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Toxicological information

Toxicity to reproduction

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Endpoint:
fertility, other
Remarks:
other: repeated dose toxicity studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours over the concentration range of 1 to 200 mg/mL and in a refrigerator for at least 10 days, Test Facility Study No. 514832
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none
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
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the start of the study under laboratory conditions
- Health inspection: upon receipt of the animals
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test item. No correction was made for purity/composition of the test item.

VEHICLE
- Rationale for vehicle: based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Purity: pure (Elix water, Millipore, France)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability for at least 5 hours at room temperature, under normal laboratory light conditions over the concentration range 1 to 200 mg/mL and in a refrigerator for at least 10 days, Test Facility Study No. 514832.
No test item was detected in the control/vehicle formulation.
The concentrations analysed in the formulations of 60, 200 and 700 mg/kg groups (week 1, 6 and 13 formulations) were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of the 60 mg/kg and 700 mg/kg groups were homogeneous (i.e. coefficient of variation below or equal 10%).
Duration of treatment / exposure:
at least 90 days; animals were dosed up to the day prior necropsy
Frequency of treatment:
once daily, 7 days per week
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
700 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Based on the results of an available 28-day gavage study with the test material in rats the dose levels for this 90-day oral gavage study were selected to be 0, 60, 200, and 700 mg/kg bw
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 515566 for logistic reasons and reported under Test Facility Study No. 514828). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly during first 4 weeks and weekly thereafter.

FOOD CONSUMPTION: Yes
- Twice weekly during first 4 weeks and weekly thereafter.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest and in week 13
- Dose groups that were examined: pretest: all animals; week 13: goups 1 and 4 animals; Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. at the end of the treatment
- Anaesthetic used for blood collection: Yes (identity: isoflurane)
- Animals fasted: Yes, overnight
- How many animals:
- The following Parameters were examined according to Guideline:
White blood cells
Differential leucocyte count
neutrophils, lymphocytes, monocytes,
eosinophils, basophils
Red blood cells
Reticulocytes
Red blood cell distribution width
Haemoglobin
Haematocrit
Mean corpuscular volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
Platelets
Clotting Potential: Prothrombin time and Activated Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. at the end of the treatment
- Animals fasted: Yes, overnight
- How many animals:
- The following Parameters were examined according to Guideline:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Total Protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate
Bile acids


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: During Week 12-13 of treatment; Tests were performed after dosing at no specific time point, but within a similar time period after dosing.
Dose groups that were examined: on the first 5 animals/sex of all groups
Battery of functions tested:
- hearing ability, pupillary reflex and static righting reflex
- fore- and hind-limb grip strength was recorded as the mean of three measurements per animal
- locomotor activity test: recording period 1 hour. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No


OTHER:
Estrous cycle determination: All females had a daily lavage from Day 72 up to and including Day 92 to determine the stage of estrous.
Spermatogenesis: From all males of Group 1 and 4 additional slides of the testes were prepared to examine staging of spermatogenesis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (according to Guideline; see list below)
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex] (7 levels)
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides *
Eyes with optic nerve [if detectable] and
Harderian gland *
Female mammary gland area
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
Larynx
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles including coagulating gland
(Skeletal muscle)
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes *
Thymus
Thyroid including parathyroid [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
* Fixed in modified Davidson's solution, prepared at Charles River Den Bosch using Formaldehyde 37-40%, Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Uterus (including cervix)
Prostate
Seminal vesicles including coagulating glands
Thyroid including parathyroid


HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- All tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- thyroid gland and the liver of all animals of Groups 2 and 3 (males and females) and adrenal glands and kidneys of Groups 2 and 3 (males), based on (possible) treatment-related changes in these organs in Group 4,
- the additional slides of the testes of Group 1 and 4 males to examine staging of spermatogenesis,
- all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.

