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Description of key information

A 90-day oral gavage study in rats was performed according to GLP and OECD 408 (1998). Based on decreased platelet count and increased incidence of follicular hypertrophy/hyperplasia in the thyroid glands in males at 250 mg/kg bw/d and above, the NOAEL was set at 75 mg/kg bw/d.

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:
Nov 2011-Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 42 +/- 1 days
- Weight at study initiation: 141.2-161.4 for males; 114.0-129.3 for females
- Fasting period before study: no
- Housing: group-housing in H-Temp polysulfonate cages with wooden bedding and for enrichment wooden gnawing blocks
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat "GLP" supplied by Provimi Kliba SA, Switzerland
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Nov 2011 To: 21-22 Feb 2012
Route of administration:
oral: gavage
Vehicle:
other: highly deionized water and 5 mg/100 ml Cremophor EL
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The emulsion was prepared by weighing out the appropriate amount of test substance for the desired concentration, filling up with highly deionized water and 5 mg/100 ml Cremophor EL to the desired volume followed by high-speed homogenizing. During administration preparations were kept homogeneous by magnetic stirring. The test substance preparations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): none given
- Concentration in vehicle: 0, 0.75, 2.5 and 10 g/100 ml
- Administration volume: 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance preparation for a period of 4 hours at room temperature was proven before the start of the administration period. Homogeneity was verified in 3 samples in the highest and lowest concentrations (was used as a concentration control at the same time) at the beginning of the administration period; additional concentration control analyses were verified in the mid concentration, and in 3 samples of each concentration at the end of teh administration period.
Method used: LCMS
Duration of treatment / exposure:
3 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
75, 250 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: none given
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality: twice daily on working days and once daily on Saturdays, Sundays and public holidays; clinical signs: daily before administration and within 2 and 5 hours after administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first administration and weekly thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, weekly over a period of 1 day

WATER CONSUMPTION:
- Time schedule for examinations: daily by visual inspection only

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start and at end of administration period
- Dose groups that were examined: 0 and 1000 mg/kg bw/d

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacirifce
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: 10/sex/group
- Parameters examined according to OECD 408 (1998).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes, overnight
- How many animals: 10/sex/group
- Parameters examined according to OECD 408 (1998) with additionallly chloride, inorganic phosphate, calcium, globulins and triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: day 81 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight during collection of urine
- Parameters examined according to OECD 408 (1998) with additionally ketones, urobilinogen, bilirubin and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: home cage observations, open field observations, sensory motor tests/reflexes and motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights: according to OECD 408 (1998) with additionally cauda epididymis, pituitary gland, prostate, seminal vesicles with coagulating glands and thyroid glands.
HISTOPATHOLOGY: Yes, according to OECD 408 (1998); organs/tissues examined from all animals: jejunum, kidneys, liver and thyroid glands.
Other examinations:
Estrous cycle: vaginal smears were prepared in the morning and evaluated for diestrous, proestrous, estrous or metestrous stage from day 63 to 83.
Sperm parameters: at sacrifice in right testis and cauda epididymis; sperm motility, morphology, head count (testis and cauda epididymis).
Statistics:
Two-sided Dunnet's test: body weight (change), cycle length, number of cycles
Non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon test (two-sided) for equal medians: faeces, rearing, grip strength forelimbs/hindlimbs, foot-splay test, motor activity, bidirectional changes in blood parameters, urine pH, volume, specific gravity, color and turbidity, organ weights
Pairwise comparison of each dose group with control group using Wilcoxon test (one-sided): for unidirectional changes in blood parameters, remaining urine parameters
Pairwise comparison of each dose group with control group using Wilcoxon test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians: sperm parameters; for % of abnormal sperms (ABNORMAL6_C) values < 6% were set to 6% (cut off 6%).
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
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
One male at 75 mg/kg bw/d was humanely sacrificed on day 86, which was not related to treatment. The animal showed hypothermia, oblique head posture, respiratory sounds and stiff gait.
Slight to moderate salivation after treatment for a short period was observed in animals at 250 and 1000 mg/kg bw/d on several days; considered to be related to bad taste of the substance or local affection of the upper digestive tract.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight of females at 1000 mg/kg bw/d was signficantly higher on day 28, 42, 56 and 63 and mean body weight changes on day 28, 42 and 63. As body weight/body weight gain was not significantly increased at the end of the administration period, the effects observed is considered to be incidental and not related to treatment. No effects were observed in females at 75 and 250 mg/kg bw/d and males at all dose levels.

FOOD CONSUMPTION
No test substance-related effects.

WATER CONSUMPTION
No test substance-related effects.

OPHTHALMOSCOPIC EXAMINATION
No test substance-related effects.

