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Administrative data

Description of key information

A well conducted 28-day and 90-day oral toxicity study were available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2007 - January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to guideline
Guideline:
other: Japanese MHLW, METI, ME
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals were housed in a controlled environment, in which optimal
conditions were considered to be approximately 15 air changes per
hour, a temperature of 21.0 ± 3.0°C (actual range: 19.7 - 23.3°C), a
relative humidity of 30-70% (actual range: 26 - 92%) and 12 hours
artificial fluorescent light and 12 hours darkness per day.
Temporary fluctuations from the light/dark cycle (with a maximum of
1 hour) occurred due to performance of functional observations in
the room. Based on laboratory historical data, these fluctuations
were considered not to have affected the study integrity.

Group housing of 5 animals per sex· in Macrolon cages (MIV type,
height 18 cm; during overnight activity monitoring individual housing
in Mill type; height 15 cm.) with sterilised sawdust as bedding
material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cageenrichment
(Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd),
Surrey, United Kingdom). No cage-enrichment was provided during
overnight activity monitoring.
Certificates of analysis were examined and then retained in the
NOTOX archives. Acclimatisation period was at least 5 days before
start of treatment under laboratory conditions.

Free access to pelleted rodent diet (SM RlM-Z from SSNIFF®
Spezialdiaten GmbH, Soes~ Germany).
Results of analyses for nutrients and contaminants of each batch
were examined and archived.

Free access to tap water. Certificates of analysis (performed
quarterly) were examined and archived.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
Once daily, 7 days per week, approximately the same time each
day with a maximum of 4 hours difference between the earliest and
latest dose.
2 mL./kg body weight.
Actual dose volumes were calculated weekly according to the
latest body weight.
At least 13 weeks. Animals were dosed up to the day prior to
necropsy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-MS
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 50, 150 and 450 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
No.
Observations and examinations performed and frequency:
Mortality / Viability: At least twice daily.
At least once daily, detailed clinical observations were made in all
animals. Once prior to start of treatment and at weekly intervals,
this was also performed outside the home cage in a standard
arena. The time of onset, degree and duration were recorded.
All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe
During week 12-13 of treatment, the following tests were
performed on all animals: (abbreviations mentioned in the
respective tables indicated between brackets):
hearing ability (HEARING), pupillary reflex (PUPIL L/R), static
righting reflex (STATIC R) and grip strength (GRIP) (Score 0
= normal/present, score 1 = abnormal/absent).
motor activity test (recording period: 12 hours during
overnight for individual animals, using a computerised
monitoring system, Pearson Technical Services, Debenham,
Stowmarket, England).
Following instillation of tropicamide solution (5 mg/mL, THEA
Pharma, Ukkel, Belgium) both eyes were examined by means of
an ophthalmoscope (Heine Beta 200):
at pretest : All animals
at week 13 : All animals
Body weights: Weekly.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no
quantitative investigation introduced as no effect was suspected.

Blood samples were collected from all animals under iso-flurane anaesthesia (Abbott
Laboratories Ltd., Hoofddorp, The Netherlands) immediately prior to scheduled post mortem
examination at the end of the treatment period, between 7.00 and 10.30 a.m .. The animals were
fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided.
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner BioOne,
Bad Haller, Austria) prepared with EDTA for haematological parameters (0.5 mL), with
citrate for cloWng tests (0.9 mL) and Li-heparin treated tubes for clinical biochemistry
parameters (0.5 mL).
Sacrifice and pathology:
Necropsy
All animals were deeply anaesthetised using iso-fiurane vapour (Abbott Laboratories Ltd.,
Hoofddorp, The Netherlands) and subsequently exsanguinated. All animals assigned to the
study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of
tissues and organs were collected from all animals at necropsy and fixed in 10%
buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The
Netherlands).

The organ weights and terminal body weight were recorded from the surviving animals
on the scheduled day of necropsy.