Statistics:
The following statistical methods were used to analyze 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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

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
Description (incidence and severity):
No toxicological relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen after dosing amongst 200 and 700 mg/kg animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals generally remained in the same range as controls over the study period (see Table 1). The treated male rats showed a slightly lower body weight at termination (-9 to -5%) that was not considered as adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight remained similar to the control level over the study period.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 3 for hematological findings:
The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
• Percentage red blood cell distribution width (%RDW) was increased in 60 and 700 mg/kg males and 700 mg/kg females.
• Haemaglobin levels were reduced in 700 mg/kg males.
• Haematocrit levels were reduced in 700 mg/kg males and females.
• Mean red blood cell volume (MCV) was reduced in 700 mg/kg males and in 200 and 700 mg/kg females.
• Mean cell hemoglobin mass per red blood cell (MCH) was reduced in 700 mg/kg males and females.
• Prothrombin time (PT) was reduced across all doses in both males and females (not dose dependent; not statistically significant in 200 mg/kg females).
Discussion:
Hematological analysis revealed alterations in red blood cell parameters, including red blood cell distribution width (% RDW), haemoglobin (only in males), haematocrit, and mean cell hemoglobin mass per red blood cell (MCH) levels in 700 mg/kg bw/day animals. The mean red blood cell volume (MCV) was reduced in 700 mg/kg bw/day males and in 200 and 700 mg/kg bw/day females. These changes were not accompanied by morphologic correlates and were within the range expected for rats of the same age and strain. Prothrombin time (PT) was reduced in all test item groups (no dose dependence, not statistically significant in 200 mg/kg bw/day females), which may be related to the morphological alterations observed in the liver (e.g. hepatocellular hypertrophy).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 4 for clinical biochemistry findings:
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals:
• Total protein levels were increased in 700 mg/kg males and females.
• Albumin levels were increased in 700 mg/kg males and females.
• Total bilirubin levels were reduced in 200 and 700 mg/kg females.
• Cholesterol levels were increased in 700 mg/kg females.
• Bile acid levels were increased in 700 mg/kg males and females. An increase was seen in 60 mg/kg males, however this effect occurred in the absence of a dose-related trend.
• Potassium and calcium levels were increased in 700 mg/kg males and females.
Discussion: Clinical biochemistry analysis revealed increased total protein, albumin, bile acid, potassium, calcium and cholesterol (only in females) levels in 700 mg/kg bw/day males and/or females, and reduced bilirubin levels in 200 and 700 mg/kg bw/day females, which may be related to the observed changes in hepatic function.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See Table 5 for functional findings:
In 700 mg/kg females, total movement levels were reduced in a statistically significant manner when compared to controls, as well as a reduced incidence of ambulatory behaviour (not statistically significant). Furthermore, 700 mg/kg females demonstrated a faster motor activity habituation profile at the third interval of the test period.
Motor activity analysis demonstrated a statistically significant reduction of total movements in 60 mg/kg males, however this effect occurred in a non-dose related trend. All male groups displayed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was unaffected in males, however in females, foregrip strength in 60 and 700 mg/kg groups, and hindgrip strength in the 700 mg/kg group were reduced in a statistically significant manner.
Discussion: Functional observation tests revealed no adverse changes. The slightly lower total movement level of females at 700 mg/kg bw/day and a faster habituation profile at one particular timepoint, were considered not to represent an adverse effect on neurobehaviour. Grip strength was unaffected in males, however in females, foregrip strength in 60 and 700 mg/kg bw/day groups, and hindgrip strength in the 700 mg/kg bw/day group were reduced in a statistically significant manner. These results were not supported by clinical observations and had no supportive morphological correlates in examined neuronal tissues.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Table 1 for organ weight findings:
Higher kidney weights were present in males at 200 mg/kg (relative to body weight) and in males and females at 700 mg/kg (absolute and relative to body weight). There was no microscopic correlate for this.
Statistically higher liver weights were present in males at 60 mg/kg (only relative to body weight, +10%), in males and females at 200 mg/kg (only relative to body weight, + 19% and +12%) and in males and females at 700 mg/kg (absolute + 56% for males and + 65% for females and relative to body weight + 62% for males and +63% for females). The microscopic correlate was hepatocellular hypertrophy only at 700 mg/kg.
In males relative heart weights were significantly higher in all dose groups and relative testes weights were significantly higher in the high dosed males. No significant changes were recorded for absolute heart and testes weights. Since the male body weight was about 9-5% lower in the treated groups the relative changes were considered to be the result of a decreased body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in the following organs:
• Kidneys, enlarged was present in 6/10 males treated at 700 mg/kg. There was no clear microscopic correlate for this finding.
• Liver, enlarged was present in 8/10 males and a single (1/10) female treated at 700 mg/kg. This corresponded microscopically with hepatocellular hypertrophy.
• Thyroid gland, enlarged was present in a single (1/10) male treated at 700 mg/kg. This corresponded microscopically with follicular cell hypertrophy.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Table 2 for histopathological findings:
Liver:
Hepatocellular hypertrophy was present in all males and females treated at 700 mg/kg up to a slight degree (9 males minimal, 1 male slight; 4 females minimal, 6 females slight). No histopathological effects were seen in the liver at lower doses or in the control group.
Thyroid:
Follicular cell hypertrophy was present at slightly increased incidence and severity in males starting at 60 mg/kg and in females at 700 mg/kg up to slight degree. Affected males in control/low/mid/high dose: 5/7/9/9; affected females in control/low/mid/high dose: 2/1/3/9
Colloid alteration was present in 9 males (up to slight) and 4 females (minimal) treated at 700 mg/kg.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Kidney males:
Hyaline droplet accumulation was present at increased incidence and/or severity in males starting at 60 mg/kg up to moderate degree. The hyaline droplets were considered to represent alpha2µ-globulin, a protein specific for male rat kidneys which undergoes reabsorption in the proximal cortical tubules.
Tubular basophilia was present at increased incidence and severity in males treated at 700 mg/kg up to moderate degree. A range of chemicals are known to increase hyaline droplet formation, which ultimately leads to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. These findings are considered adverse for male rats.
Granular casts were present in males treated at 700 mg/kg up to marked degree.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle determination:
No test item related changes were noted in estrous cycle determination.
One control female (no. 47) showed an irregular estrous cycle length and one 200 mg/kg female (no. 65) showed extended estrus. All other females showed a normal (regular) estrous cycle of 4/5 days. The incidence of irregular estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.
Spermatogenesis:
Spermatogenic staging profiles were normal for all males (control and high dose group) examined.

