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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7th June 1999 - 11th August 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
the part on neurobehavioural testing and neuropathology, which had been drafted according to OECD Guideline for the Testing of Chemicals 424 (adopted 21 July 1997)
Principles of method if other than guideline:
Some deivations from the study protocol occurred. None of these were felt to affect the validity of the study.

— All animals were weighed twice instead of once prior to the start of the study;
— Male animals were housed in macrolon cages type 4 and females animals were housed in macrolon cages type 3.5, instead of in macrolon cages type 3;
— The spinal ganglion L4 and Gasserian ganglion were dissected completely, instead of in parts and the Gasserian ganglion was disected longitudinally instead of transversally;
— M.H.K. Bos-Kuijpers, DVM, PhD and M.V.W. Wijnands, DVM were responsible for pathology instead of R.A. Woutersen PhD;
— The relative humidity in the animal room was generally higher than 70% for daily periods of 2 h to 24 h, throughout the study;
— Erroneously, the animals of the satellite 15 ppm dose-group and the satellite controls were also ophthamoscopically examined towards the end of the study;
— The fixed tissues obtained from the animals from the satellite 200 ppm dose group (group E) were stored at 2-10°C in monthly-refreshed phosphate-buffer (pH 7.2-7.4; containing 0.02% sodium azide) prior to processing for microscopy;
— Instead of ranking the morphological status of the cerebrum at each of the 4 protocol-levels separately, i.e. 1) telencephalon, 2) di-encephalon, 3) transition di- and mesencephalon and 4) mesencephalon) the results of the 4 levels were taken together and presented in the tables and appendices as 'cerebrum' to increase the precision of the ranking system on localisation, intensity and severity of the brain lesions;
— The dorsal and ventral roots at L4 were dissected, embedded and examined microscopically as indicated in the protocol. If present, the roots included in the spinal cord sections (at lumbal, thoracal and cervical levels) were examined microscopically as well. Primarily, the results of the roots attached to the lumbal spinal cord were used to complete the neuropathology data, if necessary. If not present in the section, the results of the roots attached to the thoracal or cervical spinal cord were used;
— One animal of the high-dose (200 ppm) group (no. E141) was found dead on day 27. Protocol tissues of this animal were fixed by immersion-fixation and were embedded. Sections were cut of the blocs. However, the results of the microscopical examination are not included in the report. They are, however, kept as raw data;
— In addition to the protocol, clinical signs were observed for the animals of the neuropathology subgroups, prior to perfusion fixation;
— Measurements or microscopic examinations could not always be performed on
all animals specified in the protocol; the reasons are given in the Appendices.
— The Department of Pesticides and Industrial Toxicology was renamed Department of Residue Analysis.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): MMTTC/DMDTC
- Storage condition of test material: 2-10ºC

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks old; pre-test neurobehavioural testing was to be conducted on animals of 5 weeks of age.
- Weight at study initiation: 149.8 g to 191.5 g (mean 174.3 g) for males and from 123.4 g to 154.2 g (mean 137.7 g) for females
- Housing: under conventional conditions in one room, in macrolon cages, type 3.5 (males) or type 4 (females) with sterilized wood shavings (Woody Clean, Type 3/4) as bedding material, five (main groups) or three (satellite groups) rats per cage, separated by sex. In the last week of the treatment period, the rats of the main groups were placed in stainless-steel metabolism cages (one rat per cage) for 16 hours to collect urine.
- Diet (e.g. ad libitum): Rat & Mouse No. 3 Breeding Diet, RM3 ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): At least 30%. The relative humidity exceeded 70% on most days of the study during 2 to 24 hours per day, to a maximum of 95%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