The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all gross lesions.
Based on (possible) treatment-related changes in liver and thyroid the histological examination
was extended to those particular organs of all animals of groups 2 and 3 (males and/or
females). All abnormalities were described and included in the report. An attempt was made to
correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test' (many-to-one
t-test) based on a pooled variance estimate was applied for the comparison of the
treated groups and the control groups for each sex.
The Steel-test' (many-to-one rank test) was applied when the data could not be assumed to
follow a normal distribution.
- The exact FiSher-test' was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated for
discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances. Individual values, means and standard deviations
may have been rounded off before printing. Therefore, two groups may display the same printed
means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A number of haematological changes were present in the blood at primarily 450 mg/kg/day that suggests an altered red blood cell turn over.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
enlarged liver was noted in most animals at 450 mg/kg/day and in one male at 150 mg/kg/day; reduced thymus weights of females at 450 mg/kg/day, and for the slightly increased spleen to body weight ratio in males at 450 mg/kg/day
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
enlarged liver was noted in most animals at 450 mg/kg/day and in one male at 150 mg/kg/day;low incidence of bile duct hyperplasia was noted in two males and three females 450 mg/kg/day, and in a single male at 150 mg/kg/day
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver and thyroid hypertrophy
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes in liver, kidneys and blood
Critical effects observed:
not specified

Formulation analyses confirmed that formulations of test substance in Arachid oil were prepared accurately and homogenously, and were stable over at least 5 hours.

Treatment did not result in any toxicologically significant changes during the in-life phase (I.e. clinical signs, body weights, food intake, ophthalmoscopy and functional observations).

At necropsy, an enlarged liver was noted in most animals at 450 mg/kg/day and in one male at 150 mg/kg/day. This change was accompanied by increased liver weights (absolute and/or relative) in males and females at 150 and 450 mg/kg/day (absolute liver weights were increased with approximately 33 and 65% towards controlleve[s for males and females respectively), and correlated to centrilobular (periacinar) hepatocytic hypertrophy as seen histopathologically in most animals at 150 and 450 mg/kg/day, and in a single male at 50 mg/kg/day. Centrilobular (periacinar) hepatocytic hypertrophy in the liver is commonly seen as an adaptive response associated with the metabolism of xenobiotics or their metabolites.

Although there was no microscopic evidence of any degenerative changes suggestive of hepatocytotoxicity, an increased alanine and aspartate aminotransferase activity level was measured in one male at 150 mg/kg/day, and in two males at 450 mg/kg/day. Further, cholesterol levels were increased in females at 150 and 450 mg/kg/day. Also, the lower prothrombin and partial thromboplastin time in females at 150 and/or 450 mg/kg/day Indicated an effect on liver function as most clotting factors are synthesized in the liver. Next to hepatocellular hypertrophy, a low incidence of bile duct hyperplasia was noted in two males and three females 450 mg/kg/day, and in a single male at 150 mg/kg/day.

Thyroid follicular epithelial hypertrophy was noted among males at 50 mg/kg/day and higher In a dose-related manner. This was considered to be a secondary adaptive effect associated with centrilobular hypertrophy in the liver and subsequent increased thyroid hormone elimination.

In addition to the liver effects described above, several animals at 450 mg/kg/day and a single male at 150 mg/kg/day showed enlarged kidneys, along with increased (absolute and/or relative) kidney weights in females at 150 mg/kg/day and In both sexes at 450 mg/kg/day. The enlargement/increase in weight may be related to the granular eosinophilic proximal convoluted tube epithelium noted in all males at 450 mg/kg/day. These granules were considered to represent alpha"globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to Increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular epithelial cell damage. However, no morphological evidence of tubular epithelial cell damage was obtained. The higher inorganic phosphate levels in males at 450 mg/kg/day suggested a functional renal impairment since inorganic phosphate levels are primarily regulated by the renal tubules. No concurrent clinical biochemistry changes were noted.