HAEMATOLOGY
Platelet counts were decreased in males at 250 and 1000 mg/kg bw/d (83 and 85% of control, resp.) and females at 1000 mg/kg bw/d (80% of control).
Mean haemoglobin was statistically decreased in males at 1000 mg/kg bw/d (97% of control) and in females at 250 and 1000 mg/kg bw/d (97 and 92% of control, resp.). Only the change at 1000 mg/kg bw/d in females is considered a toxicologically relevant effect as other changes were minimal. Mean red blood cell count was statistically decreased in females at all dose levels (95-93% of control). Mean haematocrit was decreased in females at 250 and 1000 mg/kg bw/d (97 and 93% of control, resp.). Relative reticulocyte count was increased in males at 1000 mg/kg bw/d (127% of control). Since all means were within historical control range (RBC 7.28-8.25 tera/L), hematocrit 0.378-0.424 L/L, reticulocytes 0.9-2.5%), these effects are not considered treatment-related.

CLINICAL CHEMISTRY
In both sexes at 1000 mg/kg bw/d ALT was increased (159% of control for males and 158% for females). Albumin was increased in females at 1000 mg/kg bw/d (109% of control). Bilirubin levels were decreased in all males (82-62% of control) and in females at 250 and 1000 mg/kg bw/d (73-55% of control) in a dose-related manner. Total bile acid was slightly decreased in females at 1000 mg/kg bw/d. These bile alterations were regarded as reflecting higher conjugation and excretion rate of bilirubin, but not toxicologically relevant.
All other changes were marginal and/or within historical control ranges.

URINALYSIS
A higher incidence of phosphate crystals occurred in the urine of both sexes at 1000 mg/kg bw/d.
In males at 250 and 1000 mg/kg bw/d specific gravity was increased (dose-related) and, although not significantly, urine volume was lower compared to controls. This is considered to reflect a lower water intake and not toxicologically relevant.

NEUROBEHAVIOUR
No treatment-related effects observed during home cage and open field observations. No treatment-related effects observed for sensorimotor tests and reflexes. Motor activity was decreased compared to control in males at 1000 mg/kg bw/d due to decreases at a few single intervals. As no time-related trend was seen, the effect was not considered treatment-related.
In females, motor activity was increased compared to control at 1000 mg/kg bw/d overall and at interval 5; also an overall increase was seen at 250 mg/kg bw/d. Since both sexes showed opposite effects, effects showed no time-related trend and these effects were caused by low values for more animals in the control group than in the higher dose animals, the effect was not considered to be treatment-related. All changes were within the historical control range.

ORGAN WEIGHTS
The increased liver weights in males (relative: 123% of control) and females (absolute and relative: 132 and 126% of control, resp.) at 1000 mg/kg bw/d as well as the increased absolute and relative weights of thyroid glands (124 and 119% of control, resp.) in females at 1000 mg/kg bw/d were considered treatment-related. The increased relative liver weight in males at 250 mg/kg bw/d was only marginal and had no histopathological correlate; therefore, was not considered treatment-related.
The terminal body weight of males at 1000 mg/kg bw/d was decreased (93% of control) resulting in a decrease of the absolute weight of epididymides (94% of control) and increased relative heart and kidney weights (both 113% of control). The increased heart weight (absolute/relative: 111%/107% of control) were not considered to be treatment-related, in the absence of a dose-response relationship and a histopathological correlate. The absolute kidney weight was increased in females at 1000 mg/kg bw/d (109% of control), but not considered treatment-related in absence of a dose-response relationship, no increase in relative kidney weight and histopathological correlate.

GROSS PATHOLOGY
Three females at 1000 mg/kg bw/d showed an enlarged liver.

HISTOPATHOLOGY: NON-NEOPLASTIC
Fatty change (cytoplasmic vacuolization) of hepatocytes was observed in 3 males and 9 females (2 with enlarged liver) at 1000 mg/kg bw/d. The fatty change was located in the peripheral periportal hepatocytes and characterized by lipid-containing vacuoles.
Dilation of lymph vessels mostly in the villi of the cranial part of the jejunum was seen in all treated animals: 0, 2, 8 and 10 males and 0, 4, 8 and 6 females at 0, 75, 250 and 1000 mg/kg bw/d. Lipid-material was shown attached plaque-like to the endothelial wall of some dilated lymph vessels. No signs of inflammation, degenerative changes or effects on lymph nodes was detectable. Therefore, although it might be related to treatment, this was not considered to be toxicologically relevant.
The incidence of minimal or slight diffuse follicular hypertrophy/hyperplasia in thyroid glands was increased in males at 250 and 1000 mg/kg bw/d (3 and 6 vs 1 in control) and females at 1000 mg/kg bw/d (4 vs 0 in control).
A variable number of eosinophilic droplets was observed in the cytoplasm of proximal tubules in male kidneys without a clear dose-response relationship. This increase was probably due to alpha 2mu-globulin and no tubular injury was noted. Therefore, the increase was not considered adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: decreased platelet count and increased diffuse follicular hypertrophy/hyperplasia in thyroid glands of males at 250 mg/kg bw/d and above
Critical effects observed:
not specified

Analysis of preparations:

The test substance preparations tested were stable and homogeneous. At the beginning of the administration period measured concentrations were within the expected range, i.e. 90.9 -100% of nominal concentrations. At the end of the administration period measured concentrations for the 75 and 250 mg/kg bw/d group revealed values within 95 -100% of nominal concentrations. However, measured concentrations for the 1000 mg/kg bw/d group showed values at 72% (mean value of all samples) of the nominal concentration with an unknown reason. This deviation did not have an influence on the validity of the study as clear signs of toxicity were noted in animals at 1000 mg/kg bw/d.

