2,2-bis(methylthio)propane

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-900-1

EC Name:

-

Cas Number:

6156-18-9

Molecular formula:

C5 H12 S2

IUPAC Name:

2,2-bis(methylthio)propane

Test material form:

liquid

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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.

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.