IN-LIFE DATES: From: 7th June 1999 To: 20th September 1999

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The test substance was incorporated in the basal diet by mixing in a mechanical blender. Four batches of test diets were prepared, viz. on 31 May, 28 June, 20 July and 20 August 1999. A 10,000 ppm premix was prepared first. The high-dose (200 ppm) diet was prepared by diluting the premix with the appropriate amount of RM3 diet. Subsequently, the 6 and 15 ppm diets were prepared by diluting the high-dose diet with the appropriate amount of RM3 diet. The 1 ppm diet was prepared by diluting the 15 ppm diet with the appropriate amount of RM3 diet. The controls received the unsupplemented RM3 diet. Directly after preparation the diets were stored in a freezer (<-18°C) until use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The diets were extracted with a methanol/water solution. The organic tin chlorides were ethylated with sodium tetraethyl borate (NaBEt4). The methyl ethyl tin compounds were subsequently extracted with hexane. The concentrations of MMTTC and DMDTC were determined using a Carlo Erba/Fisons QMD-1000 GC/MS under the following conditions:

Column: fused silica 60 m x 0.32 mm; 0.25 µm DB-1
Column temperature: After 3 minutes at 50°C at a rate of 5°/min. to 140°C; then at a rate of 10°/min. to 260°C; 10 minutes at 260°C
Carrier: Helium (70 kPa)
Injection volume: 1 µl
Injection temperature: 250°C
Injection method: splitless
Ionisation: Electron Impact, 70 eV
Mass range: 100 - 250 amu
Mass fragments used DMTDC low concentrations: m/z = 179
for quantification: DMTDC high concentrations: m/z = 193
MMTTC low concentrations: m/z = 193
MMTTC high concentrations: m/z = 205
TMTMC: m/z = 165
Internal standard (naphthalene) m/z = 102

Homogeneous distribution of the test substance in rat feed was demonstrated by using the same diet preparation protocol and concentrations that were used in the present study. Moreover, the stability of the test substance in rat feed under simulated experimental conditions (7 days at room temperature in an open container) and after 5 weeks in a freezer was demonstrated.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 6, 15 and 200 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 per sex per dose group (main study)
6 per sex per dose group (satellite groups)
Control animals:
yes, plain diet
Details on study design:
The study comprised five main groups of 10 males and 10 females each and five satellite groups (neuropathology study) of 6 males and 6 females each. One control group receiving unsupplemented basal diet and four test groups receiving diets containing different levels of the test substance for 13 weeks. These groups are intended to provide information on the subchronic oral toxicity of the test substance and to establish a no-observed-adverse-effect level (NOAEL).

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded. Any animal showing signs of severe debility or intoxication, particularly if death appeared imminent, was killed by exsanguination or by whole body perfusion fixation (satellite animals used for neuropathology), under ether anaesthesia to prevent loss of tissues by cannibalism or autolytic degeneration.

BODY WEIGHT:
The body weight of each animal was recorded twice during the acclimatization period, at initiation of treatment and once weekly thereafter. In addition, the animals were weighed on the day of scheduled necropsy (terminal body weight, for calculation of the organ to body weight ratios).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured per cage over weekly periods by weighing the feeders. The results were expressed in g per animal per day.
The intake of the test substance per kg body weight per day was calculated from the nominal concentration of the test substance in the diet, the food consumption and the body weight (mean of the body weights measured at the start and at the end of each week).

FOOD EFFICIENCY:
The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

WATER CONSUMPTION:
Water intake was measured per cage by weighing the bottles. The results were expressed in g per animal per day. The consumption was measured daily during four days in week 1, 6 and 12 for rats of the main groups only.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of the study in all rats of the main groups and towards the end of the treatment period in all rats of the main and satellite control group and the main and satellite 15 ppm dose-group. Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY:
At necropsy at the end of treatment, blood samples were taken from the abdominal aorta of all surviving rats of the main groups whilst under ether anaesthesia. K2-EDTA was used as anticoagulant.

The following parameters were examined: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count.

The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
Clinical chemistry determinations were made on all surviving rats of the main groups. Fasting glucose was determined shortly before the end of the treatment period (on nominal day 88) in blood collected from the tip of the tail after deprivation of food for 16 hours. The collection of fasting blood was combined with the collection of urine (see next section). At necropsy at the end of treatment, blood was collected from the abdominal aorta in heparinized plastic tubes and plasma was prepared by centrifugation.

The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT) , alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, albumin/globulin ratio, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate.

URINALYSIS:
In the urine samples collected from the rats in the renal concentration test, the following determinations were carried out in individual samples: appearance, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen and microscopy of the sediment.

