Cyclohexanamine, 4,4'-methylenebis[N-(1-methylpropyl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 6, 2002 - July 18, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was conducted in compliance with GLP and according to OECD Guideline 407 with acceptable restrictions (all the mandatory endpoints of histopathology not performed). The original study report is in Japanese. The translated version of the study report is not of the best quality (e.g. writing and spelling). However, the study has been conducted in a consistent manner and is scientifically acceptable.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

other: Review summary

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

, all the mandatory endpoints of histopathology has not been performed.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Toxicity test in mammals by 28-day repeated administration" specified in "Partial revision of Test methods of novel chemical substances"

Deviations:

not specified

Remarks:

, the guideline was not available for the reviewer

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine

EC Number:

679-514-8

Cas Number:

154279-60-4

Molecular formula:

C21H42N2

IUPAC Name:

N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine

Constituent 2

Reference substance name:

4,4'-methylenebis(N-secbutylcyclohexanamine)

IUPAC Name:

4,4'-methylenebis(N-secbutylcyclohexanamine)

Details on test material:

- Name of test material (as cited in study report): Bis-(4-N-sec-butyl amino cyclohexyl)-methane (NH-2)
- Physical state: Clear liquid
- Analytical purity: => 99%
- Lot No.: 811176189
- Storage condition of test material: Room temperature
- Density: 0.90 g/cm3 (20°C)
- Solubility: Soluble in oil
- Dissolution: Water: Less than 32.3 mg/mL; DMSO: Less than 50 mg/mL; Acetone: 323 mg/mL or more
- Supplier: Canon Inc.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan (Hino Breeding Center, 735 Shimokomazuki, Hino-cho, Gamo-gun, Shiga 529-1633)
- Age at study initiation: 5 weeks
- Weight at study initiation: Males: 134.7 to 160.1 g; Females: 114.3 to 138.5 g
- Housing: Before grouping 5 animal/stainless steel mesh floor cage (260 W x 380 D x 180 H mm, Tokiwa Kagaku Kiki KK); ; After grouping: individually in stainless steel mesh floor cage (165 W x 300 D x 150 H mm, Tokiwa Kagaku Kiki KK)
- Diet: Solid diet (MF, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): tap water of Hita City, chlorinated water by means of automatic watering machine; ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2 °C
- Humidity: 55±10%
- Air changes: 10-15 times/hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was precisely weighed, and dissolved in olive oil, and 0.3 w/v% was prepared. Diluting from 0.3 w/v%, 0.1 and 0.03 w/v% solutions were prepared.
The test material was prepared once a week.

VEHICLE
- Lot No.: 016OAA, (Fujimi Pharmaceutical)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of test material preparing solution was measured by gas chromatograph (GC), right after preparation of solution by taking 3 samples each from top, middle and bottom layers, and diluted in 2-propanol. Uniformity was verified from relative standard deviation of test material concentration in sampling positions.

As for stability, the solution was kept in cool dark place, and 3 samples were taken from the middle layer 3 days and 7 days later, and diluted in 2-propanol, and the test material concentration was measured by GC, and presence or absence of change from the concentration right after preparation (result of uniformity test) was verified.

GC analysis conditions:
Apparatus used (HP6890)
- Data processor: HP GC-Chemstation (Hewlett Packard)
- Gas chromatograph: HP6890 Series (Hewlett Packard)
- Controller: G1512A (Hewlett Packard)
- Injector: 18593B (Hewlett Packard)

Measuring conditions:
- Column: HP-INNOWax (F.T. 0.25 µm) 0.25mm I.D.×30m
- Column temperature: 120°C (0 min) - 40°C - 240°C (5 min)
- Injection port temperature: 250°C
- Detector: FID
- Detector temperature: 250°C
- Injection amount: 1 µL
- Injection method: Splitless
- Carrier gas: Helium
- Carrier gas flow rate: 1.2 mL/min

Duration of treatment / exposure:

28 days

Frequency of treatment:

Every day in the morning.

Doses / concentrations

Remarks:

Doses / Concentrations:
3, 10 and 30 mg/kg/day
Basis:
other: nominal in olive oil

No. of animals per sex per dose:

6 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Repeated oral administration toxicity test for 14 days was conducted in three dose groups, 50, 250, and 1,000 mg/kg/day. Deaths were observed in dose groups 250 and 1000 mg/kg. In surviving animals in 250 mg/kg group, general symptoms were extremely aggravated, and animals were sacrificed on day 6. In 50 mg/kg group, changes suspected of effects of test material were found in general symptoms, body weight, hematology, serum chemistry, organ weight, and pathology. Hence, the high dose in the present test was set at 30 mg/kg/day, and lower dose groups were administered by 10 and 3 mg/kg/day. Recovery groups were provided in 30, 10 mg/kg group, and medium control group.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Three times a day from day 1 to day 28, before administration (in the morning), during and after administration, and in the afternoon; In recovery period, general symptoms were observed once a day in the morning from day 1 to day 14

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: on day -2 (when grouping) before administration, days 1, 3, 8, 12, 17, 21, 26, and 28 in administration period, and days 1 (recovery), 5, 10, and 14 in recovery period.