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse findings in the kidneys of male rats at 700 mg/kg bw/day, taking also into account the high liver weight increase in males and females and additional observations in the kidneys of females at this dose
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
other: kidney, alpha2µ-globulin nephropathy in males
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Treatment with the test substance in general was well tolerated. No treatment related mortality occurred and no toxicologically relevant clinical signs were noted. Body weight and food consumption were not affected by treatment. A slightly lower body weight was recorded for treated males (-9 to -5%) that was not considered as adverse (see Table 1).

Adverse test item-related morphologic alterations were present in the kidneys of males (see Table 2). This included hyaline droplet accumulation ( increased incidence and/or severity in males starting at 60 mg/kg bw/day up to moderate degree), which is considered to represent alpha2uglobulin, a protein specific for male rat kidneys which undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation, which ultimately leads to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. These findings are considered adverse for male rats. Furthermore, increased kidney weight (see Table 2; also observed as enlarged kidney), and changes in potassium and calcium levels in blood accompanied these findings (see Table 4). Taking into account that alpha2uglobulin formation is a specific effect in male rats, i.e. this effect is not present in female rats or in higher mammals, including humans (Sahota et al, Toxicologic Pathology - Nonclinical Safety Assessment. CRC,Press, Tailor& Francis Group, Boca Raton, pp 450-451, 472, 2013), this effect should not be considered relevant for human risk assessment. In high-dose females, increased kidney weight was observed, accompanied by changes in blood potassium and calcium levels. Since no morphological changes were noted in the kidneys of females, the adversity of these changes was unclear.

