mg/kg/day. There were also higher mean spleen weights in females at 160 and 640 mg/kg/day.

 

Treatment-related organ weight differences (expressed in %)

Sex	Male			Female
Dose-level (mg/kg/day)	40	160	640	40	160	640
- Kidneys
.absolute	+6	+16**	+32**	0	+7	+7
.relative	+9	+15**	+33**	0	+7	+12*
- Liver
.absolute	-3	+11	+44**	+2	+5	+19*
.relative	+1	+9	+46**	+2	+6	+26**
- Spleen
.absolute	-12	+3	+4	-5	+33	+70**
.relative	-9	+1	+5	-4	+34**	+80**

Statistically significant from controls: *: p<0.05, **: p<0.01.

 

Liver and kidneys were enlarged in three and two animals respectively at 640 mg/kg/day and tan discoloration of the kidney was also noted in three males. At microscopic examination, there were centrilobular hepatocellular hypertrophy in all males and females at 640 mg/kg/day, along with minimal vacuolation in two males and pigment-laden Küpffer cells in seven females. In the absence of associated microscopic degenerative changes, the liver changes, previously observed in the 28-day study and associated with increased in P450 enzyme proteins (CYP2B1/2 and CYP3A1/2), are usually seen as an adaptive response and considered not to be adverse. Moderate to mark tubular hyaline droplets were noted in the kidneys of all males, with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells). These effects are considered to be rat-specific and with no relevance for human risk assessment [1] [2].

At this dose-level, there was also minimally increased extramedullary hematopoiesis in the spleen of females. At 160 mg/kg/day, higher incidence and severity of tubular hyaline droplets were noted in the kidney of males, along with foci of tubular basophilia. Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males.

Based on the experimental conditions of this study, and taking into consideration the clinical signs and the adaptive effects on the spleen, it can be concluded that the dose-level of 640 mg/kg/day was the effect level and 160 mg/kg bw/d was the No Observed Adverse Effect Level (NOAEL).

 

7-day range finding study

The potential toxicity of BMTP was evaluated following daily oral administration (gavage) to rats for 7 days. On completion of thetreatment period, the animals were not treated for 6 days before sacrifice. Three treated groups of four male and four female Sprague-Dawley rats received BMTP by gavage at the dose-levels of 100, 300 or 1000 mg/kg/day for 7 days. One other group received the vehicle alone (corn oil) under the same experimental conditions and acted as a control. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight was recorded once before the beginning of the treatment period, on the first day of treatment and then on days 4, 7 and 11. Food consumption was recorded twice a week. Six days after completion of the treatment period, all animals were sacrificed and submitted to a full macroscopicpost-mortemexamination. Designated organs were weighed and selected tissues were preserved. No microscopic examination was performed.

No unscheduled deaths occurred during the study. At 1000 mg/kg/day, mostly on the first day of treatment, males and females showed loss of balance, hypoactivity, lacrimation, half-closed eyes, staggering gait and loud breathing. Hypersalivation and piloerection were also recorded during the study in males and females treated at 1000 mg/kg/day. At 300 mg/kg/day, 1/4 males and 2/4 females showed hypersalivation on day 7. No clinical signs were recorded in animals treated at 100 mg/kg/day. Body weight and food consumption were not affected by treatment with the test item. No treatment-related effects on organ weights were recorded and no treatment-related necropsy findings were noted.

Conclusion

In all studies, the liver changes were seen as a metabolic adaptive response and not considered to be adverse in the absence of associated microscopic degenerative changes. As well, the kidney effects in the male rats were considered to be male rat-specific and with no relevance for human risk assessment [1] [2]. Therefore, based on these studies and the minimal effects on the red blood cell parameters of the females and the associated adaptive effects on the spleen, the No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/d.

 

[1] According to EU regulation No 1272/2008 (CLP) of 16 December 2008, section 3.9.2.9.9., “nephrotoxicity seen only in male rats of a particular strain known to be susceptible to this effect may result in the decision not to classify”.

[2] Swenberg JA and Lehman-McKeeman LD (1999) Alpha 2 Urinary globulin associated nephropathy as a mechanism of renal tubule cell carcinogenesis in male rats (Species Differences in Thyroid, Kidney and urinary bladder carcinogenesis), IARC, 147, 95-118.

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

Reference 1

Endpoint:

sub-chronic toxicity: oral

Remarks:

This study was performed for a chemical registration dossier in China.

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: The males had a mean body weight of 205 g (range: 183 g to 232 g) and the females had a mean body weight of 153 g (range: 135 g to 205 g).
- Fasting period before study: no
- Housing: two, by sex and group, in Individually Ventilated Cages (IVC) (polysulfone 904 cm2, Tecniplast)
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (ad libitum): tap water (filtered with a 0.22 µm filter).
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet and sawdust were analyzed by the suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 8 to 15 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations transferred on the eve of dosing to the animal unit and stored within an air-flow safety hood at room temperature and protect from light (see § Dose formulation supply) were administered by gavage, using a glass syringe fitted with a metal gavage tube, once a day, in the morning. Due to the foul odor of the test item, the animals were dosed within an air-flow safety hood.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were provided by the Sponsor as ready-to-use vials of 50 or 100 mL of capacity, at the concentrations of 0 (corn oil), 2, 20 and 60 mg/mL. Formulations were prepared and sent to CiToxLAB France every 3 weeks approximately (from 23 December 2014 to 16 March 2015).

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 2, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): Sigma-Aldrich, MKBQ9948V

Analytical verification of doses or concentrations:

yes

Remarks:

GC/FID

Details on analytical verification of doses or concentrations:

Results have shown that all measured concentrations are included in the acceptability interval (± 10% of nominal concentrations) and that dose formulations are stable throughout at least a 5 weeks period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Control and high doses: 16
Low and mid doses: 10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-level selection
The dose-levels were selected by the Sponsor, following the results of two previous studies:
¿ OECD 407 rat study (CiToxLAB France/Study No. 32643 TSR): the test item (batch No. 2KS149) was given by gavage to Sprague-Dawley rats in corn oil at 80, 240 or 720 mg/kg/day for 4 weeks.
Hypersalivation was observed at 720 mg/kg/day and minimal disturbances of red blood cell parameters suggesting increased red blood cell turn over were recorded in females given 720 or 240 mg/kg/day.
Treatment-related organ weight changes were recorded in the kidney of males treated from 240 mg/kg/day, the liver of males and females treated from 240 mg/kg/day and the spleen of females treated from 240 mg/kg/day.
Treatment-related lesions were observed in the kidneys of male rats from 80 mg/kg (eosinophilic hyaline droplets with tubular degeneration/necrosis), the liver (hepatocellular hypertrophy) and the spleen (extramedullar hematopoiesis or EMH) at 720 and/or 240 mg/kg/day. None of these morphological changes were considered as adverse. EMH and hemosiderosis was considered to be a compensatory effect, secondary to the test-item related effect on red blood cells.
The dose-level of 80 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL),
¿ OECD 421 rat study (CiToxLAB France/Study No. 38744 RSR): the test item (batch No. OP3/2010) was given by gavage to Sprague-Dawley rats in corn oil at 40, 160 or 640 mg/kg/day for 2 weeks before mating, during mating, gestation and until Day 5 p.p.
At 640 mg/kg/day, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and/or abdominal breathing were observed at the beginning of the treatment period and towards the end of gestation. At 160 mg/kg/day, there were also hypoactivity and mainly loss of balance at the beginning of the treatment period. Ptyalism was recorded in a dose related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females.
Treatment-related organ weight changes were recorded in the kidney from 160 mg/kg/day, the liver at 640 mg/kg/day and the spleen of females treated from 160 mg/kg/day. Liver and kidneys were enlarged in a few animals at 640 mg/kg/day and tan discoloration of the kidney was noted in a few males.
At microscopic examination at 640 mg/kg/day, there were centrilobular hepatocellular hypertrophy in all animals, along with minimal vacuolation in few males and pigment laden Küpffer cells in several females. Tubular hyaline droplets were noted in the kidneys of all males, with foci of tubular basophilia (occasionally with degenerated cells). There was also minimally increased extramedullary hematopoiesis in the spleen of females.
At 160 mg/kg/day, higher incidence and severity of tubular hyaline droplets were noted in the kidney of males, along with foci of tubular basophilia. Minimal centrilobular hepatocellular hypertrophy was also noted in few males.
The dose-level of 160 mg/kg/day was considered to be the NOAEL.

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
- Morbidity and mortality
Each animal was checked for mortality and morbidity once a day during the acclimation period, at least twice a day during the treatment period and at least once a day during the treatment-free period, including weekends and public holidays.
- Animal care
At several occasions during the study, medical care (draining) was given to the right ear of female D25453 following the formation of an othematoma (hematoma of the ear).

- Clinical signs
Each animal was observed once a day, at approximately the same time each day, for the recording of clinical signs.

- Detailed clinical examination
Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) and bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

- Body weight
The body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and then once a week until the end of the study.