FOOD CONSUMPTION
- Food consumption was measured once before administration, and on days 3, 8, 15, 22, and 28 in administration period, and days 4 (recovery), 8, and 14 in recovery period.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After overnight fasting (16 to 20 hours) at the end of administration period (excluding recovery group) and at the end of recovery period, blood was sampled from the abdominal aorta
- Anaesthetic used for blood collection: Yes/ Etherization
- Animals fasted: Yes
- Parameters examined: See attached background material named, "Hematology parameters"

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After overnight fasting (16 to 20 hours) at the end of administration period (excluding recovery group) and at the end of recovery period
- Animals fasted: Yes
- Parameters examined: See attached background material named, "Serum chemistry"

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were analyzed once (day 28) in administration period (excluding recovery group), and once (day 14 (recover)) in recovery period.
- In urine (collected in about 16 hours) samples in individual rearing cages (150 W x 200 D x 263 H mm), urine volume and color were measured, together with pH, protein, ketone body, bilirubin, occult blood, sugar and urobilinogen by using test paper (N-Martistics®, Bayer Medical).

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see attached background material named, Pathology and Histopathology)
HISTOPATHOLOGY: Yes (see attached background material named, Pathology and Histopathology)

Statistics:

Results of body weight, food uptake, hematology, serum chemistry, urine volume, and organ weight were tested by equal variance of Bartlett's method, and when equal variance is noted at level of significance of 5%, data were analysed by one-way layout variance. When significant difference was noted in variance analysis, the medium control group and each dose group were tested by Dunnett's method.
When equal variance was not recognized, Kruskal-Wallis test was conducted, and when significant difference was noted, the medium control group and each dose group were tested by nonparametric Dunnett's method.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No death occurred during study at tested dose levels of 3, 10 and 30 mg/kg/day. Clinical signs are reported in a section "Details on results" below.

Mortality:

mortality observed, treatment-related

Description (incidence):

No death occurred during study at tested dose levels of 3, 10 and 30 mg/kg/day. Clinical signs are reported in a section "Details on results" below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results".

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results".

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results".

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortality on any of the tested dose level.
- General Symptoms: During administration period
Male: Salivation was observed in all dose groups (5/6 animals in 3 mg/kg group, 11/12 animals in 10 mg/kg group and 12/12 animals in 30 mg/kg group) and in the control group (8/12 animals). In all the groups, the symptom was observed sporadically or consecutively from right after administration, but the starting day varied. In the control group, the symptom was observed from day 8 to day 28, in 3 mg/kg group from day 4 to day 28, in 10 mg/kg group from day 4 to day 28 and in 30 mg/kg group from day 2 to day 28.

In the highest dose group (30 mg/kg) lowering of spontaneous motion was observed in all animals (12/12) sporadically or consecutively during observation period before administration, right after administration, and in the afternoon, from day 4 to day 28. Other observed symptoms in 30 mg/kg group were staining of nasal and peroral area (3/12), staining of lower abdomen (3/12) and rough hair (1/12). Missing of right upper incisor was noted in one case in medium control group.

Female: Salivation was observed in all dose groups (2/6 animals in 3 mg/kg group, 11/12 animals in 10 mg/kg group and 12/12 animals in 30 mg/kg group) and in the control group (6/12 animals) In all the groups, the symptom was observed sporadically or consecutively from right after administration, but the starting day varied. in the control group, the symptom was observed from day 5 to day 28, in 3 mg/kg from day 4 to day 28, in 10 mg/kg group from day 4 to day 28 and in 30 mg/kg group from day 2 to day 28.

In the highes dose group (30 mg/kg) lowering of spontaneous motion was observed in 6/12 animals sporadically or consecutively during observation period before administration, right after administration, and in the afternoon, from day 4 to day 27. Other symptoms were cyclopean eye (1/12), lacrimation (2/12), and staining of nasal and peroral area (1/12) in 30 mg/kg group.
Depilation: 1/12 animals in medium control group; 1/12 animals in 10 mg/kg group and 2/12 animals in 30 mg/kg group.

- General Symptoms: During recovery period
Male: In 30 mg/kg group, lowering of spontaneous motion (1/6), and staining of nasal and peroral area (1/6) were noted.
Female: No abnormality was found.
The summary of clinical signs is presented in Table 1.