Test item related findings were also identified in the liver which included minimal to slight hepatocellular hypertrophy in all animals at 700 mg/kg bw/day (see Table 2). This was accompanied by increased relative liver weight (see Table 1) at 200 mg/kg bw (+19% in males, +12% in females) and absolute/relative liver weight at 700 mg/kg bw (+56/62% in males, +65/63% in females), also observed as enlarged liver at 700 mg/kg bw in both sexes. These large increases in liver weight at 700 mg/kg bw can be considered adverse, however in the absence of any other indicator of hepatocellular toxicity no clear adversity could be ascribed to these changes in the liver.

Non-adverse test item-related morphologic alterations were also present in the thyroid glands of males at 60, 200 and 700 mg/kg bw/day, and in females at 700 mg/kg bw/day. This included follicular cell hypertrophy, which is regarded as an adaptive response to the induction of hepatic enzymes and considered non-adverse at the incidences and severities reported. The colloid alteration present at 700 mg/kg bw/day in both sexes is often associated with thyroid stimulation, occurs spontaneously, and increases gradually with age, but is accelerated by follicular cell hypertrophy. No test substance related effects were observed in the adrenals.

Hematological analysis (see Table 3) revealed alterations in red blood cell parameters, including red blood cell distribution width (% RDW), haemoglobin (only in males), haematocrit, and mean cell hemoglobin mass per red blood cell (MCH) levels in 700 mg/kg bw/day animals. The mean red blood cell volume (MCV) was reduced in 700 mg/kg bw/day males and in 200 and 700 mg/kg bw/day females. These changes were not accompanied by morphologic correlates and were within the range expected for rats of the same age and strain.  Prothrombin time (PT) was reduced in all test item groups (no dose dependence; not statistically significant in 200 mg/kg bw/day females), which may be related to the morphological alterations observed in the liver. Clinical biochemistry analysis (see Table 4) revealed increased total protein, albumin, bile acid, potassium and calcium levels and cholesterol (only in females) levels in 700 mg/kg bw/day males and/or females, and reduced bilirubin levels in 200 and 700 mg/kg bw/day females, which may be related to the abovementioned changes in hepatic function.

Functional observation tests revealed no adverse changes (see Table 5). The slightly lower total movement level of females at 700 mg/kg bw/day and a faster habituation profile at one particular timepoint were considered not to represent an adverse effect on neurobehaviour. Grip strength was unaffected in males, however in females, foregrip strength in 60 and 700 mg/kg groups, and hindgrip strength in the 700 mg/kg group were reduced in a statistically significant manner. These results were not supported by clinical observations and had no supportive morphological correlates in examined neuronal tissues.

Reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs) revealed no treatment-related changes.

Table 1: body weight at termination and absolute and relative organ weights (restricted to organs that showed statistical significant changes)

 dose (mg/kg bw/day)  0 (males)  60 (males)  200 (males)  700 (males)  0 (females)  60 (females)  200 (females)  700 (females)
 body weight at termination (g)  404  370* (-9%)  380 (-6%)  386 (-5%)  235  223  226  236
 liver (absolute, in g)  8.98  9.05 (+11%)  10.13 (+13%)  13.98** (+56%)  5.68  5.41  6.13 (+8%)  9.36** (+65%)
 liver (relative, in %)  2.23  2.45** (+10%)  2.66** (+19%)  3.61** (+62%)  2.43  2.43  2.71* (+12%)  3.96** (+63%)
 kidney (absolute, in g)  2.25  2.28  2.40 (+7%)  2.90** (+29%)  1.52  1.55  1.60 (+5%)  1.85** (+22%)
 kidney (relative, in %)  0.56  0.62 (+11%)  0.63* (+13%)  0.75** (+34%)  0.65  0.70 (+8%)  0.71 (+9%)  0.78** (+20%)
 heart (absolute, in g)  0.976  0.962  0.990  1.047  0.706  0.669  0.683  0.763
 heart (relative, in %)  0.242  0.261*  0.261*  0.272**  0.301  0.300  0.302  0.323
 testes (absolute, in g)  3.61  3.54  3.43  3.79        
 testes (relative, in %)  0.90  0.96  0.91  0.98*        