RENAL CONCENTRATION TEST:
On nominal days 87 and 88, all surviving rats of the main groups were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were kept in metabolism cages (one rat per cage) and urine was collected. The concentrating ability of the kidneys was investigated by measuring the urinary volume and density of the individual samples.

NEUROBEHAVIOURAL EXAMINATION:
Before the start of the study and in weeks 1, 4, 8 and 13, spontaneous motor activity measurements and Functional Observational Battery (FOB) tests were performed in four animals of both sexes of each main group (two animals with the lowest identification numbers from each cage) and in all animals of each satellite group.
Sacrifice and pathology:
GROSS PATHOLOGY:
Based on the severe clinical signs and mortality observed in the high-dose (200 ppm) group, it was decided to sacrifice the main and satellite high-dose groups after approximately one month of treatment. The surviving animals of the main and satellite high-dose groups were considered moribund and were killed by exsanguination from the abdominal aorta under ether anaesthesia or by whole body perfusion fixation, under ether anaesthesia, respectively.

After completion of the 13 week treatment period, all surviving animals of the main groups were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia and then examined macroscopically for pathological changes. A thorough autopsy was also performed on all animals that died intercurrently or that had to be killed because they were moribund.

The following organs of all surviving animals of the main groups were weighed: adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid (with parathyroids), liver and uterus.

Samples of the following tissues and organs of all animals of the main groups were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin): adrenals, aorta, * axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, epididymides, * eyes, * exorbital lachrymal glands, * femur with joint, heart, kidneys, liver, lungs, mammary glands (females), *mandibular (cervical) lymph nodes, mesenteric lymph nodes, nerve-peripheral (sciatic nerve), oesophagus, ovaries, pancreas, parathyroids, parotid salivary glands, pituitary, prostate, rectum, * seminal vesicles with *coagulating glands, * skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (three levels), spleen, sternum with bone marrow, stomach (glandular, non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchii, urinary bladder, uterus (with cervix), * vagina and all gross lesions.

* The tissues marked with an asterisk were preserved but not processed for histopathological examination, unless histopathological examination was considered necessary on the basis of the results of gross observations.

HISTOPATHOLOGY:
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed on all tissues and organs listed above of all animals of the main control group and the main high-dose (200 ppm) group and of all animals that died during the study. Based on the clinical findings in the high-dose animals, the premature termination of the high-dose group and after consultation with the sponsor, it was decided to extend the histopathology to the main 15 ppm dose-group. Gross lesions were examined microscopically in all rats of all main groups.
Other examinations:
NEUROPATHOLOGY:
The animals of the satellite high-dose (200 ppm) group were sacrificed on 7, 8 and 9 July 1999 by whole body perfusion fixation, under ether anaesthesia. Based on the neurotoxic findings in the high-dose animals, the premature termination of the high-dose group and after consultation with the sponsor, it was decided to extend the neuropathology to the satellite 15 ppm dose-group.

The surviving animals of the satellite groups were killed on 13 to 17 September 1999. They were subjected to whole body perfusion fixation, under ether anaesthesia, with buffered glutaraldehyde/formaldehyde mixture (pH 7.2 - 7.4; 12 minutes; pressure: 130 mm Hg), preceded by saline solution (approximately 30 seconds; pressure: 130 mm Hg). This was done in such a sequence that the average time of killing was approximately the same for each group. After perfusion, relevant tissues were taken from the skull (the brain), the spinal column (spinal cord with roots and spinal ganglia) and the left hind limb (the sciatic nerve, the tibial nerve, the sural nerve and the plantar nerve). These tissues were stored overnight in the fixative. The remaining tissues and the intact right hind limb were also stored in the fixative. Subsequently, the tissue were rinsed in phosphate-buffer (pH 7.2-7.4) for a period of at least 48 hours at 2-10°C.

For embedding in paraffin, the following tissues from the satellite control and the 15 and 200 ppm groups were further dissected:
— transversal slabs from the cerebrum:
— telencephalon
— diencephalon
— transition zone diencephalon/mesencephalon
— transversal slab from mesencephalon (pons cerebri)
— transversal slab from cerebellum and medulla oblongata
— transversal slab from medulla oblongata

These tissues were dehydrated in ethanol and embedded in paraffin. Sections (2-5 µm thick) were cut from these blocks and sections were stained with either haematoxylin and eosin (H&E). Sections were examined by light microscopy.

