Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study according to OECD guideline and GLP. The core of the test report is in Japanese language, but the figures, tables and appendices are in English. An translation to English is available. An English abstract is published by SCCS, a scientific body of the EU.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2003
Reference Type:
secondary source
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Fulfilling version of 1998.
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Oily, slightly viscous liquid at room temperature.
Specific details on test material used for the study:
Used lot number: WAL 4150
Made by: Wako Pure Chemical Industries, Ltd.
Purity. 99.9 %

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Details on species / strain selection:
From: Japan Charles River Co., Ltd. (Atsugi breeding center).
Sex:
male/female
Details on test animals and environmental conditions:
75 males and 75 females were introduced at 4 weeks of age, after quarantine and acclimatization for each week. They were growing smoothly and no abnormality was recognized. From these animals, 60 males and 60 females, close to the median weight (weight range at the time of administration, male: 118 - 134 g, female: 94 - 105 g) were selected and used for the test.
The animals were kept in the whole breeding period including the quarantine period in the barrier zone (AC - 1 air conditioning area).

The animals were kept at the set temperature 23 ± 2 ° C. (measured value 22.7 ± 0.4 ° C.) throughout the whole period.
Setting humidity 55 ± 15% (measured value 54 ± 2%),
Light / dark cycle: 12 hours on (8:00 - 20:00) / 12 hours off (20:00 - 8:00),
Ventilation frequency 15 - 17 times / hour
The animals were housed in individual cages (stainless steel double cage, W 170 D 294 H 176 mm).
Feed: CRF-1 of Oriental Yeast Co., Ltd. Chiba Plant (8-2, Shin-port, Mihama-ku, Chiba-shi, Chiba-shi). 30 kGy - gamma-irradiated sterilized feed. Provided during the entire breeding period from a solid feeder. Free intake.

During the quarantine period, drinking water is filtered by filtering city water (supplied by Hatano City Waterworks Bureau) and irradiated with ultraviolet rays. It was provided free by an automatic water supply device. During the acclimatization period, deionized water was allowed to be taken free by a water supply bottle. During the administration period, test drink mixed drinking water adjusted to a predetermined concentration was allowed to be ingested freely by a water supply bottle.
For the control group, only deionized water was given as during the acclimatization period. In addition, the water bottle exchange was twice a week.

Analyses of feed and drinking water was performed by the supplier.

Administration / exposure

Route of administration:
oral: drinking water
Details on route of administration:
The test substance was dissolved in drinking water and adjusted to the set concentration in a brown glass water supply bottle. The animals were allowed to take in ad libitum-
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
As a control group, city water was filtered, irradiated with ultraviolet light. A group of only ionized and filtered water (hereinafter referred to as deionized water) was provided.
The test substance was added to deionized water and stirred. The test substance was dissolved so that each set concentration was obtained. The indication of concentration is expressed in ppm (weight to weight ratio). The preparation frequency was twice weekly according to the replacement frequency of the water bottle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the mixed drinking water was measured at three points from the preparation container of each concentration at the initial preparation. The prepared concentrations of each group was in the range of 101 to 102% with respect to the set concentrations.
The frequency of exchange of test substance mixed drinking water was twice a week.

The stability of the test substance in the mixture was evaluated in a preliminary test. There, 25000 ppm and 100 ppm test substance mixed drinking water were filled in a water supply bottle for rats, stored at room temperature (5 days) and were analyzed using HPLC. It was confirmed by comparing the respective measurement results. As a result, when the concentration at the time of preparation was taken as 100%, on the fifth day, 104% at 25000 ppm, and 100 ppm were obtained..
Duration of treatment / exposure:
13 weeks (95 to 96 days).
Frequency of treatment:
The mixture was administered continuously until just prior to regular dissection.
Doses / concentrationsopen allclose all
Dose / conc.:
1 250 mg/L drinking water
Dose / conc.:
2 500 mg/L drinking water
Dose / conc.:
5 000 mg/L drinking water
Dose / conc.:
10 000 mg/L drinking water
Dose / conc.:
20 000 mg/L drinking water
No. of animals per sex per dose:
10 m + 10 f.
Control animals:
yes
yes, concurrent no treatment
Details on study design:
Concentrations:in drinking water: 0, 1250, 2500, 5000, 10000, and 20000 mg/L Basis:nominal in water.
The concentrations in drinking water corresponded to doses of :0, 96, 185, 369, 687, and 1514 mg/kg/day in males and 0, 163, 313, 652, 1000, and 1702 mg/kg/day in females on the basis of actual ingested test substance.
Dose concentrations were determined based on the results of the 2 week preliminary test.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
All animals were checked for life and death and moribund once daily.
Detailed observation of the general condition of animals was checked once weekly after administration.

The body weight was measured once a week.
The water and feed intake were determined during the administration period is once a week.