    *: P<0.05, **: P<0.01                          

 

Table 2: Summary of test item related microscopic findings: kidney in males and liver/thyroid in males and females

 dose (mg/kg bw/day)  0 (10 males)  60 (10 males)  200 (10 males)  700 (10 males)  0 (10 females)  60 (10 females)  200 (10 females)  700 (10 females)
 kidney hyaline droplet accumulation                
 - minimal / slight / moderate / no. examined  3 / 2 /-  3 / 5 / 1  3 /2 / 2  1 /3 / 6  -      -
 kidney basophilia tubular                
 - minimal / slight / moderate  3 / - / -  4 / 1 / -  4 / - / -  1 / 4 / 5  -      -
 kidney granuar cast                
 - minimal / slight / moderate / marked  -  -  - 1 / 3 / 2 / 2  -      -
 liver - hepatocellular hypertrophy                
 - minimal / slight  -  -  -  9 / 1  -  -  -  4 / 6
 thyroid glands - hypertrophy follicular cell                
 - minimal / slight  5 / -  6 / 1  7 / 2  7 / 2  2 / -  1 / -  3 / -  6 / 3
 thyroid glands - alteration colloid                
 - minimal / slight  1 / -  -  -  6 / 3  -  -  -  4 / -

Table 3: Summary of hematological (statistically significant) findings

 dose (mg/kg bw/day)  0 (10 males)  60 (10 males)  200 (10 males)  700 (10 males)  0 (10 females)  60 (10 females)  200 (10 females)  700 (10 females)
red blood cell distribution width (RDW, %)  12.4  13.0*  12.9 13.8**  11.1  11.3  11.5  11.7*
 haemoglobin (mmol/L)  10.2  10.2  10.0  9.5**  9.4  9.4  9.4  9.1
 haematocrit (L/L)  0.508  0.509  0.498  0.474**  0.471  0.474  0.468  0.451*
 mean red blood cell volume (MCV, fL)  53.9  52.6  52.6  50.8**  56.3  56.0  54.7*  54.1**

 mean cell hemoglobin mass per red

blood cell (MCH, fmol)

 1.09  1.05  1.06  1.02**  1.12  1.11  1.09  1.09*
 prothrombin time (PT, s)  18.3  16.6**  16.3**  16.6**  17.7  16.0**  16.7  15.3**

*: P<0.05, **: P<0.01     

Table 4: Summary of clinical biochemistry (statistically significant) parameters

 dose (mg/kg bw/day)  0 (10 males)  60 (10 males)  200 (10 males)  700 (10 males)  0 (10 females)  60 (10 females)  200 (10 females)  700 (10 females)
 total protein (g/L)  66.1  66.9  67.5  72.1**  68.8  68.0  70.6  77.0**
 albumin (g/L)  33.1  34.3  34.1  36.6**  36.7  36.4  37.3  40.9**
 total bilirubin (µmol/L)  1.9  2.1  1.6  1.5  2.6  2.2  1.9**  1.9**
 cholesterol (mmol/L)  2.15  2.06  1.97  2.29  1.83  1.71  2.04  2.62**
 bile acids (µmol/L)  25.1  13.1*  15.4  40.2**  17.5  21.0  30.2  43.5**
 potassium (mmol/L)  3.78  3.72  3.88  4.02*  3.50  3.47  3.45  3.70*
 calcium (mmol/L)  2.56  2.55  2.57  2.63*  2.63  2.60  2.64  2.76**