- Food consumption
The quantity of food consumed by the animals in each cage was recorded once a week, over a 7 day period, during the study (see § Study plan adherence).
Food consumption was calculated per animal and per day.
When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of food consumption.

- Ophthalmology
Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on all control and high-dose animals sacrificed at the end of the treatment period on one occasion at the end of the treatment period.
The pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont¿Ferrand, France). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy (Oméga 500, Heine, Herrsching, Germany).

- Monitoring of estrous cycle
The estrous cycle stage was determined for each female planned to be sacrificed at the end of the treatment period, from a fresh vaginal lavage (stained with methylene blue), daily for the last 4 consecutive days (in the morning between 8.00 and 10.00 AM) of the treatment period (see § Study plan adherence).

LABORATORY INVESTIGATIONS
- Blood collection
Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours.
Blood samples were taken from the orbital sinus of the animals (before the daily treatment), under light isoflurane anesthesia, and collected into appropriate tubes (see following tables).

- Hematology
Peripheral blood
The following parameters were determined at the end of the treatment period for all animals planned to be sacrificed at the end of the treatment or treatment-free period.
Blood was collected into BD Microtainer® (K2EDTA) tubes:
Erythrocytes (RBC), Mean cell volume (MCV), Packed cell volume (PCV), Hemoglobin (HB), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell
count with cell morphology, Reticulocytes (RTC).
A blood smear for possible determination of the differential white cell count (with cell morphology) was prepared for each animal and stained with May Grünwald Giemsa. As all blood samples were successfully analyzed by the ADVIA 120, blood smears were archived without further investigation.
A blood smear (stained with blue cresyl) for possible determination of the reticulocyte count was prepared for each animal. As all blood samples were successfully analyzed by the ADVIA 120, blood smears were archived without further investigation.
Blood was collected into sodium citrate tubes: Prothrombin time (PT)

- Bone marrow
Two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal sacrificed on completion of the treatment or treatment-free period and stained with May Grünwald Giemsa.
As no adverse abnormalities were observed during the hematological investigations, the bone marrow differential cell count was not determined and smears were archived.

- Blood biochemistry
The following parameters were determined at the end of the treatment period for all animals planned to be sacrificed at the end of the treatment or treatment-free period.
Blood was collected into lithium heparin tubes: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total cholesterol (CHOL), Triglycerides (TRIG),

- Thyroid hormones
At the same time as blood sampling for hematology and blood biochemistry, an additional blood sample (approximately 0.8 mL) was taken from each animal sacrificed at the end of the treatment or treatment-free period into lithium heparin tubes.
The blood was centrifuged (approximately 3000 g for 10 minutes at +4°C). The plasma (approximately 400 µL, see § Study plan adherence) was frozen in individual tubes at 20°C pending possible analysis. The levels of the thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) were not determined in the absence of indication for an effect on the pituitary-thyroid axis.

Sacrifice and pathology:

PATHOLOGY
Precautions used for necropsy were documented in a Specific Operating Procedure.

- Sacrifice
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
Control female D25422 was prematurely sacrificed in the same way (no fasting).

- 2.7.2 Organ weights
The body weight of each animal was recorded before scheduled sacrifice at the end of the treatment or treatment-free period. The organs of those animals specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

- Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

- Preservation of tissues
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Two bone marrow smears for potential determination of the bone marrow differential cell count (see § Bone marrow) were prepared from the femur of each animal sacrificed on completion of the treatment or treatment-free period.

- Preparation of histological slides
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax (after 96 hours maximum in formalin for kidneys), sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (see § Study plan adherence).
Kidneys of all recovery males were embedded after a fixation of 96 hours maximum in formalin.
One additional kidney slide of all males sacrificed at the end of the treatment and treatment-free periods was immunostained with an antibody for Alpha 2µ-globulin protein.

- Microscopic examination
A microscopic examination was performed:
¿ on all tissues listed in the Tissue Procedure Table from the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and sacrificed prematurely,
¿ on liver and spleen from all low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
¿ on kidneys (males), liver (both sexes) and spleen (both sexes) from the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment-free period,
¿ on all immunostained kidneys from all males (groups 1 to 4) sacrificed at the end of the treatment and treatment-free periods,
¿ on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Other examinations:

SEMINOLOGY
At the end of the treatment period, just before sacrifice, each male was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and the left epididymis was removed. Animals were then sacrificed (see § Sacrifice). These examinations were not carried out at the end of the treatment-free period.
-Epididymal sperm
Sperm from the cauda of the left epididymis was sampled for motility and morphology investigations (see § Epididymal sperm motility and § Epididymal sperm morphology).
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at ¿20°C pending further investigation (see § Epididymal sperm count).

- Epididymal sperm motility
Sperm motility was evaluated on a slide, after appropriate dilution when needed. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40¿fold magnification. Results are expressed as the proportion of motile and non-motile spermatozoa.

- Epididymal sperm morphology
Sperm morphology was determined from a smear, after eosin staining and counting of 100 spermatozoa per slide. Results are expressed as the proportion of spermatozoa in each of the following categories:
¿ normal,
¿ normally shaped head separated from flagellum,
¿ abnormal head separated from flagellum,
¿ abnormal head with normal flagellum,
¿ abnormal head with abnormal flagellum,
¿ normally shaped head with abnormal flagellum.

- Epididymal sperm count
After thawing, the left epididymis cauda was weighed, minced and homogenized in a saline-triton solution using a Polytron. An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber. Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

- Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, it was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber. Results are expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10 which represents the duration of spermatogenic cycle of homogenization-resistant testicular spermatids; Blazak et al., 1993).

Statistics:

Citox software was used to perform the statistical analyses of body weight, food consumption, seminology, hematology and blood biochemistry.
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period
There were no test item treatment-related clinical signs.
All clinical signs recorded in test item-treated groups (abnormal color of body parts, difficulties in using limbs, alopecia/thinning of hair, scabs, wounds, locomotory difficulties, abnormal growth of teeth, ptyalism, reflux at dosing, dyspnea, chromorhynorrhea, increase in size of body parts, thin appearance, hunched posture) were of comparable incidence of controls, not dose-related and/or common in this strain of rats under laboratory conditions.

Treatment-free period
There were no test item treatment-related clinical signs.
The only clinical sign recorded in the high-dose group during the treatment-free period was alopecia having started in the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no premature deaths in test item-treated groups.
The only premature death noted during the study was not related to the test item treatment: control female D25422 was sacrificed prematurely for ethical reasons in Week 4 (bad health condition). Perforation of the oesophagus seen at necropsy was indicative of a gavage trauma.

Treatment-free period
There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 1.
Treatment period
There were no test item-treatment related effects on mean body weight and mean body weight gain during the treatment period.
Occasional statistical differences from controls were observed in mean body weight and mean body weight gain at all dose-levels but they were considered to be incidental (transient, rarely dose-related, in one sex).
Treatment-free period
There were no test item-treatment related effects on mean body weight and mean body weight gain during the treatment-free period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Table 2.
Treatment period
In males treated at 300 mg/kg/day, there was higher mean food consumption during the treatment period from week 5. This was considered to be of limited toxicologically significance in view of the slight changes from controls (up to +13%) and in absence of relevant effects on mean body weights.
The statistically significantly higher mean food consumption noted in Week 5 at 100 mg/kg/day was considered of no toxicological relevance (isolated).
In females, there were no test item-treated effects during the treatment period. Occasional statistical differences from controls were noted at 10 mg/kg/day in Weeks 3 and 5 and were not considered to be test item treatment-related (no dose-relationship).

Treatment-free period
There were no toxicologically significant effects on mean food consumption during the treatment-free period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse findings in any group.

Treatment period
There were no test item-related effects in males. The slightly shortened mean prothrombin time noted at 300 mg/kg/day (19.4 s vs. 21.5 s in controls) was due to 2/10 animals. In view of the low incidence and absence of similar trend in females, this was considered to be fortuitous.
In females, the most relevant findings in hematology at the end of the treatment period are summarized in the table 3.
In females given 300 mg/kg/day, mean red blood cell parameters were statistically significantly affected when compared with controls. Most of the individual data were comparable to what can usually be observed in this type of study. This decrease in red blood cell mass (-7%) was regenerative [increase in reticulocyte count (1.6-fold) and increased hematopoiesis noted at microscopy in the spleen] and was considered as non adverse.
There were no toxicologically significant effects in females of the other dose-levels.

Treatment-free period
In males, no changes that could be related to test item treatment were observed.
In females, statistical difference from controls in hematology at the end of the treatment-free period is summarized in the table 3.
Differences in red blood cell mass were no longer noted in females given 300 mg/kg/day (hemoglobin concentration was even higher (+3%) than in controls at the end of the treatment-free period).

Description (incidence and severity):

There were no adverse findings in any group.