BODY WEIGHT AND WEIGHT GAIN
- During administration period
Male: In 30 mg/kg group, low values were noted from day 21 to 28, and a lowering tendency was noted on day 17.
Female: In 30 mg/kg group, low values were noted on day 28.

- During recovery period
Male: In 30 mg/kg group, a lowering tendency was noted from day 1 (recovery) to day 14 (recovery).
Female: In 30 mg/kg group, low values were noted on day 5 (recovery), and a lowering tendency was noted on day 1 (recovery), day 10 (recovery), and day 14 (recovery).
Summary of body weights is presented in Table 2.

FOOD UPTAKE
- During administration period
Male: In 30 mg/kg group, low values were noted on day 3, day 22, and day 28.
Female: In 30 mg/kg group, low values were noted on day 28.

- During recovery period
Male: In 30 mg/kg group, a lowering tendency was noted from day 4 to day 14.
Female: In 30 mg/kg group, a lowering tendency was noted from day 4 to day 14.

The summary of food intakes is presented in Table 3.

OPHTHALMOSCOPIC EXAMINATION
- Not examined

HAEMATOLOGY
- End of administration period
Male: In 30 mg/kg group, increasing tendency of platelets and segmented neutrophils in differentiation of leukocytes and a decreasing tendency of lymphocytes. In 3 mg/kg group, the activated portion thromoboplastin time was extended.
Female: In 10 mg/kg and higher dose groups, increase of monocytes in differentiation of leukocytes was noted, and in 30 mg/kg group, an increasing tendency of platelets and segmented neutrophils in differentiation of leukocytes and a decreasing tendency of lymphocytes were observed.

- End of recovery period
Male: In 10 mg/kg group, platelets decreased.
Female: In 30 mg/kg group, increase of segmented neutrophils in differentiation of leukocytes and decrease of lymphocytes were observed.
The summary of hematological examinations is presented in Table 4.

CLINICAL CHEMISTRY
- End of administration period
Male: In 30 mg/kg group, increase of GPT and inorganic phosphorus, decrease of alkaline phosphatase, total protein, creatinine, and total bilirubin, and increasing tendency of GOT and blood urea nitrogen were observed.
Female: In 10 mg/kg and higher dose groups, decrease of total cholesterol was observed, and in 30 mg/kg group, increase of GPT and blood urea nitrogen, decrease alkaline phosphatase, and increasing tendency of GOT were noted. Besides, increase of sodium was noted in 3 and 30 mg/kg groups, and decrease of potassium in 3 mg/kg group.

End of recovery period
Male: In 30 mg/kg group, decrease of alkaline phosphatase and increasing tendency of GOT were noted.
Female: In 30 mg/kg group, increase of GOT and GPT, and decrease of alkaline phosphatase were noted.

The summary of blood chemical examinations is presented in Table 5.

URINALYSIS
-No abnormality was observed in either male or female animals at the end of the administration period and end of recovery period.
NEUROBEHAVIOUR
-Not examined
ORGAN WEIGHTS
- End of administration period
Male: In 30 mg/kg group, relative weight increase of liver, kidney, testis, brain and adrenal, and absolute weight increase of adrenal were observed.
Female: In 30 mg/kg group, relative weight increase of liver, kidney, and adrenal, and absolute weight increase of adrenal were observed.

- End of recovery period
Male: In 30 mg/kg group, absolute and relative weight increase of adrenal was observed.
Female: In 30 mg/kg group, relative weight increase of liver, brain, and adrenal was
observed.
Summaries of absolute and relative organ weights are presented in Tables 7 and 8, respectively.

HISTOPATHOLOGY
- End of administration period
Male: In 10 mg/kg group, observed changes include foam cells (+, 1/6) of lung, foamy changes (+, 1/6) of bile duct epithelium in liver, diffuse hyperplasia (+, 2/6) of transitional epithelium of urinary bladder, and fine vacuolation (±,2/6; +, 2/6) of transitional epithelium; and in 30 mg/kg group, observed changes include foam cells (+, 3/6; ++, 3/6) of lung, foamy changes (+, 5/6; ++, 1/6) of bronchial epithelium, complex keratinization (+, 1/6) of proventriculus, foam cells (±, 1/6; +, 3/6) in tunica mucosa propria of jejunum, foam cells (+, 1/6) in tunica mucosa propria of ileum, fine vacuolation (+, 6/6) of hepatocytes in liver, foam cells (+, 2/6) in sinusoid, foamy changes (++, 6/6) of bile duct epithelium, localized myocarditis (+, 3/6) of heart, fine vacuolation (±, 2/6; +, 4/6) of transitional epithelium of kidney, diffuse hyperplasia (+, 3/6; ++, 2/6) of transitional epithelium of urinary bladder, fine vacuolation (+, 6/6) of transitional epithelium, foamy changes (+, 6/6) of high epithelial cells in axillary lymph nodes, foam cells (±, 1/6; +, 5/6) in lymphatic sinus of mesenteric lymph nodes, foamy changes (+, 6/6) of high epithelial cells, foamy changes (+, 6/6) of high epithelial cells in agmen peyerianum, foam cells (±, 3/6; +, 3/6) in red splenic pulp of spleen, and fine vacuolation (+, 6/6) and cortical hypertrophy (+, 1/6) of adrenal cord bands. Besides, in medium control group, observed changes include pyelectasis (+, 1/6) of kidney, solitary cyst (+, 1/6) of medulla, solitary cyst (+, 1/6) under capsule, localized necrosis of spleen (++, 1/6), and extramedullar hyperhemopoiesis (+, 1/6).