For embedding in epoxy resin, the following tissues from the satellite control and the 15 and 200 ppm groups were further dissected:
— transversal slabs from spinal cord:
— the cervical spinal cord
— the thoracic spinal cord
— the lumbar spinal cord
— the dorsal spinal nerve root at the level of L4
— the ventral spinal nerve root at the level of L4
— the spinal ganglion at the level of L4
— Gasserian ganglion

— transversal slabs from nerve tissue from left hind limb:
— the sciatic nerve
— the tibial nerve
— the sural nerve
— the plantar nerve

These tissues were post-fixed with osmiumtetroxide and were dehydrated in a series of increasing ethanol solutions, followed by propylene oxide before embedding in epoxy resin.

Semi-thin sections were cut from these blocks. The semi-thin sections were stained with toluidine blue and were examined light-microscopically. All epoxy-resin embedded tissues of the perfusion-fixed animals of the satellite control group and the satellite 15 and 200 ppm groups were examined.

The fixed tissues obtained from the animals from the satellite 200 ppm dose group were stored at 2-10°C in monthly-refreshed phosphate-buffer (pH 7.2- 7.4; containing 0.02% sodium azide) prior to processing for microscopy. The fixed tissues obtained from the animals from the satellite 1 and 6 ppm dose groups were stored at 2-10°C in monthly-refreshed phosphate-buffer (pH 7.2-7.4; containing 0.02% sodium azide; awaiting possible processing for neuropathology) and were archived in buffered formalin after completion of the study. During storage, the fixed tissues in phosphate-buffer were visually inspected weekly for contamination.
Statistics:
The statistical procedures used in the evaluation of data were as follows:

- body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests;
- water intake: one-way analysis of variance (Anova) followed by least significant difference tests;
- food consumption/efficiency, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, urinary volume and density, and organ weights: one-way Anova followed by Dunnett's multiple comparison tests;
- relative differential white blood cell counts, and semi-quantitative urinary determinations: Kruskal-Wallis non-parametric Anova followed by Mann-Whitney U-tests;
- histopathological changes: Fisher's exact probability test.

Probability values of p<0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Three females of the high-dose (200 ppm) group died towards the end of the first month of treatment. Almost simultaneously, most of the females and a number of males of the high-dose group started to show severe neurological signs, including tremors and convulsions. Based on the death of three females and the severe nature of the clinical signs, all animals of the high-dose group were considered moribund and killed for humane reasons.

The daily observations did not reveal treatment-related clinical signs among the animals of the other groups. The few signs that were observed in these animals are common findings in Wistar rats and were randomly distributed among the remaining groups.

BODY WEIGHT AND WEIGHT GAIN
Generally, from day 7 to day 28 the body weight of the animals of the high-dose (200 ppm) group was lower than that of the controls, except on day 14 for male rats. On days 7 and 28 the difference reached the level of statistical significance for male rats.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was statistically significantly decreased in high-dose (200 ppm) animals on day 7 (males and females) and increased on day 28 (females). Food consumption was similar in the other groups throughout the study.

Due to the well-known decrease in food consumption per kg body weight with increasing age of rats, the intake of test substance per kg body weight decreased in the course of the study. The mean intakes of MMTTC/DMTDC in the groups receiving 1, 6, 15 or 200 mg per kg diet were 0.06, 0.39, 0.98 and 16.81 mg/kg bw/day in males and 0.07, 0.41, 1.02 and 17.31 mg/kg bw/day in females, respectively.

FOOD EFFICIENCY
Food conversion efficiency values tended to be decreased in high-dose animals on days 21 (males) and 28 (males and females). This decrease was statistically significant in high-dose males on day 21. Food conversion efficiency values were similar in the other groups throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption was generally similar among the groups throughout the study. The water consumption was statistically significantly decreased in females dosed with 6 ppm or with 200 ppm on day 1 only.