Treatment period
Statistically significant differences from controls in blood biochemistry at the end of the treatment period are summarized in the table 4.
In females treated at 300 mg/kg/day, mean glucose concentration was lower (-18%) than in controls. Higher mean phosphorus level (+17 and 19%, respectively) was also observed in a dose-related manner from 10 mg/kg/day. As the variations were not severe, these findings were not considered to be adverse, and of no toxicological significance at 10 mg/kg/day (not statistically significant).
Female D25453, treated at 300 mg/kg/day (this female had a hematoma of the external ear, unilaterally, during the study, and chondropathy with bone formation and bone marrow was observed at microscopy), had higher total bilirubin level and ASAT and ALAT activities, as well as very low glucose level. As it was the only animal affected in the high-dose group (ASAT, ALAT), this was considered to be incidental.
In males and when compared with controls, there was higher mean cholesterol concentration (+37%) at 300 mg/kg/day. In view of the slight amplitude of difference, this was not considered to be adverse.
Variations in sodium, chloride and glucose levels and decrease in alkaline phosphatase activity in males were considered to be incidental (no dose-relationship) or not biologically significant (direction and amplitude of changes).

Treatment-free period
Blood biochemistry findings recorded at the end of the treatment period were no longer observed after the 6-week treatment-free period.

Urinalysis findings:

not examined

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):

Animals sacrificed at the end of the treatment period
At necropsy, the mean terminal body weight was lower in females treated at 300 mg/kg/day.
When compared with controls, the following changes were observed in the mean organ weights:
- the absolute and relative kidney weights were statistically significantly higher in males treated at 300 mg/kg/day (p<0.01). In females the relative weights were statistically significantly higher at 100 and 300 mg/kg/day (p<0.01). In males, this correlated histologically with increased incidence and severity of hyaline droplets and was considered to be test item-related,
- the absolute and relative liver weights were statistically significantly higher in males treated at 300 mg/kg/day (p<0.01). In females, only the relative weight was statistically significantly higher (p<0.01). This correlated histologically with hepatocytic hypertrophy and was considered to be test item-related,
- the relative weight of spleen was statistically significantly higher in females treated at 100 mg/kg/day (p<0.05) and the absolute and relative weights in females treated at 300 mg/kg/day (p<0.01). This may correlate partially with the higher severity of hematopoiesis seen histologically,
- the absolute and relative uterus weights were lower in females treated at 100 or 300 mg/kg/day without reaching a statistical significance. As individual values remained within these of the controls and in the absence of histopathological correlates, this was considered to be due to physiological variations in the estrous cycle and any relationship to the test item was considered to be unlikely,
- the absolute and relative adrenal weights were higher in males but without reaching a statistical significance. This was mainly due to a single animal (D25370) for which the adrenals were also noted enlarged at necropsy. In the absence of histopathological correlates, any relationship to the test item was excluded.
Other changes in the mean organ weights were considered either to reflect the lower terminal body weight in females or part of the normal variation in rats.
Changes in the mean organ weights are summarized in the table 6.

Animals sacrificed at the end of the treatment-free period
When compared with controls, the following changes were observed in the mean organ weights:
- the mean absolute and relative kidney weights were slightly higher in males previously treated at 300 mg/kg/day but without reaching a statistical significance suggesting pronounced recovery,
- the mean absolute and relative liver weights were higher in both sexes previously treated at 300 mg/kg/day, reaching a statistical significance for both values in females (p<0.05).
Other changes in the mean organ weights were considered to be part of the normal variation between animals of this age.
Changes in the mean organ weights are summarized in the table 6.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled death
Female D25422 from the control group was sacrificed in a poor condition on day 22. At necropsy, there was a perforation of the esophagus with a white mass and a pouch correlating histologically with abscess. The subcutaneous tissue was gelatinous in the thoracic region and a pouch was also present in the axillary region. All these changes were consistent with a gavage trauma.

Animals sacrificed at the end of the treatment period
Test item-related changes were observed in the liver.
Accentuated lobular pattern and tan discoloration were seen at a slightly higher incidence in males treated with the test item than in controls. As there was some correlation with centrilobular hypertrophy and/or hepatocytic vacuolation at the histopathological examination, these observations were considered to be probably related to the test item.
Other changes were considered to be either incidental or part of the normal background commonly seen in rats of this age.
The incidence of these findings is summarized in the table 7.

Animals sacrificed at the end of the treatment-free period
There were no test item-related changes.
The few observations recorded at necropsy were considered to be part of the normal background of changes commonly seen in rats of this age.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Unscheduled death (control female D25422)
Perforation of the esophagus was observed along with an abscess. Edematous subcutaneous tissue and abscess were noted in the axillary/thoracic region.
Moderate thymic atrophy and minimal cortical hypertrophy in adrenals were considered to be non specific and probably stress-related.
Slight increased granulocytes numbers in the bone marrow was considered to be secondary to abscesses.

- Animals sacrificed at the end of the treatment and recovery periods
Test item-related changes were observed in the liver (both sexes), kidneys (males) and spleen (both sexes but mainly in females).

. Liver
The incidence and severity of these changes are summarized in the table 8.

Animals sacrificed at the end of the treatment period
Minimal to moderate centrilobular hepatocytic hypertrophy was observed in the liver of males treated at 100 mg/kg/day and in both sexes at 300 mg/kg/day, particularly in males.
Minimal or slight midzonal vacuolation occasionally extending to periportal region and consisting of macrovacuoles often associated with small vacuoles, was observed at a higher incidence in test item treated males than in controls but without a clear dose-related trend.
In females, periportal vacuolation was observed in a few animals including controls, however in the absence of a dose related trend, any relationship to treatment was excluded.
These findings correlated with necropsy changes (accentuated lobular pattern and tan discoloration).
Pigment-laden Kupffer cells were observed in one control female, 3/10 females treated at 100 mg/kg/day and 7/10 females treated at 300 mg/kg/day. In the control female, the 3 females treated at 100 mg/kg/day and 6/7 animals treated at 300 mg/kg/day, this was graded minimal, involving only a few isolated cells. In one high dose animal it was graded slight as it was more diffuse.
Hematopoiesis was marginally higher in females treated at 100 or 300 mg/kg/day than in controls or other test item-treated groups. As similar observation was done in the spleen at 100 or 300 mg/kg/day, a relationship to the test item cannot be excluded, although of minor toxicological significance.

Animals sacrificed following a 6-week treatment-free period
Minimal hepatocytic hypertrophy was observed in the liver of 1/6 males previously treated at 300 mg/kg/day.
Midzonal vacuolation was observed in 1/6 control males (graded minimal) and 2/6 males previously treated at 300 mg/kg/day (graded minimal to slight).
No pigment was seen in the Kupffer cells of females.
These histological changes were considered to be within these observed in controls at the end of the treatment period.

. Kidneys (males only)
Changes in kidneys of males are summarized in the table 9.

Animals sacrificed at the end of the treatment period
Minimal to moderate hyaline droplets (dense eosinophilic granules) were observed in the cytoplasm and/or the lumen of tubular cells of proximal tubules of male kidneys with a dose-related trend. This was associated with a higher incidence and/or severity of tubular basophilia, mononuclear cell infiltrate and occasionally with granular casts.

A specific immunohistochemical staining using an antibody for Alpha 2µ-globulin stained positively these droplets. As this staining was more sensitive than hematoxylin/eosin, there were some differences in the incidence or severity when compared with the routine staining, particularly in controls. However this immunohistochemical staining confirmed the dose-related trend observed in hematoxylin/eosin.

Two minimal unilateral foci of tubular basophilia were observed in females treated at 300 mg/kg/day. Despite the absence of similar change in controls, in the absence of other test item-related changes, these isolated findings were considered to be incidental.
There were no histopathological correlates with the higher relative kidney weight noted at necropsy at 100 or 300 mg/kg/day which may be related to the lower female terminal body weight.

Animals sacrificed following a 6-week treatment-free period
In animals previously treated at 300 mg/kg/day, hyaline droplets, confirmed with the immunohistological staining were observed in the epithelium of tubules of males at a lower incidence and severity when compared to the the animals sacrificed at the end of the treatment period. Minimal or slight tubular basophilia was present multifocally in 2/6 males previously treated with the test item at 300 mg/kg/day when it was seen only focally in 2/6 controls. In addition mononuclear cell infiltrate was observed at a slightly higher incidence in previously treated males (5/6) than in controls (2/6). A slight tubulo-interstitial inflammation was observed in one previously treated male. Its exact toxicological significance remained unclear, however, in the absence of similar change at the end of the treatment period, any relationship to the test article was considered to be unlikely.

. Spleen
Changes in the spleen are summarized in the table 10.

Animals sacrificed at the end of the treatment period
There was a trend towards a slightly higher severity of hemopoiesis in females treated at 100 or 300 mg/kg/day, and a higher severity of pigment in the spleen (suggestive of hemosiderin) in these groups. Considering the high variation of pigment in the spleen and the absence of adverse changes in the hematological parameters, the minimal difference observed between controls and treated males was not considered to reflect a toxicological significance.
Changes in spleen of males and females are summarized in the table 10.
Other changes were considered to be either incidental or part of the normal background commonly seen in animals of this age.