Hematological examination: At the time of periodic dissection, EDTA-2-blood from the abdominal aorta was taken under ether anesthesia immediately before necropsy
Blood was drawn into potassium-containing blood collection tube and sodium citrate collection tube. The animals to be examined were fasted for more than 18 hours from the day before the dissection. Analyses performed: red blood cell count, hemoglobin concentration, hematocrit value, mean red blood cell volume (MCV), mean red blood cell hemoglobin level (MCH), average red blood cell hemoglobin concentration (MCHC), platelet count, reticulocyte ratio, prothrombin time, activated partial thromboplasti time (APTT), white blood cell count, white blood cell differentiation.

Blood biochemical examination: Heparin blood was taken immediately before necropsy, under anesthesia with ether, from the abdominal aorta. Blood was drawn into a blood collection tube containing lithium and centrifuged, and the plasma obtained was used for the examination. The animals to be examined were fasted for more than 18 hours from the day before the dissection. Analyses performed: total protein, albumin, A / G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, GOT, GPT, LDH, ALP, gamma-GTP, CPK, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus.

Urinalysis: Collected fresh urine for all the animals in the last week (13 weeks): Determinations: pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen.







Sacrifice and pathology:
Necropsy of all animals.

Organ weight for all animals at regular dissection: Thymus, adrenal glands, testes, ovaries, heart, lung, kidney, spleen, liver, brain, thyroid.

Histopathology for all animals: Excised organs as indicated below, fixed in 10% neutral phosphate-buffered formalin solution. After, paraffin-embedded, sliced, and hematoxylin and eosin staining, histopathological was performed by an optical microscope. It was examined: Skin, nasal cavity, nasopharynx, larynx, trachea, lung, bone marrow, lymph nodes, thymus, spleen, heart, tongue, salivary glands, esophagus, stomach, small intestine (including the duodenum), large intestine, liver, pancreas, kidney, bladder, pituitary gland, thyroid gland, epithelial bodies, adrenal, testes, epididymis, seminal vesicle, prostate, ovary, uterus, vagina, mammary gland, brain, spinal cord, peripheral nerves, eyeball, Harder gland, muscle, bone.