*: P<0.05, **: P<0.01     

Table 5: Summary of functional obsevations (statistically significant) and motor activity

 dose (mg/kg bw/day)  0 (10 males)  60 (10 males)  200 (10 males)  700 (10 males)  0 (10 females)  60 (10 females)  200 (10 females)  700 (10 females)
 foregrip strength (g)  1130  1113  1191  926  1098  830*  939  803*
 hindgrip strength (g)  565  487  649  591  511  422  428  358*
 total movements (mean, at wk 12 -13)  3872  4458*  3314  2597  5528  5104  3912  2569*
 ambulations (mean, at wk 12 -13)  665  870  692  634  1667  1521  1202  923

*: P<0.05, **: P<0.01     

Executive summary:

A repeated dose 90-day oral gavage toxicity study on 10 male and 10 female Wistar rats per dose group following OECD 408 was performed with the test substance. Dose levels for this study were selected based on available toxicological data for the substance (28-day study in rats) at 0, 60, 200 and 700 mg/kg bw/day. As vehicle water was used.

Treatment with the test substance in general was well tolerated. No treatment related mortality occurred and no toxicologically relevant clinical signs were noted. Body weight and food consumption were not affected by treatment. Ophthalmoscopy also showed no toxicologically significant changes.

Adverse test item-related morphologic alterations were present in the kidneys of males. This included hyaline droplet accumulation, which is considered to represent alpha2µ-globulin, a protein specific for male rat kidneys which undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation, which ultimately leads to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. These findings are considered adverse for male rats. Furthermore, increased kidney weight (also observed as enlarged kidney), and changes in potassium and calcium levels in blood accompanied these findings. Taking into account that alpha2uglobulin formation is a specific effect in male rats, i.e. this effect is not present in female rats or in higher mammals, including humans (Ref. 6), this effect should not be considered relevant for human risk assessment. In high-dose females, increased kidney weight was observed, accompanied by changes in blood potassium and calcium levels. Since no morphological changes were noted in the kidneys of females, the adversity of these changes was unclear.

Test item related findings were also identified in the liver, which included minimal to slight hepatocellular hypertrophy in all animals at 700 mg/kg bw/day. This was accompanied by increased relative liver weight at 200 mg/kg bw (+19% in males, +12% in females) and absolute/relative liver weight at 700 mg/kg bw (+56/62% in males, +65/63% in females), also observed as enlarged liver at 700 mg/kg bw in both sexes. These large increases in liver weight at 700 mg/kg bw can be considered adverse, however in the absence of any other indicator of hepatocellular toxicity no clear adversity could be ascribed to these changes in the liver.

Non-adverse test item-related morphologic alterations were also present in the thyroid glands of males at 60, 200 and 700 mg/kg bw/day, and in females at 700 mg/kg bw/day. This included follicular cell hypertrophy, which is regarded as an adaptive response to the induction of hepatic enzymes and considered non-adverse at the incidences and severities reported. The colloid alteration present at 700 mg/kg bw/day in both sexes is often associated with thyroid stimulation, occurs spontaneously, and increases gradually with age, but is accelerated by follicular cell hypertrophy. No test substance related effects were observed in the adrenals.

Hematological analysis revealed alterations in red blood cell parameters, including red blood cell distribution width (% RDW), haemoglobin (only in males), haematocrit, and mean cell hemoglobin mass per red blood cell (MCH) levels in 700 mg/kg bw/day animals. The mean red blood cell volume (MCV) was reduced in 700 mg/kg bw/day males and in 200 and 700 mg/kg bw/day females. These changes were not accompanied by morphologic correlates and were within the range expected for rats of the same age and strain.  Prothrombin time (PT) was reduced in all test item groups (no dose dependence; not statistically significant in 200 mg/kg bw/day females), which may be related to the morphological alterations observed in the liver.