Animals sacrificed following a 6-week treatment-free period
Hematopoiesis was observed at a slightly lower severity in females previously treated at 300 mg/kg/day than in controls.
Pigment was seen at a slightly higher severity in females previously treated at 300 mg/kg/day. Considering the high variation of pigment in the spleen and the absence of changes in the hematological parameters, the minimal difference observed between controls and treated males was not considered to reflect a toxicological significance.

Pathology discussion
Centrilobular hepatocellular hypertrophy correlated with the higher liver weight and accentuated lobular pattern particularly in males. In a previous 28-day oral toxicity study (CiToxLAB France/Study No.32643 TSR), the hepatocellular hypertrophy in the liver was shown to be associated with increased in P450 enzyme proteins (CYP2B1/2 and CYP3A1/2). In the absence of associated microscopic degenerative changes, this finding is usually considered as an adaptative response and considered not to be adverse.

Hepatocellular midzonal vacuolation was seen at a higher incidence in treated males than in controls but without dose related trend correlating with accentuated lobular pattern and/or tan discoloration. As present in controls, this may be due to the vehicle (corn oil), with a possible enhancement by the test item.

Presence of hyaline droplets in kidneys of males correlated with increase in kidney weights at necropsy. These hyaline droplets corresponded to Alpha 2µ-globulin (positive staining with antibody for this globulin). These were observed with a dose-related trend and were associated at 100 and 300 mg/kg/day with a higher incidence and/or severity of tubular basophilia and mononuclear cell infiltrate. Alpha-2µ globulin is known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2007). The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which may explain the tubular basophilia seen at 100 and 300 mg/kg/group. These findings were therefore considered to be adverse for rats at these dose levels. Although human excretes proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human.

In the spleen of females, although marginally increased, the extramedullary hemopoiesis was considered to be secondary to the minimal decrease in red blood cell mass noted clinically and may correlate with the higher weight at necropsy. Due to its low magnitude, it was considered to be non-adverse.
Increased severity of pigment in the spleen in females at the end of the treatment and treatment-free period was considered to be also secondary to the minimal decrease in red blood cell mass observed in this sex.

Following a 6-week treatment-free period, signs of recovery were seen in the liver, kidneys (males) and spleen.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Estrous cycle
Overall, there was a correct correlation between vaginal cytology and vagina microscopic examination at the end of the treatment period in control and high-dose groups.

SEMINOLOGY
Mean sperm analysis data at the end of the treatment period are described in the table 5.
There were no test item treatment-related effects on mean epididymal sperm motility and morphology.
At 300 mg/kg/day and when compared with controls, there were lower mean testicular sperm head count and daily sperm production rate (-15%, p<0.05), and a tendency towards higher mean epididymal counts (+14 and +15%, as number/cauda and number/g cauda, respectively). The relationship with test item treatment was questionable but considered as no toxicologically relevant in rats in view of the low amplitude of changes and as individual values are comparable to what can be observed in Sprague-Dawley rats in laboratory conditions (see above listed Reference control data).
The lower mean values at 10 mg/kg/day were due to males D25337 and D25345 which had at pathology reduced-in-size testes and/or epididymides, testis atrophy/degeneration and/or reduced sperm content in epididymis. In absence of dose relationship, no link with the test item treatment was suspected.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

excluding the male rat-specific kidney effects

Critical effects observed:

no

Table 1: Body weight

Treatment period

 

Sex	Male					Female
Dose-level (mg/kg/day)		0	10	100	300		0	10	100	300
Body weight
. Week 1		205	205	204	206		154	159	152	149
. Week 4		351	365	366	365		208	224*	209	206
. Week 14		522	542	548	542		282	294	274	266
Body weight change
. Weeks 1-14		+317	+337	+343	+336		+129	+135	+122	+116
. Weeks 1-2		+59	+60	+62	+65**		+22	+22	+23	+22
. Weeks 3-4		+37	+41	+42	+40		+14	+20*	+17	+18
. Weeks 5-6		+24	+29	+30*	+27		+13	+14	+9	+11
. Weeks 11-12		+12	+13	+17*	+13		+7	+5	+4	+5

Statistically significant from controls: *: p<0.05, **: p<0.01.

  

Treatment-free period

 

Sex	Male					Female
Dose-level (mg/kg/day)		0	10	100	300		0	10	100	300
Body weight
. Week 14		528	-	-	550		271	-	-	274
. Week 20		555	-	-	568		284	-	-	295
Body weight change
. Weeks 14-20		+27	-	-	+18		+14	-	-	+21

-: not applicable.

Table 2: Food consumption

Treatment period

 

Sex		Male
Dose-level (mg/kg/day)	0		10	100	300
. Weeks 5-6	22.3		23.7	24.5*	24.6*
. Weeks 6-7	21.1		23.0	22.9	23.4*
. Weeks 7-8	20.8		22.2	22.4	23.3**
. Weeks 9-10	21.6		21.8	22.7	23.3*
. Weeks 11-12	20.9		21.6	22.3	23.1**
. Weeks 12-13	19.7		20.2	20.3	22.3**
. Weeks 13-14	18.6		18.3	18.8	20.7*

Statistically significant from controls: *: p<0.05, **: p<0.01.

 

Table 3: Hematology

Treatment period

Sex	Female
Dose-level (mg/kg/day)	0	10	100	300
Red blood cells (T/L)	8.54	8.65	8.32	7.95*
Hemoglobin (g/dL)	15.1	15.3	14.7	14.0**
Mean cell volume (fL)	54.4	54.5	55.0	56.9*
Mean cell hemoglobin concentration (g/dL)	32.6	32.6	32.2	31.0**
Reticulocytes (%)	1.86	1.83	2.28	3.27**
Reticulocytes (T/L)	0.16	0.16	0.19	0.26**

Statistically significant from controls: *: p<0.05, **: p<0.01.

 

 

Treatment-free period

Sex	Female
Dose-level (mg/kg/day)	0	10	100	300
Hemoglobin (g/dL)	15.1	-	-	15.6*

-: not applicable.

Table 4: Blood biochemistry

There were no adverse findings in any group.

Treatment period

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	10	100	300	0	10	100	300
Cholesterol (mmol/L)	1.50	1.64	1.69	2.05**	1.92	1.70	2.11	2.15
Glucose (mmol/L)	6.84	5.59**	5.44**	6.68	5.14	5.37	5.32	4.23**
Phosphorus (mmol/L)	1.88	1.66	1.82	1.78	1.41	1.60	1.65*	1.68**
Sodium (mmol/L)^a	146	147*	147	146	146	146	146	146
Chloride (mmol/L)^a	106	105	104**	106	106	106	105	105
Alkaline phosphatase (U/L)	302	247**	226**	228**	130	140	125	123

Statistically significant from controls: *: p<0.05; **: p<0.01;^a:values rounded to three significant digits.

Table 5: Seminology

 Treatment period

Dose-level (mg/kg/day)	0	10	100	300	Reference data
% of motile epididymal sperm	95.2	78.8	97.8	99.1	[86.0-100]
% of morphologically normal epididymal sperm	94.7	76.2	94.6	95.7	[90.0-98.1]
Mean number of epididymal sperm (10^6/cauda)^a	143	132 (160^b)	157	163	[99.8-182]
Mean number of epididymal sperm (10^6/g cauda)^a	419	400 (468^b)	460	481	[319-591]
Mean number of testicular sperm heads (10^6/g testis)^a	126	93.2 (114^b)	116	107*	[96.2-151]
Daily sperm production rate (10^6/g testis/day)	20.6	15.3 (18.8^b)	19.0	17.5*	[15.8-24.7]

Statistically significant from controls: *: p<0.05;

^a:values rounded to three significant digits;^b: when excluding values from malesD25337 and D25345.