Estrous cycle: No treatment-related effect on cycle length and number of cycle was noted.

Sperm parameters: No treatment-related effect was seen on sperm motility, sperm count (testis/cauda epididymis) and percentage of abnormal sperms.

Conclusions:
Based on decreased platelet count and increased diffuse follicular hypertrophy/hyperplasia in thyroid glands of males at 250 mg/kg bw/d and above, the NOAEL was set at 75 mg/kg bw/d.
Executive summary:

In a 90-d oral gavage study performed according to GLP Wistar rats were given 0, 75, 250 and 1000 mg/kg bw/d. Next to parameters according to OECD 408 (1998) also sperm parameters and estrous cycle were determined.

No treatment-related effects on mortality, clinical signs, body weight, food consumption, ophthalmoscopy, neurobehaviour were noted. Platelet count was decreased in both sexes at 1000 mg/kg bw/d and males at 250 mg/kg bw/d. In both sexes at 1000 mg/kg bw/d ALT was increased and albumin was increased in females at 1000 mg/kg bw/d. A higher incidence of phosphate crystals occurred in the urine of both sexes at 1000 mg/kg bw/d. The increased liver weights in males and females at 1000 mg/kg bw/d as well as the increased absolute and relative weights of thyroid glands in females at 1000 mg/kg bw/d were considered treatment-related. Three females at 1000 mg/kg bw/d showed an enlarged liver. Fatty change (cytoplasmic vacuolization) of hepatocytes was observed in 3 males and 9 females (2 with enlarged liver) at 1000 mg/kg bw/d. The fatty change was located in the peripheral periportal hepatocytes and characterized by lipid-containing vacuoles. The incidence of minimal or slight diffuse follicular hypertrophy/hyperplasia in thyroid glands was increased in males at 250 and 1000 mg/kg bw/d (3 and 6 vs 1 in control) and females at 1000 mg/kg bw/d (4 vs 0 in control).

Based on decreased platelet count and increased incidence of follicular hypertrophy/hyperplasia in the thyroid glands in males at 250 mg/kg bw/d and above, the NOAEL was set at 75 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1: The study was performed according to GLP and OECD 408 (1998).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 90-d oral gavage study performed according to GLP Wistar rats were given 0, 75, 250 and 1000 mg/kg bw/d. Next to parameters according to OECD 408 (1998) also sperm parameters and estrous cycle were determined.

No treatment-related effects on mortality, clinical signs, body weight, food consumption, ophthalmoscopy, neurobehaviour were noted. Platelet count was decreased in both sexes at 1000 mg/kg bw/d and males at 250 mg/kg bw/d. In both sexes at 1000 mg/kg bw/d ALT was increased and albumin was increased in females at 1000 mg/kg bw/d. A higher incidence of phosphate crystals occurred in the urine of both sexes at 1000 mg/kg bw/d. The increased liver weights in males and females at 1000 mg/kg bw/d as well as the increased absolute and relative weights of thyroid glands in females at 1000 mg/kg bw/d were considered treatment-related. Three females at 1000 mg/kg bw/d showed an enlarged liver. Fatty change (cytoplasmic vacuolization) of hepatocytes was observed in 3 males and 9 females (2 with enlarged liver) at 1000 mg/kg bw/d. The fatty change was located in the peripheral periportal hepatocytes and characterized by lipid-containing vacuoles. The incidence of minimal or slight diffuse follicular hypertrophy/hyperplasia in thyroid glands was increased in males at 250 and 1000 mg/kg bw/d (3 and 6 vs 1 in control) and females at 1000 mg/kg bw/d (4 vs 0 in control).

Based on decreased platelet count and increased incidence of follicular hypertrophy/hyperplasia in the thyroid glands in males at 250 mg/kg bw/d and above, the NOAEL was set at 75 mg/kg bw/d.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable repeated dose toxicity study according to OECD 408 and in accordance with GLP principles.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: blood coagulation; glandular: thyroids

Justification for classification or non-classification

Based on the available data on repeated dose toxicity, no classification is warranted for GPM-800 according to Regulation 1272/2008 (and amendments) on Classification, Labelling and Packaging of Substances and Mixtures.