A mild mucosal haemorrhage was noted in the glandular stomach of two males at 450 mg/kg/day (mild degree) which correlated to the red foci noted at macroscopic examination. This suggests that the test substance has Irritating properties.

Brown pigmented cells were noted in the liver (centriacinar), hepatic lymph nodes (syncytial macrophages) and kidneys (tubules) at 450 mg/kg/day, and at lower incidence in males at 50 and/or 150 mg/kg/day. This was supported macroscopically by black/red-brown discolouration. The nature of the pigmentation could not be ascertained based on results obtained in this study.

A number of haematological changes were present in the blood at primarily 450 mg/kg/day that suggests an altered red blood cell turn over. These changes included reduced haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin, and increased red cell distribution width and reticulocyte counts in males and/or females. At 150 mg/kg/day, lower red blood cell counts, haemoglobin, haematocrit, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were noted in males or females. At 50 mg/kg/day, these haematologlcal changes were confined to lower mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration in males. The increased reticulocyte counts in males at 450 mg/kg/day indicated that the bone marrow Is actively responding by producing an Increased number of red cells (regenerative anaemia). However, in females of this dose group no clear increase in reticulocyte counts was noted.

Also, no dose-related reduction in red blood cell counts was noted, and there was no evidence of red blood cell production outside the bone marrow (extramedullary haematopolesis). This suggests that the effects on red blood cell turn over were relatively minor.

The higher total bilirubin levels in males at 450 mg/kg/day and in females at 150 and 450 mg/kg/day may be the result of haemolysis as bilirubin is mainly formed from breakdown of haemoglobin.

No histopathological support was obtained for the slightly reduced thymus weights of females at 450 mg/kg/day, and for the slightly increased spleen to body weight ratio in males at 450 mg/kg/day.

Conclusions:
Findings at 50 mg/kg/day were of a minimal nature and did not indicate an adverse effect on
functional integrity. Based on the findings in the liver, kidneys and blood at 150 and 450
mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for TRIGONOX 301 of 50 mg/kg/day
was established.
Executive summary:

Title: Repeated dose 90-day oral toxicity study with TRIGONOX 301 by daily gavage in the rat.

Guidelines: The study was based on the following guidelines. - EC Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001. OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998. - OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998. - Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No.2) and ME (No. 031121002).

Rationale for dose levels: Dose levels for this 90-day oral gavage study were selected to be 0, 50, 150 and 450 mg/kg/day. The dose levels were based on a twenty-eight day repeated dose oral (gavage) toxicity study with Trigonox 301 in Sprague-Dawley rats in which five animals/sex/group were exposed at dose levels of 15, 150 and 1000 mg/kg bw/day. At 1000 mg/kg, toxicity consisted of clinical signs, reduced body weight gain, reduced food intake, increased water consumption, affected haematology and clinical biochemistry parameters, increased liver and kidneys weight, enlarged and/or dark liver, dark kidneys, and microscopic changes in the liver and kidneys. The microscopic changes observed at 15 and 150 mg/kg (minimal degree of hepatocellular hypertrophy at 15 and 150 mg/kg/day, and minimal to moderate degree of bile duct hyperplasia and pigment in bile ducts at 150 mg/kg/day) were considered not to be an indication of marked target organ dysfunction.

Study outline: The test substance, formulated in vehicle, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Evaluated parameters: Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability over 5 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Findings at 50 mg/kg/day were of a minimal nature and did not indicate an adverse effect on functional integrity. Based on the findings in the liver, kidneys and blood at 150 and 450 mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for TRIGONOX 301 of 50 mg/kg/day was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

The 90 -day study showed a NOAEL of 50 mg/kg bw. Changes at the next higher level tested of 150 mg/kg bw did not indicate an adverse effect on functional integrity.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Apparently well conducted GLP study.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based on a NOAEL of 50 mg/kg bw and changes that did not indicate an adverse effect on functional integrity at the next higher level of 150 mg/kg bw following gavage treatment for 90 days, no classification is needed.