Reference control data based onCiToxLAB/Study No. 40814TCR, 41748TCR, 38872TCR,36834TCR: individual data percentiles [5%-95%]

Table 6: Organ weights

Animals sacrificed at the end of the treatment period

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	10	100	300	10	100	300
Number of animals	10	10	10	10	10	10
- Final body weight	+5	+6	+4	+1	-6	-12**
- Kidneys
.absolute	+8	+10	+25**	-1	+1	+1
.relative	+3	+4	+20**	-1	+8**	+15**
- Liver
.absolute	+5	+7	+25**	-2	-1	+4
.relative	-1	+1	+20**	-2	+6	+19**
- Spleen
.absolute	+9	+5	+3	+2	+12	+24**
.relative	+3	-1	-1	+1	+20*	+41**
- Uterus
.absolute				-12	-31	-30
.relative				-11	-27	-21

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Animals sacrificed at the end of the treatment-free period

 

Sex	Male	Female
Group	4	4
Dose-level (mg/kg/day)	300	300
Number of animals	6	6
- Final body weight	+1	+5
- Kidneys
.absolute	+11	+5
.relative	+10	+1
- Liver
.absolute	+11	+17*
.relative	+9	+12*

Statistically significant from controls: *: p<0.05

Table 7: Macroscopic post-mortem examination

Animals sacrificed at the end of the treatment period

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	10	100	300	0	10	100	300
Number of animals	10	10	10	10	9	10	10	10
Accentuated lobular pattern in liver	4	9	8	9	3	0	3	4
Tan discoloration	2	6	5	0	0	0	0	0

Microscopic examinations

Table 8: Liver

Animals sacrificed at the end of the treatment period

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	10	100	300	0	10	100	300
Number of animals	10	10	10	10	9	10	10	10
Hypertrophy; hepatocytes (centrilobular)
Minimal	1	-	6	-	-	-	-	6
Slight	-	-	-	6	-	-	-	3
Moderate	-	-	-	4	-	-	-	-
Vacuolation; hepatocytes; midzonal
Minimal	1	4	7	3	-	-	-	-
Slight	1	3	1	4	-	-	1	-
Vacuolation; hepatocytes; periportal
Minimal	-	-	-	-	1	3	1	1
Slight	-	-	-	-	1	-	1	-
Pigment; Kupffer cells
Minimal	-	-	-	-	1	-	3	6
Slight	-	-	-	-	-	-	-	1
Hematopoiesis
Minimal	3	3	2	3	7	9	8	9
Slight	-	-	-	-	-	-	2	1

-: Not observed in this group

 

Table 9: Kidney (males only)

Animals sacrificed at the end of the treatment period

Group	1	2	3	4
Dose level (mg/kg/day)	0	10	100	300
Number of animals	10	10	10	10
Hyalin droplets in tubular epithelium
Minimal	-	2	2	1
Slight	-	3	4	2
Moderate	-	-	4	7
Alpha 2 µ globulin staining
Minimal	4	2	-	-
Slight	-	3	5	3
Moderate	-	-	5	7
Basophilia; tubules
Minimal	4	5	5	4
Slight	-	2	4	5
Mononuclear cell infiltrate
Minimal	5	6	2	8

-: not observed in this group

Animals sacrificed following a 6-week treatment-free period

Group	1	4
Dose level (mg/kg/day)	0	300
Number of animals	6	6
Hyalin droplets in tubular epithelium
Minimal	1	-
Slight	1	1
Alpha 2 µ globulin staining
Minimal	2	-
Slight	-	1
Basophilia; tubules
Minimal	2^(1)	1^(2)
Slight	-	1^(2)
Infiltrate; mononuclear inflammatory cells
Minimal	3	5

 -: Not observed in this group;⁽¹⁾: focal;⁽²⁾multifocal

Table 10:Spleen

Animals sacrificed at the end of the treatment period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	10	100	300	0	10	100	300
Number of animals	10	10	10	10	9	10	10	10
Hematopoiesis
Minimal	5	7	8	7	2	1	1	2
Slight	5	3	2	2	5	8	5	4
Moderate	-	-	-	-	2	1	3	4
Marked	-	-	-	-	-	-	1	-
Pigment (suggestive of hemosiderin)
Minimal	7	5	5	4	3	2	-	-
Slight	3	5	5	6	4	7	6	1
Moderate	-	-	-	-	2	1	4	9

- : Not observed in this group;

Animals sacrificed following a 6-week treatment-free period

Sex	Males		Females
Group	1	4	1	4
Dose level (mg/kg/day)	0	300	0	300
Number of animals	6	6	6	6
Hematopoiesis
Minimal	2	3	-	2
Slight	4	3	3	2
Moderate	-	-	3	2
Pigment
Minimal	2	1	1	-
Slight	3	3	3	-
Moderate	1	2	2	4
Marked	-	-	-	2

 -: Not observed in this group

Conclusions:

BMTP was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats. At the dose-levels of 100 and 300 mg/kg/day, adverse test item treatment-related effects were observed in the kidneys of male rats: the hyaline droplets in the tubular cells were associated with Alpha 2µ-globulin accumulation as demonstrated by immunohistochemistry and with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. Alpha 2µ-globulin nephropathy of male rats is a common finding following the administration of hydrocarbons (Khan et al., 2002) and has already been associated with karyomegaly within the proximal tubules (Williams et al., 2001). It is considered to be a rat-specific effect with no relevance for human risk assessment (Swenberg et al., 1999). No other adverse effects were observed in the study. Therefore, excluding the male rat-specific kidney effect, a No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was established for human risk assessment and classification

Executive summary:

The potential repeated dose toxicity of bismethylthiopropane (BMTP) was evaluated following daily oral administration (gavage) to rats in a study conducted according to OECD guidelines no. 408. Three groups of 10 (low- and mid-dose groups) or 16 (high-dose group) male and female Sprague-Dawley rats each received BMTP by daily oral gavage administration for at least 13 weeks. BMTP was administered at 10, 100 and 300 mg/kg/day as a solution in the vehicle (corn oil) under a constant dosage-volume of 5 mL/kg/day. A control group of 16 animals per sex received the vehicle alone under the same experimental conditions. On completion of the treatment period, the animals in each group were sacrificed, except for the first six animals per sex in the control and high-dose groups which were kept for a 6 week treatment-free period. The concentration of the dose formulations was checked at each preparation (thus 5 times during the study) before the administration to the animals. The animals were checked at least twice daily during the treatment period or once daily after the treatment period for mortality and morbidity, and once daily for clinical signs. In addition, detailed clinical examinations were performed weekly. The body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as well as food consumption. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals at the end of the treatment period. Estrous cycle stage was determined daily for the last 4 consecutive days of the treatment period. Hematology and blood biochemistry were performed towards the end of the treatment and treatment-free periods. Just before sacrifice, under deep anesthesia, left epididymides and testes were sampled from all males for sperm analyses (motility, morphology and counts). Animals were euthanized and submitted to a full macroscopic post mortem examination. Designated organs were weighed and several tissues were preserved. A microscopic examination was performed on selected tissues from animals of the control and high-dose groups sacrificed at the end of the treatment period, on liver and spleen (both sexes) from animals of the low-and mid-dose groups sacrificed at the end of the treatment period, on liver and spleen (both sexes) and on kidneys (males) from recovery animals of the control and high-dose groups, and on all macroscopic lesions. Males kidney slides immunostained with an antibody for Alpha 2µ-globulin protein were also microscopically examined at the end of treatment and treatment-free periods.

BMTP concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%) and no test item was detected in control formulations. There were no test item treatment-related deaths or clinical signs. Mean body weight and mean body weight gain were not affected by the test item treatment. In males treated at 300 mg/kg/day, there was higher mean food consumption during the treatment period from week 5, considered to be of limited toxicologically significance. In females, there were no test item treated effects on mean food consumption. There were no toxicologically significant effects on mean food consumption during the treatment-free period. There were no ophthalmological findings and no test item-treated effects on estrous cycle data at the end of the treatment period. At hematology at the end of the treatment period, when compared with controls, mean red blood cell count and hemoglobin concentration were statistically significantly decreased along with higher mean cell volume and reticulocyte count in females given 300 mg/kg/day. These variations were no longer observed at the end of the treatment-free period and there were no test item-related effects in males. These findings were not considered to be adverse. At blood biochemistry in females treated at 300 mg/kg/day, mean glucose concentration was lower than in controls. Higher mean phosphorus levels were recorded at 100 and 300 mg/kg/day. In males and when compared with controls, there was higher mean cholesterol concentration at 300 mg/kg/day. All these findings were not considered to be adverse and recovery was complete at the end of the treatment-free period. At sperm analyses, there were no test item treatment-related effects on mean percent of motile epididymal sperm cells and their morphology. The relationship of the test item treatment with the variations observed in mean epididymal sperm cells count (+14/15% from controls) and mean testicular sperm head count and daily sperm production rate (-15%) at 300 mg/kg/day was questionable but considered to be of no toxicological significance in rats. At necropsy at the end of the treatment period, there were higher mean kidney and liver (absolute and/or relative-to body weight) weights in both sexes at 300 mg/kg/day and higher mean relative kidney weights in females at 100 mg/kg/day. There were also higher mean spleen weights in females at 100 and 300 mg/kg/day. At microscopic examination, presence of hyaline droplets (Alpha 2µ-globulin) in kidneys of males from all test item-treated groups correlated with increases in mean kidney weights at necropsy; it was observed with a dose-related trend and associated at 100 and 300 mg/kg/day with a higher incidence and/or severity of tubular basophilia and mononuclear cell infiltrate (300 mg/kg/day). These findings were considered to be adverse at these dose-levels but non-relevant for human considering the male rat-specific mechanism. Centrilobular hepatocellular hypertrophy at 100 (males) and 300 (both sexes but mainly males) mg/kg/day correlated with the higher mean liver weight, and the accentuated lobular pattern in males; it is usually seen as an adaptative response and in the absence of associated degenerative changes considered not to be adverse. Midzonal vacuolation was seen at a higher incidence in all test item-treated group males than in controls, without dose-related trend, correlating with tan discoloration and/or accentuated lobular pattern noted at necropsy. At 100 and 300 mg/kg/day, there was a trend towards a minimally increased extramedullary hematopoiesis in the spleen of females considered to correlate with the decrease in red blood cell mass noted clinically and considered to be non-adverse. This was accompanied with a slightly higher severity of pigment deposit (suggestive of hemosiderin) which was also considered to be secondary to the lower red blood cell mass noted clinically. Following a 6-week treatment-free period signs of recovery were seen in the liver, kidneys (males) and spleen.