Statistics:
Body weight, water consumption, food consumption, hematology, blood biochemical test and organ weights were analysed by first Bartlett method and then ANOVA followed by Dunnett's multiple comparison or by the rank test of Kruskal-Wallis. Incidences of lesions and urinalysis were analyzed by Chi-square test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Soiled fur around genitalia was observed in all animals administered with 20000 mg/L test substance and in a few animals exposed to 5000 and 10000 mg/L.
Mortality:
mortality observed, treatment-related
Description (incidence):
One male of the highest dosed group died in the last week. All females survived.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, a significant decreased body weight was observed in the highest dosed group in both sexes of the 20000 mg/L dose group (±19% lower compared to the control group). Decrease in body weight was also observed in females of the 10000 and 20000 mg/L groups (±8% lower compared to the control group).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the entire study a decrease in food consumption was observed in males and females in the 20000 mg/L dose groups (±20% relative to controls) and in females in the 10000 mg/L dose group (±10% relative to controls).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of males was lower for 10000 and 20000 mg/L groups until weeks 7 - 10. For 5000 mg/L group a lower water consumption was observed also for the first 3 weeks.
During the entire study water consumption was decreased in females administered with 10000 and 20000 mg/L. The decrease in females was approximately 30-40%.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Taken from the SCCS 2016 evaluation: "Haematological changes were observed including statistically significant reductions in red blood cells (at ≥10,000 ppm in males and females) and haemoglobin (at ≥10,000 ppm in females and at 20,000 ppm in males) and increases in MCV and MCH (at ≥10,000 ppm in males and at 20.000 ppm in females). Reticulocyte count was increased only in females at the top-dose group (20,000 ppm). These changes are consistent with slight anaemia at doses of ≥10,000 ppm. Statistically significant decreases in platelet counts were observed in females at doses ≥10,000 ppm and in males at doses ≥ 5,000 ppm. However, the decrease at 5000 ppm was <10% change from controls and the decreased platelets were not accompanied by evidence of a functional effect since no changes were observed in coagulation parameters (prothrombin and APPT). Furthermore, no effects on platelet counts were seen in the 104-week study in rats (see Section 3.3.7). Therefore, the observation for platelet counts in this 13-week study is considered to be of questionable toxicological significance."
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males exposed to 20000 mg/L test substance a reduction in total protein, glucose, sodium, calcium and an increase in A/G ratio, total cholesterol, phospholipids, urea nitrogen, potassium was observed. In the 10000 mg/L dose group only the effects on total protein, cholesterol, phospholipids, sodium and potassium were present. No effects were observed at lower dose levels.
In female exposed to 20000 mg/L increases in A/G ratio, ALP, and urea nitrogen was observed. In the 10000 mg/L dose group total protein was decreased and urea nitrogen was increased.
Taken from the SCCS (2016): "With the exception of urea nitrogen in both sexes and ALP in females, these statistically significant differences in relation to controls were slight (generally less than 10%) and in several cases were limited to one sex. The ALP elevation was observed only at the highest dose level in only one sex (females) and was not accompanied by other changes in liver enzymes or histopathological findings in liver or bone."
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in pH was observed after administration of 20000 mg/L in both sexes. This could be the result of excretion of 2-phenoxyacetic acid, the major metabolite of the test substance.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 10000 mg/L dose groups, the relative liver weight was increased in males and females. In addition in females absolute adrenal weight was decreased and relative kidney and brain weights were increased. In males of the 20000 mg/L dose group a decrease in absolute thymus, testes, heart, lung, and spleen was observed. In addition an increase in relative testes, lung, kidney, liver, brain, and thyroid was determined. In females of the 20000 mg/L dose group decreases in absolute thymus, adrenals, ovaries, heart, lungs, and spleen weights were determined. Relative kidney, liver, and brain weights were increased.
Taken from SCCS (2016): "While increases in relative organ weights were observed for several organs (thyroid, lung, brain, testis), most notably in males at 20,000 ppm, these observations were not accompanied by increases in absolute organ weight and were likely related to the decrease in body weight observed at 20,000 ppm. Decreases in absolute organ weights were observed in the thymus, heart, lung, spleen, testis, and ovary at 20,000 ppm and in the adrenal at ≥10,000 ppm. Since there were no histopathological findings pointing to any treatment-related effect in these organs, these findings were considered not to be indicative of organ-related toxicity.
There was a dose-related increase up to 15% in relative liver weight in both sexes at 10,000 and 20,000 ppm, but no increase in absolute liver weight in either sex. Biochemical changes in the serum (increased total cholesterol and phospholipids in males ≥ 10,000 ppm, decreased total protein in males at ≥ 10,000 ppm and in females at 10,000 ppm only, and increased albumin/globulin ratio in both sexes at 20,000 ppm) were slight and did not provide a clear pattern of toxicity, and GOT and GPT were unchanged. Therefore the relative liver weight changes may also be a reflection of the decrease on body weights. No treatment-related histopathological findings were reported in the liver which also supports the conclusion that the liver was not a target organ for toxicity. An increase in relative
kidney weight of 10% was seen in females at doses of ≥ 10,000 ppm and in both sexes up to 27% at 20,000 ppm. Absolute kidney weights were not significantly increased in either males or females."
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Herniation of liver; nodule in glandular stomach; dry subcutis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In 2 males and 1 female dosed with 10000 mg/L, slight urothelial hyperplasia of the renal pelvis was observed. Slight to moderate urothelial hyperplasia was observed in 6 males of the highest dose group. In 2 and 7 females dosed with 10000 and 20000 mg/L test substance, respectively slight to moderate urinary bladder transitional epithelial hyperplasia was observed. Slight urinary bladder transitional epithelial hyperplasia was also observed in one male of the high dose group.
Taken from the SCCS (2016): "Slight to moderate urothelial hyperplasia of the renal pelvis was observed at 10,000 ppm in males (n=2 of 10 animals) and females (n=1 of 10 animals) and at 20,000 ppm in males (n=6 of 10 animals). Slight to moderate urinary bladder transitional epithelial hyperplasia was observed at 10,000 ppm in females (n=2 of 10 animals) and at 20,000 ppm in females (n=7 of 10 animals). Slight urinary bladder transitional epithelial hyperplasia was observed in one male at 20,000 ppm. As mentioned above, urea nitrogen was increased in both sexes at 20,000 ppm and at 10,000 ppm in females. These observations are all considered treatment-related toxicologically significant effects."
Histopathological findings: neoplastic:
no effects observed
Details on results:
Taken from the SCCS (2016):
"Conclusion
The critical effects in this study pertinent to the establishment of the NOAEL are the effects on red blood cell parameters and the histopathological changes in the kidney and urinary bladder which occurred at doses ≥ 10,000 ppm. The decrease in platelets at 5000 ppm was not considered relevant for the establishment of the NOAEL due to the minimal degree of the effect and the absence of any functional impact on coagulation at any dose. Based on the overall weight of the evidence, the NOAEL is considered to be 5000 ppm corresponding to 369 mg/kg/day in males and 652 mg/kg/day in females. The LOAEL is 687 mg/kg/day in males and 1000 mg/kg/day in females."

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
369 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
652 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/L drinking water
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/L drinking water
System:
haematopoietic
Organ:
erythrocyte development
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The NOAEL in this 90-days toxicity study with rats and dosing by mixture to drinking water is 369 mg/kg bw per day, based on effects in males in the kidney, bladder and the haematopoietic system.
Executive summary:

A subchronic toxicity test with rats was performed according to OECD-Guideline 408. Dosing was performed via drinking water. The NOAEL is 369 mg/kg bw per day, based on effects in males in the kidney, bladder and the haematopoietic system.