NEUROBEHAVIOUR
In the first week of administration of the test substance, no effects of treatment were observed. In week 4 of treatment, increased activity was observed in females of the high-dose (200 ppm) group, together with increased rearing in the open field. Also convulsive activity was observed in a number of females from this group. These effects were not observed in males at this test time point. In males, increased landing footsplay was measured in the 200 ppm group. These effects were considered treatment-related and toxicologically relevant. Shortly after week 4, the high-dose (200 ppm) animals were excluded from the study, so no behavioural observations were recorded at later test time points for this group.
In weeks 8 and 13, no toxicologically relevant effects were observed.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmoscopic examination of rats of the 15 ppm dose-group and the controls in week 13 of the study did not reveal any treatment-related ocular changes.

HAEMATOLOGY
No statistically significant changes in red blood cell and coagulation variables or total and differential white blood cell counts were observed.

CLINICAL CHEMISTRY
Clinical chemistry values showed statistically significantly increased alanine amino transferase and aspartate amino transferase activities in males of the 1 ppm dose-group. In the absence of similar changes at higher dose -levels, the increases at the 1 ppm level are considered fortuitous findings.
No other statistically significant changes in clinical chemistry values were observed between treated rats and controls.

URINALYSIS AND RENAL CONCENTRATION
The urinary density was statistically significantly increased in females of the 6 ppm dose-group. In the absence of a similar change at the next higher dose-level, this is considered a fortuitous finding. No other statistically significant changes were observed in urinary volume or density, semi-quantitative urinary observations or microscopy of the sediment.

GROSS PATHOLOGY
Some high-dose animals demonstrated gross skin changes, which, at least in part, were probably related to treatment with the test material. Apart from that, no treatment-related gross changes were observed at autopsy. No treatment-related changes were observed in absolute or relative organ weights

HISTOPATHOLOGY: NON-NEOPLASTIC
At microscopic examination treatment-related histopathological changes were observed in the brain, the kidneys and the thymus of the animals treated with 200 ppm of the test substance. The histopathological changes in the brain comprised cell death, especially in the Ammon's horn, the dentate gyrus and the piriform cortex, and submeningeal oedema. In females, the extensiveness of the cell death was slightly higher than in males. The kidneys demonstrated treatment-related tubular dilatation in the cortical area. The tubules involved were the last parts of the distal tubules and the first part of the collecting tubules. In general, the tubular dilatation was not accompanied by degenerative or inflammatory changes. In one control and some treated females very slight to slight nephrocalcinosis in the kidneys was found. Nephrocalcinosis is a common finding in female rats. The distribution of 1 in the control group, 0 in the 1 ppm group, 4 in the 15 ppm group and 4 in the 200 ppm group was not considered to be an indication of a relationship with treatment but a fortuitous finding. Two control and six 15 ppm females showed minimal mineralisation in the ovaries. The difference in incidence in these groups was not statistically significant. Mineralisation in ovaries of 200 ppm females was not observed. However, those animals were exposed to the test substance for only one month. Speculations with respect to a possible incidence of this change after 3 months exposure cannot be founded. Therefore, the mineralisation in the ovaries was not considered to be treatment-related. In females treated with 200 ppm an increased incidence of corticomedullary haemorrhage in the thymus was observed. Most of the high-dose females also showed cortical lymphoid depletion in the thymus. This was not considered a direct toxic effect of the test substance, but an indirect effect caused by stress, due to the severe effects of the test material. All females and males treated with 200 ppm showed decreased accumulation of brown pigment in the spleen. Accumulation of brown pigment is a normal phenomenon in rats, which gradually increases with age. The 200 ppm animals were killed at an early stage of the study, at which they had not yet accumulated pigment in the spleen. Therefore, the decreased splenic pigment was not an effect of the treatment, but was the consequence of the difference in age between these animals and those of the other treatment groups and the controls, that were killed two months later. The aforementioned treatment-related histopathological changes were not observed in dose-group treated with 15 ppm. It was concluded that, based on the microscopic examination, 15 ppm was the no observed adverse effect level under the given conditions. Therefore, the animals of
the lower dose groups were not examined microscopically.