BMTP was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats. At the dose-levels of 100 and 300 mg/kg/day, adverse test item treatment-related effects were observed in the kidneys of male rats: the hyaline droplets in the tubular cells were associated with Alpha 2µ-globulin accumulation as demonstrated by immunohistochemistry and with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. Alpha 2µ-globulin nephropathy of male rats is a common finding following the administration of hydrocarbons (Khan et al., 2002) and has already been associated with karyomegaly within the proximal tubules (Williams et al., 2001). It is considered to be a rat-specific effect with no relevance for human risk assessment (Swenberg et al., 1999). No other adverse effects were observed in the study. Therefore, excluding the male rat-specific kidney effect, a No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was established for human risk assessment and classification.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The preparation of the dosage forms (groups 1 to 4) was not conducted as fully compliant with GLP principles.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: 222 g (range: 213 g to 236 g) for the males and 191 g (range: 178 g to 203 g) for the females.
- Fasting period before study: no
- Housing: in two in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast) containing sawdust
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (ad libitum): tap water (filtered with a 0.22 µm filter).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 6 December 2007 to 10 January 2008

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The Sponsor supplied the solutions prepared at the concentrations of 0 (corn oil), 16, 48 and 144 mg/mL intended for use of study groups 1, 2, 3 and 4. The detail of this preparation is summarized as follow:
. solubilisation of weighed amounts of Bismethylthiopropane (batch 2KS149) in corresponding volumes of corn oil (Sigma ref. C8267 batch 126K0117). After introduction of convenient amounts of Bismethylthiopropane, each flask is placed on a stirring plate and mixed using a magnetic bar until good homogeneisation (visual checking). When solutions are considered as homogeneous, corresponding series are prepared as previously indicated, and registered as
GLP samples,
. repartition of the solutions in 40 vials of 25 mL and sent to the laboratoru for dosing of animals purposes,
. remaining preparations (11 x 20 mL) of each formulation (including the vehicle group) were kept by the sponsor to determine the concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability tests and the check of the concentration in the dosage forms were carried out at Sponsor’s facility.
The analyses using GC/FID were repeated before instituting any significant change in the dosage form preparation (e.g. change in the range of concentration).
During the course of the treatment period, the concentration of the test item in solutions prepared for use in the study was determined.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor on the basis of the following results:
Acute toxicity study performed in female Sprague-Dawley rat:
. no mortality occurred after a single administration of the test item at 300 or 2000 mg/kg,
. at 2000 mg/kg, 2/3 animals experienced decubitus, sedation and dyspnea on the day of administration and 1/3 was in coma. On day 2, all the animals had completely recovered. No effect on body weight gain was noted at 300 or 2000 mg/kg. There were no necropsy findings.
7-day toxicity study:
BMTP was given (batch No. 2KS149) by the oral route (gavage) to Sprague-Dawley rats for 7 days at the dose-levels of 100, 300 and 1000 mg/kg/day:
. at 1000 mg/kg/day, males and females had clinical signs, mostly on the first day of treatment (loss of balance, hypoactivity, lacrimation, half-closed eyes, staggering gait and loud breathing). Hypersalivation and piloerection were also recorded during the study,
. at 300 mg/kg/day, isolated cases of hypersalivation were noted in males and females,
. at 100 mg/kg/day, no relevant findings were observed,
. there were no treatment-related macroscopic abnormalities during the autopsy performed on day 13.
- Post-exposure recovery period in satellite groups: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays, and once a day on other days.
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made on all animals outside the home IVC cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded

BODY WEIGHT: Yes
The body weight of each animal was recorded once before group allocation, on the first day of treatment, and then on a weekly basis.

FOOD CONSUMPTION: Yes
The quantity of food consumed by the animals in each cage was recorded once a week until the end of the study.
Food consumption was calculated per animal and per day.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy (week 5).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy (week 5).
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: end of the treatment period
- Dose groups that were examined: all
- Battery of functions tested: detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 3)
A microscopic examination was performed on:
all tissues listed in the Tiable 3 for animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period, all macroscopic lesions and the kidneys, the liver and the spleen of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period.

Additional investigation (immunohistochemistry)
Additional examination of the liver for CAB and CYP 1A1/2, 2B1/2, 3A1/2 and 4A1 of a subset of males and females and kidneys of all males were performed on animals from group 1 (controls) and group 4 (treated at 720 mg/kg/day.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No signs of toxicological importance were observed, hypersalivation (recorded as ptyalism), not considered as adverse, was the only sign noted from day 4 among all animals given 720 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Neither mean body weight nor mean body weight change were affected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not affected.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When compared to the control mean values, red blood cell parameters recorded in females given 720 or 240 mg/kg/day were decreased (red blood cell count, hemoglobin concentration, pack cell volume, mean cell hemoglobin concentration or increased (mean cell volume, mean cell hemoglobin). These differences were minimal, not always clearly dose-related and the value were still in the range of the historical controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

None of the blood biochemical parameters were disturbed by the test item treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects on the central nervous system were evidenced by the FOB tests.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no relevant differences in organ weights in the 80 mg/kg/day group.
High organ weights that were considered to be treatment-related were recorded at 240 and 720 mg/kg/day groups, in the male kidneys, the liver (males and females) and the female spleen, as shown (data expressed as percentage):

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlargement of the liver noted in a single male treated at 720 mg/kg/day correlated with the hypertrophy of hepatocytes observed at microscopic examination.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were noted in the kidney of males from 80 mg/kg/day, liver of males and females from 240 mg/kg/day and spleen of males at 720 mg/kg/day and females from 240 mg/kg/day.

Minimal to marked increase in eosinophilic hyaline droplets was noted in the cytoplasm of tubular cells of the proximal convoluted tubules of the kidney of males from 80 mg/kg/day. It was accompanied by minimal to moderate, multifocal tubular degeneration and necrosis of the cortical cells and proximal tubular cells of the outer stripe, mainly at the junction of the outer and inner stripes and by an increase in incidence and severity of tubular basophilia, due to increased tubular regeneration of the proximal convoluted tubules of the outer stripe adjacent to the inner stripe. Affected tubules exhibited single cell necrosis, eosinophilic granular casts, increased mitotic figures and flattened tubular cell basophilia indicating tubular regenerative attempts. No such changes were noted in females at any dose levels. Altogether, these changes indicated hyaline droplet nephropathy of the male rat, an adverse lesion specific to male rats and of no relevance to humans, which is due to an exacerbated production of alpha2µ globulin and increased reabsorption in the proximal convoluted tubules (reviewed in Handbook of Toxicologic Pathology, 1991, page 340-342).

Minimal-to-moderate centrilobular-to-midzonal hepatocellular hypertrophy was noted in the liver of males and females treated from 240 mg/kg/day. Hepatocellular hypertrophy was slightly more pronounced in males than in females and this finding correlated with higher absolute and relative liver weights (see text table 1). Along the main finding of hepatocytes with increased size, there were minimal extramedullary hematopoiesis in males from 240 mg/kg/day and/or minimal increase in the number of hepatocytes in mitoses (males only at 720 mg/kg/day) or minimal greenish pigment-laden Kupffer’s cells (females only). Kupffer’s cells containing greenish pigments were few and scattered within the parenchyma. Extramedullary hematopoiesis was characterized by sparsely scattered islets of erythropoiesis.