Clinical biochemistry analysis revealed increased total protein, albumin, bile acid, potassium and calcium levels and cholesterol (only in females) levels in 700 mg/kg bw/day males and/or females, and reduced bilirubin levels in 200 and 700 mg/kg bw/day females, which may be related to the abovementioned changes in hepatic function.

Functional observation tests revealed no adverse changes. The slightly lower total movement level of females at 700 mg/kg bw/day and a faster habituation profile at one particular timepoint were considered not to represent an adverse effect on neurobehaviour. Grip strength was unaffected in males, however in females, foregrip strength in 60 and 700 mg/kg groups, and hindgrip strength in the 700 mg/kg group were reduced in a statistically significant manner. These results were not supported by clinical observations and had no supportive morphological correlates in examined neuronal tissues.

Reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs) revealed no treatment-related changes.

Conclusion

From the results presented in this report, based on the presence of adverse findings in the kidneys of high dosed male rats and taking also into account the highly increased liver weight of unclear adversity in male and female rats and additional observations in the kidneys of females, the overall No Observed Adverse Effect Level (NOAEL) for Triethyl phosphate was established with 200 mg/kg bw/day for male and female rats.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Cited as Directive 84/449/EEC,B.7
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 10, 100, 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
6/sex/dose
Control animals:
yes
Details on study design:
Post-exposure period: no
Positive control:
no data
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):
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
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
Details on results:
see: Remarks on results
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Critical effects observed:
not specified

Bodyweights (g)

 Males           Females         
 Week  0  10  100  1000  0  10  100  1000
 0  133  132  131  130  121  122  126  124
 1  163  163  166  152  132  134  144+  132
 2  211  211  215  193  144  151  161+  150
 3  227  224  229  204 ++  146  152  163+  145
 4  263  258  263  235+  161  170  180 ++  165
Executive summary:

Six male and female Wistar rats per sex received TEP (triethyl phosphate) by gavage for 28 days in a subacute oral toxicity study performed according to =ECD 407. The dose groups tested were: 0, 10, 100 and 1000 mg/kg bw/day.

There were no compound related effects on survival in males or females at any dose.

Body weight was only influenced in the male 1000 mg/kg bw dose group (ca. 10% lower than control group at weeks 3 and 4; body weight gain was not siginificantly affected at any time point). A slight increase in body weight in the female mid dose (100 mg/kg bw/day) was not considered biologically significant due to the lack of dose response and the slightly higher body weight of females in this group at the start of the experiment. The water consumption was higher in the female 1000 mg/kg bw/day dose group compared to the control animals.

Adaptive effects on the liver of high dose animals were observed. Absolute liver weights were significantly increased in mid and high dose females and relative liver weight was significantly increased in males and females at the highest dose. Histopatological examinations revealed a hepatocellular hypertrophy in the 1000 mg/kg bw groups (all surviving animals were affected: female 4; male 6). Based on the slight hypertrophy the increase in liver weight is regarded as an adaptive effect and considered to be not adverse.

There are no compound related observations on any other organ weight or histopathology.

There are no compound related observations on ophthalmology, hematology, clinical chemistry or urine analysis reported.

The observations at 1000 mg/kg bw/day (liver weight increase with slight hypertrophy and slightly reduced body weight) is considered to be an adaptive response; NOEL 100 mg/kg bw. Overall, the study did not reveal any adverse effects up to the highest dose tested. The NOAEL was 1000 mg/kg bw.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Principles of method if other than guideline:
OECD Guideline 407 and 408 Study
GLP compliance:
yes
Limit test:
no
Justification for study design:
repeated dose toxicity studies including reproductive parameters can be used in a weight of evidence approach for the assessment of reproductive toxicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Triethyl phosphate
Molecular formula: C6H15O4P
Molecular weight: 182.15 g/mol
Appearance: clear colourless liquid
Batch: CH1/30036
Purity/Composition: 99.6 %
Test item storage: At room temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CH1/3003
- Lab specific test material number: 207699/A
- Expiration date of the lot/batch: 2018-03-09
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours over the concentration range of 1 to 200 mg/mL and in a refrigerator for at least 10 days. Test facility No. 514832
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
see endpoint study records listed under 'cross-reference'