NEUROPATHOLOGY:
Clinical signs:
One animal of the high-dose (200 ppm) subgroup for neuropathology was found dead on day 27. The other animals of this high-dose subgroup were killed moribund on day 31. Convulsions, blepharospasm and tremors were observed in these animals prior to whole body perfusion fixation. One animal showed hypothermia and four animals showed a hunched posture.

Inspection of the remaining high-dose animals, the control animals and animals of the 15 ppm subgroup, assigned for neuropathological examination did not reveal treatment-related changes prior to fixation.

Macroscopic observations:
No treatment-related gross changes were observed at necropsy.

Microscopic observations:
Microscopic examination of the cerebrum of the rats of the high dose (200 ppm) group showed pronounced neuronal death in a number of areas. This effect was most pronounced in female rats. Predominantly affected in both sexes were the hippocampal pyramidal neurons in the CA2/CA1 area and the CA4 neurons, embraced by the polymorphous layer of the dentate gyrus. Strikingly, neuronal death was not observed in the hippocampal CA3 area. The dying neurons typically showed karyorhexis and karyolysis and intense eosinophilic cytoplasm. Little or no glial reaction was observed. Occasionally, the surrounding neuropil showed very slight edema. Also the granular neurons of the dentate gyrus were locally affected (mainly in female rats), although the extent of damage was far less compared to the CA 1/2 and CA4 pyramidal cells. In addition, neuronal death was observed in the (pre- / para-)subiculum. Other predominantly lesioned areas in the cerebrum were the piriform and entorhinal cortices and the amygdala. The adjacent perirhinal cortex showed dying neurons as well.

In the fore-brain (telencephalon) the tenia tecta and adjacent area of the olfactory nuclei were affected, as was the frontal part of the piriform cortex.

In general, the morphological status of the neocortex was good, particularly in the male rats. In female rats, the results of the 4 brain levels, taken together, indicated that incidentally a single or just a few dying neurons were observed in the orbital cortex (one animal), the insular, retrosplenial and temporal cortices (five animals), the parietal cortex (three animals), the occipital cortex (two animals) and frontal cortex (one animal). These dying neurons, which showed a pathological appearance as described for those in the predominantly lesioned areas, were located mainly in those parts of these cortical regions neighbouring one of the severely affected predilection areas. In the male rats single cell necrosis of in the neocortex was observed only in the cingulate cortex of one animal and in the orbital, cingulate and parietal cortices of another animal.

One animal showed a slight dilatation of the lateral ventricle, a phenomenon which was observed to a lesser extent in another animal.

Microscopic examination of other protocol organs for neuropathology revealed neuronal micro-vacuolization in both the spinal ganglion (L4) and Gasserian ganglion in the control and in both dose groups. Neuronal microvacuolization may be induced by processes like hypoxia or ischemia but also by certain toxins. In.this study, neuronal micro-vacuolization was observed in the lumbal spinal ganglion in 4/5 females and 6/6 males of the high-dose (200 ppm) group, in 6/6 females and 6/6 males of the 15 ppm group, in 6/6 females and 6/6 males of the control group, and in the Gasserian ganglion in 2/4 females and 5/6 males of the high-dose group, in 4/6 females and 3/6 males of the 15 ppm group, in 4/5 females and 4/6 males of the control group. In short, no dose-response relationship was found and, therefore, the observed neuronal micro-vacuolization was not considered to be treatment-related.

Incidentally, degenerating axons (1 to 3) were observed in peripheral nerves, and dorsal and ventral roots of animals of the high-dose (200 ppm) group, but in animals of the other groups as well. This incidental axonal degeneration was not considered treatment-related, because the finding was within the range of the normal background pathology for rats of this strain and age.

Also in the spinal cord (3 levels) degenerating axons were present. In addition, small numbers of apparent 'swollen axons' (i.e. the axon is retracted from the relatively thinned and widened myelin sheet), were observed in animals of all groups. These swollen axons were sparsely distributed over the white matter with minor preference for the ventral part of the spinal cord. In the lumbal spinal cord their number was slightly higher in the high-dose (200 ppm) group (in 4/5 females and 4/6 males of the 200 ppm group, in 1/6 females and 0/6 males of the 15 ppm group, in 1/6 females and 2/6 males of the control group). If at all treatment-related, these effects were very mild.