Increased extramedullary hematopoiesis was noted in the spleen of males treated at 720 mg/kg/day and females treated from 240 mg/kg/day, along with markedly increased hemosiderosis in females treated at 720 mg/kg/day. In females, these findings correlated with hematological minimal changes and increased absolute and relative spleen weights observed from 240 mg/kg/day.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Immunohistochemistry
- Liver CYP immunohistochemical staining
CYP 1A1/2
Males or females: No significant increase in staining when comparing high-dose and controls.
CYP 2B1/2
Significant increase in staining - from minimal faint centrilobular in controls (all controls with grade 1) to centrilobular/midzonal in high-dose females (all moderate or grade 3) and almost diffuse in high-dose males (all marked or grade 4), leaving only a thin rim of periportal hepatocytes - was noted in females (slight increase) and males (moderate increase).
CYP 3A1/2
Significant minimal increase in staining (from centrilobular staining in controls – minimal to slight, median grade 2 – to moderate to marked in treated males and females at 720 mg/kg/day – median grade 3) was noted in males and females. Females had a minimally higher expression, both in controls and treated animals.
CYP 4A1
Males or females: No significant increase in staining was noted when comparing high dose and controls.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

haematology

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 4: Red blood cell parameters in females

 

Dose-level (mg/kg/day)		0		80		240		720
RBC (T/L)		8.15		8.12		7.53 **		7.29 **
HB (g/dL)		15.9		15.5		14.7 **		14.9 **
PCV (L/L)		0.42		0.41		0.40 **		0.41
MCV (fL)		52		51		52.6		56.2 **
MCH (pg)		19.5		19		19.6		20.5 *
MCHC (g/dL)		37.4		37.3		37.3		36.4 **
*: p<0.05 ; **: p<0.01 RBC: red blood cell count; HB: hemoglobin concentration; PCV: packed cell volume; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration

Table 5: Treatment-related deviation from controls (expressed in %) in the absolute and relative organ weights

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	80	240	720	80	240	720
- Kidneys
. absolute	+3	+15*	+24**	0	+11	+8
. relative	+3	+21**	+24**	+1	+5	+11*
- Liver
. absolute	+3	+9	+39**	+5	+22**	+26**
. relative	+3	+15**	+39**	+6	+15	+29
- Spleen
. absolute	-9	-8	+1%	+16	+50**	+50**
. relative	-10	-3	0	+16	+41**	+52**

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

 

Conclusions:

Hypersalivation was observed at 720 mg/kg/day and minimal disturbances of red blood cell parameters suggesting increased red blood cell turn over were recorded in females given 720 or 240 mg/kg/day.
Treatment-related organ weight changes were recorded in the kidney of males treated from 240 mg/kg/day, the liver of males and females treated from 240 mg/kg/day and the spleen of females treated from 240 mg/kg/day.
Treatment-related lesions were observed in the kidneys of male rats from 80 mg/kg (eosinophilic hyaline droplets with tubular degeneration/necrosis), the liver (hepatocellular hypertrophy) and the spleen (extramedullar hematopoiesis or EMH) at 720 and/or 240 mg/kg/day. None of these morphological changes were considered as adverse. EMH and hemosiderosis was considered to be a compensatory effect, secondary to the test-item related effect on red blood cells.

Based on the experimental conditions of this study, and taking into consideration the treatment related effects on red blood cell parameters, it can be concluded that the dose-level of 80 mg/kg/day was the No Observed Adverse Effect Level (NOAEL).

Executive summary:

The potential toxicity of BISMETHYLTHIOPROPANE (BMTP) was evaluated following daily oral administration (gavage) to rats for 4 weeks. This study was conducted according to OECD guideline No. 407. Three groups of six male and six female Sprague-Dawley rats were administered BMTP, daily by the oral route (gavage) at the dose-level of 80, 240 or 720 mg/kg/day for 4 weeks. An additional group of six males and six females received the vehicle alone (corn oil) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. Chemical analysis of the dosage forms supplied ready for use and stability data were given by the Sponsor. The animals were housed in two in Individually Ventilated Cages (IVC). Clinical signs and mortality were checked respectively once a day. A detailed clinical observation was performed prior to start the dosing and once a week throughout the study. At the end of the treatment period, effects on the central nervous system were evaluated by means of a Functional Observation Battery (FOB), including motor activity testing. Body weight was recorded once before group allocation, on the first day of treatment, then once a week during the treatment period. Food consumption was recorded once a week during the treatment period. Hematological and blood biochemical were performed on all animals prior to the necropsy.

On completion of the treatment period, all surviving animals were sacrificed and subjected to a full macroscopic postmortem examination. Designated organs of were weighed and selected tissue specimens were preserved. Microscopic examination were performed on designated tissues from the control and high-dose group animals sacrificed at the end of the treatment period and on macroscopic lesions, the kidneys, the liver and the spleen from all animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period. Additional immunohistochemistry was carried out on liver and kidneys.

A satisfactory agreement was observed between the theoretical and measured concentrations of BMTP in the formulations since the deviations varied from -8% and +4%. The dosage forms prepared at 16, 48 and 144 mg/mL were stable over 4 weeks. No deaths occurred during the study. No signs of toxicological importance were observed, hypersalivation (recorded as ptyalism), not considered as adverse, was the only sign noted from day 4 among all animals given 720 mg/kg/day. No effects on the central nervous system were evidenced by the FOB tests. Neither mean body weight nor mean body weight change were affected. Food consumption was not affected. When compared to the control mean values, red blood cell parameters recorded in females given 720 or 240 mg/kg/day were decreased (red blood cell count, hemoglobin concentration, pack cell volume, mean cell hemoglobin concentration or increased (mean cell volume, mean cell hemoglobin). These differences were minimal, not always clearly dose-related and the values were still in the range of the historical controls. None of the blood biochemistry parameters were disturbed by BMTP treatment.

There were no relevant differences in organ weights in the 80 mg/kg/day group. High organ weights that were considered to be treatment-related were recorded at 240 and 720 mg/kg/day groups, in the male kidneys, the liver (males and females) and the female spleen, as shown (data expressed as percentage):

Sex	male		female
Dose-level (mg/kg/day)	240	720	240	720
- Kidneys
. absolute	+15*	+24**	+11	+8
. relative	+21**	+24**	+5	+11*
- Liver
. absolute	+9	+39**	+22**	+26**
. relative	+15**	+39**	+15	+29
- Spleen
. absolute	-8	+1%	+50**	+50**
. relative	-3	0	+41**	+52**

*: p<0.05, **: p<0.01.

Enlargement of the liver noted in a single male treated at 720 mg/kg/day correlated with the hypertrophy of hepatocytes observed at microscopic examination. Findings in the kidneys of males from 80 mg/kg/day (eosinophilic hyaline droplets in the cytoplasm of tubular cells of tubules, accompanied by tubular degeneration/necrosis of the cells) were considered to be rat-specific and with no relevance for human risk assessment. In the liver of males and females given 720 and 240 mg/kg/day, hepatocellular hypertrophy was noted along with (males only) increase in extramedullary hematopoiesis. Increased extramedullary hematopoiesis was noted in the spleen of rats treated from 240 mg/kg/day. An increased hemosiderosis was observed in females given the high dose. At immunohistochemistry, the hepatocellular hypertrophy in the liver was associated with increased in P450 enzyme proteins (CYP2B1/2 and CYP3A1/2).

Based on the experimental conditions of this study, and taking into consideration the treatment-related effects on red blood cell parameters, it can be concluded that the dose-level of 80 mg/kg/day was the No Observed Adverse Effect Level (NOAEL).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 March 2012 - 09 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 9 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 347 g (range: 329 g to 367 g) and the females had a mean body weight of 221 g (range: 195 g to 253 g)
- Fasting period before study: no
- Housing: at receipt, the animals were housed by three or five from the same sex in suspended wire-mesh cages. A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
From randomization, the animals were individually housed, except during pairing and lactation, in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast). Individual housing was chosen because of software limitations and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) was provided to females and their litters as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: : the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 22 March 2012 to 17 May 2012.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The Sponsor supplied the solutions prepared at the concentrations of 0 (corn oil), 8, 32 and 128 mg/mL intended for use in study groups 1, 2, 3 and 4, respectively. This preparation was carried out as follows: a total of 25 brown vials of at least 50 mL each, plus one vial of 200-250 mL, were prepared three times during the study (at a 3 week-interval approximately). The 25 vials were received by the labs on 20 March 2012, 11 April 2012 and 02 May 2012 for dosing of animals purposes. The remaining vials of 200-250 mL were kept by the sponsor to determine the concentration.
The density of formulation was not measured for safety precautions.
The dose formulations were received by the labs at room temperature and protected from light. From receipt, the formulation was stored at room temperature and protected from light for up to 3 weeks of use. Each eve of dosing, the volume required for the forthcoming day was stored in a safety cabinet in the animal unit, at room temperature and protected from light.
High precautions were taken to avoid the heating of the solutions.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in corn oil
- Concentration in vehicle: 8, 32 and 128 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: not assessed, dose formulation is a solution
Stability: stable for 4 weeks

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (36 days in total),
In the females:
- 2 weeks before mating,
- during the mating period,
- during pregnancy,
- during lactation until day 5 post-partum inclusive (at least 44 days, up to 58 days),
- until sacrifice for females which had not delivered.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected by the Sponsor, following the results of a previous 4-week toxicity study.
In the study, the test item was given by gavage to Sprague-Dawley rats in corn oil at 80, 240 or 720 mg/kg/day for 4 weeks.
Ptyalism was observed at 720 mg/kg/day and minimal disturbances of red blood cell parameters suggesting increased red blood cell turn over were recorded in females given 720 or 240 mg/kg/day.
Treatment-related organ weight changes were recorded in the kidney of males treated from 240 mg/kg/day, the liver of males and females treated from 240 mg/kg/day and the spleen of females treated from 240 mg/kg/day.
Treatment-related lesions were observed in the kidneys of male rats from 80 mg/kg (eosinophilic hyaline droplets with tubular degeneration/necrosis), the liver (hepatocellular hypertrophy) and the spleen (extramedullar hematopoiesis or EMH) at 720 and/or 240 mg/kg/day. None of these morphological changes were considered as adverse. EMH and hemosiderosis was considered to be a compensatory effect, secondary to the test item-related effect on red blood cells.
Based on the aforementioned results, the high dose-level of 640 mg/kg/day was selected in order to produce signs of toxicity. The two others were selected according to a 4-fold interval.