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
see endpoint study records listed under 'cross-reference'
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see endpoint study records listed under 'cross-reference'
Duration of treatment / exposure:
28 day and 90 day study
Frequency of treatment:
once daily
Details on study schedule:
see endpoint study records listed under 'cross-reference'
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
doses of 10, 100, and 1000 mg/kg bw/day were tested in the 28d oral toxicity study
Dose / conc.:
700 mg/kg bw/day
Remarks:
doses of 60, 200, and 700 mg/kg bw/day were tested in the 90d oral toxicity study
No. of animals per sex per dose:
5 per sex/dose in the 28 day study and 10 per sex/dose in the 90 day study
Control animals:
yes, concurrent vehicle
Details on study design:
see endpoint study records listed under 'cross-reference'

Examinations

Parental animals: Observations and examinations:
Weight determinations in the 90d oral toxicity study: ovaries, testes, uterus (including cervix), prostate, seminal vesicles including coagulating glands
Macroscopic and microscopic in the 90d oral toxicity study: cervix, epididymides, female mammary gland area, ovaries, pituitary gland prostate gland, seminal vesicles incuding coagulation gland, testes, uterus vagina
Oestrous cyclicity (parental animals):
All females in the 90d oral toxicity study had a daily lavage from Day 72 up to and including Day 92 to determine the stage of estrous.
Sperm parameters (parental animals):
From all control and high dosed males of the 90d oral toxicity study additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Results and discussion

Results: P0 (first parental animals)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test item related changes were noted in estrous cycle determination in the 90d oral toxicity study. One control female (no. 47) showed an irregular estrous cycle length and one 200 mg/kg female (no. 65) showed extended estrus. All other females showed a normal (regular) estrous cycle of 4/5 days. The incidence of irregular estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined in the control and high dose group in the 90d oral toxicity study.

Details on results (P0)

Weight determinations in the 90d oral toxicity study showed no treatment-related effects on the reproductive organs of male and female rats (ovaries, testes, uterus (including cervix), prostate, seminal vesicles including coagulating glands)
Macroscopic and microscopic investigtions in the 90d oral toxicity study showed no treatment related alterations that differ in prevalence, severity, or histologic character to incidental tissue alterations (cervix, epididymides, female mammary gland area, ovaries, pituitary gland prostate gland, seminal vesicles incuding coagulation gland, testes, uterus vagina)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 90 day repeated dose toxicity study
Dose descriptor:
NOAEL
Remarks:
reproductive organ toxicity
Effect level:
>= 700 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 90 day repeated dose toxicity study

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Repeated dose toxicity of triethyl phosphate was investigated in two comprehensive guideline studies performed according to OECD TG 407 and 408 and conducted in compliance with GLP. In the 28 day study (performed 1992) the test item was given via oral gavage in doses of 0, 10, 100, and 1000 mg/kg bw/day to 5 male and 5 female Wistar rats per dose group. No treatment-related effects on reproductive organs were noted.

In the 90 day study (performed 2017) the test item was given via oral gavage in doses of 0, 60, 200 or 700 mg/kg bw/day to 10 male and 10 female Wistar rats per dose group.

The overall NOAEL for repeated dose toxicity is established at 200 mg/kg bw/day based on effects on liver weight in the 90 day study. The NOAEL for effects to reproductive organs is established with >= 700 mg/kg bw/day, the highest dose in 90 day study. Reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging) and morphology of gonads and accessory reproductive organs revealed no treatment-related changes.There were no effects on the weight of the gonads (males: testes and epididymes, seminal vesicles including coagulating glands; females ovaries, uterus including cervix) or the microscopic examined integrity of the gonads (males: epididymides, prostate, seminal vesicles and testes; females: uterus and ovaries, vagina) at any dose tested.

Applicant's summary and conclusion