Summarizing, the results of the neuropathology showed treatment-related neuronal death in a number of predilection areas in the tel-and mesencephalon. Predominantly lesioned areas in the mesencephalon were the hippocampal region in broader sense (i.e. including subiculum and dentate gyrus), the piriform, entorhinal and perirhinal cortices and amygdala. In the telencephalon, neuronal death was present in the olfactory nuclei and tenia tecta. Neuronal death was observed in both sexes but was most pronounced in female rats and was, for both sexes, restricted to animals of the high-dose (200 ppm) group.

In the spinal cord, incidental swollen axons were observed in the white matter and their number was slightly increased in animals of the high-dose (200 ppm) group. This effect, if at all treatment related, was very mild. No treatment -related effects were observed in the peripheral nerves, spinal roots and spinal ganglia.

It was concluded that treatment of rats with 200 ppm MMTTC/DMTDC (30/70) induced marked neural lesions in a number of areas in the cerebrum, whereas no neuropathology was found in the 15 ppm MMTTC/DMTDC (30/70) group and in the control group.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
0.98 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Neurological effects.
Dose descriptor:
NOAEL
Effect level:
1.02 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Neurological effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The achieved concentrations in the present study were close to the nominal levels and were all in accordance with the criteria for content of the test substance (the acceptable maximum mean concentration).

Applicant's summary and conclusion

Conclusions:
On the basis of the neurotoxic effects observed in the high-dose group the No-Observed-Adverse-Effect Level in the present study was placed at 15 ppm. This level was equivalent to 0.98 (males) and 1.02 (females) mg of MMTTC/DMTDC (30/70) per kg body weight per day.
Executive summary:

The objective of this study was to examine the possible sub-chronic oral toxicity of the test substance MMTTC/DMTDC (30/70). The test substance was administered to main groups of 10 male and 10 female Wistar rats and satellite groups (for neuropathology) of 6 male and 6 female Wistar rats at constant concentrations of 0, 1, 6 or 15 ppm in the diet for 13 consecutive weeks or at a constant concentration of 200 ppm for approximately one month.

After about one month of treatment, neurotoxic effects of the test substance became evident at the highest dose-level. Three high-dose females died and a large number of high-dose animals of both sexes started to develop signs of neurotoxicity, including tremors and convulsions. On account hereof, all animals of the main and satellite high-dose groups were sacrificed after approximately one month of treatment. High-dose (200 ppm) males and females showed slight growth retardation, which was associated with decreased food intake during the first week and with slightly lower food conversion efficiency in weeks three and four. Body weight, food consumption or food conversion efficiency were not affected by the test substance at the lower dose levels. Neurobehavioural and motor activity testing showed clear treatment-related neurotoxic effects in animals of the 200 ppm group in week 4 of the study. In week 1 of the study these effects were not yet observed, which is in accordance with the onset of the neurological clinical signs in the 200 ppm group after about 4 weeks. No toxicologically relevant effects were observed in any of the other dose-groups, at any of the other time-points investigated. Ophthalmoscopy, haematology, clinical chemistry, urinalysis or absolute and relative organ weights did not reveal any treatment-related changes after dietary exposure to concentrations up to 15 ppm for 13 weeks. These parameters were not measured for animals of the 200 ppm group, because of the premature termination of this group. Microscopic examinations (general pathology) demonstrated clear treatment-related changes in the kidneys, thymus and brain of animals of the 200 ppm group. The effect on the thymus was not considered a direct toxic effect, but an effect caused by stress. The effects on the kidneys and brain were considered toxicologically relevant. None of the effects was present in the next lower dose-group treated with 15 ppm. Microscopic examinations (neuropathology) demonstrated treatment-related pathological changes in several parts of the brain (neuronal death) of animals of the 200 ppm group. No significant treatment-related changes were observed in the peripheral nervous system. The effects occurred in both sexes, but were most pronounced in female rats.

On the basis of the neurotoxic effects observed in the high-dose group the No-Observed-Adverse-Effect Level in the present study was placed at 15 ppm. This level was equivalent to 0.98 (males) and 1.02 (females) mg of MMTTC/DMTDC (30/70) per kg body weight per day.