- Rationale for animal assignment: computerized stratification procedure.

Positive control:

no (not required)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day (mortality) or once a day (clinicals signs) during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule:
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 p.p.
During the pairing period, the food consumption was not measured for males or females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

Sacrifice and pathology:

SACRIFICE
- Male animals: all animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 or 26 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

GROSS NECROPSY
Macroscopic post-mortem examination of principal thoracic and abdominal organs.

HISTOPATHOLOGY
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period , and for the prematurely female (Group 4) with total litter loss,
- all macroscopic lesions of all groups,
- on kidneys (males), spleen (females) and liver (both sexes) from animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed at the end of the treatment period.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS: epididymes, prostate, testes, kidneys, liver and spleen.

Statistics:

Body weight, food consumption and reproductive data
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

Organ weight
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 640 mg/kg/day, the clinical signs, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and abdominal breathing, except ptyalism, were observed during the first 4 days of treatment. They reappeared in females towards the end of the gestation period and generally disappeared in the lactation period. Two females experienced again staggering gait and/or hypoactivity on day 5 p.p.
Although these clinical signs laste 4 days only at the beginning of the treatment period and reappeared in pregnant females only at the end of gestation, they were particularly strong and likely induced in one female an abnormal maternal behaviour leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day.
Hypoactivity and loss of balance were also noted at 160 mg/kg/day for up to 4 or 5 days at the beginning of the treatment period.
As these clinical signs were less severe, they were considered as non adverse at this dose-level.
Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered to be non adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 640 mg/kg/day, one female was sacrificed prematurely on day 3 p.p. because of the death of its litter.
There were no other unscheduled deaths during the study.
A total of five females (1, 2 and 2 given 40, 160 or 640 mg/kg/day, respectively) were sacrificed on day 25 or 26 p.c. for absence of delivery. All these females were non pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean body weight and mean body weight gain.
In absence of any dose relationship, the statistically significant low mean body weight change in week 3 noted at 40 mg/kg/day in males was considered to be fortuitous.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean food consumption.
There was a slighty lower mean food consumption noted at the beginning of the lactation period at 640 mg/kg/day (–11% vs. controls). This finding was considered to be of non toxicological significance (no statistical significance and low amplitude).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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):

Higher mean absolute and relative-to-body liver weights were noted in males and females treated at 640 mg/kg/day.
Higher mean absolute and/or relative-to-body kidney weights were observed in males treated at 160 mg/kg/day and in both sexes at 640 mg/kg/day.
Higher mean absolute and/or relative-to-body spleen weights were seen in females treated at 160 or 640 mg/kg/day.
These organ weight differences were attributed to treatment with the test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Premature decedents
One female treated at 640 mg/kg/day was euthanized on day 40 following the death of its litter. No gross findings were observed.

Terminal sacrifice
Liver and kidney enlargements were observed respectively in 3/10 and 2/10 males treated at 640 mg/kg/day.
Tan discoloration of the kidney was also noted in 3/10 males and liver enlargement in 1/10 females at this dose level.
As these gross findings correlated with higher weights and microscopic findings, they were attributed to treatment with the test item.
Other macroscopic findings were of those commonly reported in the rat and therefore a relationship to treatment was considered to be unlikely.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Premature decent
Minimal centrilobular hepatocellular hypertrophy and moderate extramedullary hematopoiesis in the spleen were noted in the female treated at 640 mg/kg/day.
These microscopic findings were attributed to treatment with the test item.

Terminal sacrifice
Liver
Centrilobular hepatocellular hypertrophy was noted in all males and surviving females treated at 640 mg/kg/day, along with minimal vacuolation in 2/10 males and pigment-laden Kupffer cells in 7/9 females.
Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males treated at 160 mg/kg/day.
In the absence of associated microscopic degenerative changes, this finding is usually seen as an adaptative response and considered not to be adverse.

Kidney
Increased incidence and severity of tubular hyaline droplets were noted in the kidneys of males treated at 160 or 640 mg/kg/day, along with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells).

This correlated with increases in kidney weights and was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets are consistent with a2µ globulin and are known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2007). The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which explains tubular basophilia seen in groups 3 and 4. These findings were therefore considered to be adverse. Although human excretes proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human.

Spleen
Minimally increased extramedullary hematopoiesis was observed in the spleen of females treated at 640 mg/kg/day.

Other microscopic findings were of those commonly reported in the rat and therefore a relationship to treatment was excluded.
More specifically, there were no evidences of effects in the testis or in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 40, 160 and 640 mg/kg/day.
Based on the experimental conditions of this study:
- the dose-level of 160 mg/kg/day was considered to be an Adverse Effect Level in rats as renal hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. However, as renal hyaline droplets are considered to be specific to male rats and therefore not relevant for human, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 160 mg/kg/day (because of the strong clinical signs and the effects on the female spleen seen at 640 mg/kg/day).

Executive summary:

The potential toxic effects of Bismethylthiopropane was evaluated in a study, performed following the OECD guideline No. 421 and the principles of Good Laboratory Practices.Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, daily, by oral (gavage) administration, before pairing and through pairing and, for the females, through gestation until day 5p.p.The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 40, 160 and 640 mg/kg/day. Another group of 10 males and 10 females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The concentration of the dose formulations was checked at each preparation (thus three times during the study) before the administration to the animals.The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Body weight and food consumption were recorded once a week. The males were sacrificed after completion of the pairing period and the dams on day 6p.p.Final body weights and selected organs weights (epididymides, prostate, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (epididymides, ovaries, testes, kidneys, liver, spleen) from the control- and high-dose groups, on kidneys (males), spleen (females) and liver (both sexes) from the low- and intermediate-dose groups and on all macroscopic lesions.

The test item concentrations in the administered dose formulations analyzed at each preparation were within the acceptance criteria (± 10%).

There were no test item-related deaths in parents. At 640 mg/kg/day, hypoactivity, loss of balance, staggering gait, hypotonia, locomotory difficulties and abdominal breathing were observed at the beginning of the treatment period. They reappeared towards the end of gestation and disappeared in the lactation period. All animals of that group were affected by at least one of these clinical signs, females more than males. Although these clinical signs lasted the first 4 days and reappeared at the end of gestation only, they were particularly strong and likely induced in one female an abnormal maternal behaviour leading to cannibalism of its entire litter. They were therefore considered as adverse at 640 mg/kg/day. At 160 mg/kg/day, there were also hypoactivity and mainly loss of balance in males and especially in females at the beginning of the treatment period only. These clinical signs were considered as non adverse at this dose-level as less severe. Ptyalism was recorded in a dose-related manner from 40 mg/kg/day in males or at 640 mg/kg/day in females and was considered not to be adverse. There were no test item-related effects on mean body weight and on mean food consumption at any dose-level. At necropsy, there were higher mean kidney and liver weights in both sexes at 640 mg/kg/day and higher mean kidney weights in males at 160 mg/kg/day. There were also higher mean spleen weights in females at 160 and 640 mg/kg/day. Liver and kidneys were enlarged in three and two animals respectively at 640 mg/kg/day and tan discoloration of the kidney was also noted in three males. At microscopic examination at 640 mg/kg/day, there were centrilobular hepatocellular hypertrophy in all males and females, along with minimal vacuolation in two males and pigment-laden Küpffer cells in seven females. Moderate to marked tubular hyaline droplets were noted in the kidneys of all males, with foci of tubular basophilia (minimal to slight; occasionally with degenerated cells). At this dose-level, there was also minimally increased extramedullary hematopoiesis in the spleen of females.At 160 mg/kg/day, higher incidence and severity of tubular hyaline droplets were noted in the kidney of males, along with foci of tubular basophilia. Minimal centrilobular hepatocellular hypertrophy was also noted in 4/10 males.  In the absence of associated microscopic degenerative changes, the liver changes are usually seen as an adaptative response and considered not to be adverse. In addition, the adverse kidney effects in the male rats were considered to be rat-specific and with no relevance for human risk assessment.

In conclusion, bis methylthiopropanewas administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of40, 160 and 640mg/kg/day.

Based on the experimental conditions of this study:

. the dose-level of 160 mg/kg/day was considered to be an Adverse Effect Level in rats as renal hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. However, as renal hyaline droplets are considered to be specific to male rats and therefore not relevant for human, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 160 mg/kg/day (because of the strong clinical signs and the effects on the female spleen seen at 640 mg/kg/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline studies

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

Justification for classification or non-classification

None of the CLP criteria which trigger the STOT-RE classification is met with BMTP. The effects observed on the liver, spleen and kidneys are either an adaptive response or of no relevance for human risk assessment. There is no significant toxic effect supporting classification in the classification concentration range of 10-100 mg/kg/d based on the subchronic oral study. Therefore, STOT-RE classification is not warranted for BMTP.