Female: In 10 mg/kg group, observed changes include fine vacuolation (+, 2/6) of hepatocytes in liver, foamy changes (+, 6/6) of bile duct epithelium, and microgranuloma (+, 1/6); and in 30 mg/kg group, observed changes include foam cells (+, 5/6; ++, 1/6) of lung, foamy changes (+, 5/6; ++, 1/6) of bronchial epithelium, fine vacuolation (+, 6/6) of hepatocytes in liver, foam cells (+, 2/6) in sinusoid, foamy changes (++, 6/6) of bile duct epithelium, microgranuloma (+, 1/6; ++, 2/6), fine vacuolation (±, 2/6; +, 3/6) of transitional epithelium of kidney, diffuse hyperplasia (+, 4/6) of transitional epithelium of urinary bladder, fine vacuolation (±, 2/6; +, 4/6) of transitional epithelium, foamy changes (±, 2/6; +, 4/6) of high epithelial cells in axillary lymph nodes, foam cells (+, 5/6) in lymphatic sinus of mesenteric lymph nodes, foamy changes (+, 6/6) of high epithelial cells, foamy changes (±, 4/6; +, 1/6) of high epithelial cells in agmen peyerianum, foam cells (±, 1/6; +, 5/6) in red splenic pulp of spleen, and fine vacuolation (+, 6/6) and cortical hypertrophy (+, 5/6) of adrenal cord bands. Besides, in medium control group, observed changes include solitary cyst (+, 1/6) under capsule of kidney, and follicle decrease (+, 1/1) in skin; and also follicle decrease (+, 1/1) in skin was also observed in 10 mg/kg group.

- End of recovery period
Male: In 10 mg/kg group, diffuse hyperplasia (+, 1/6) of transitional epithelium of urinary bladder was observed; and in 30 mg/kg group, observed changes include foam cells (+, 3/6; ++, 1/6) of lung, foamy changes (+, 2/6) of bronchial epithelium, foamy changes (+, 6/6) of bile duct epithelium of liver, fine vacuolation (±, 1/6) of transitional epithelium of kidney, diffuse hyperplasia (+, 3/6) of transitional epithelium of urinary bladder, fine vacuolation (±, 1/6) of transitional epithelium, and fine vacuolation (+, 2/6) and cortical hypertrophy (+, 1/6) of adrenal cord bands. Besides, in medium control group, localized myocarditis (+, 1/6) of heart was observed.

Female: In 30 mg/kg group, observed changes include foam cells (+, 3/6) of lung, foamy changes (+, 1/6) of bronchial epithelium, foamy changes (+, 4/6; +,2/6) of bile duct epithelium of liver, microgranuloma (++, 4/6), diffuse hyperplasia (+, 1/6) of transitional epithelium of urinary bladder, foamy changes (±, 5/6) of high epithelial cells in axillary lymph nodes, foamy changes (±, 6/6) of high epithelial cells in mesenteric lymph nodes, foamy changes (±, 1/6) of high epithelial cells in agmen peyerianum, and fine vacuolation (+, 2/6) and cortical hypertrophy (+, 1/6) of adrenal cord bands.

The summary of histopahological examinations is presented in Table 10.
AUTOPSY
- End of administration period
Male: In medium control group, pyelectasis (1/6) of kidney, swelling (1/6) and whitening (1/6) of spleen were observed.
Female: Depilation (1/6) of skin was noted in medium control group, depilation (1/6) of skin in 10 mg/kg group, and depilation (1/6) of skin in 30 mg/kg group.

- End of recovery period
No abnormality was observed in either male or female animals.

The summary of macroscopic examinations is presented in Table 9.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

in olive oil

Sex:

male/female

Basis for effect level:

other: Vacuolative changes in various organs of rats from the 10 mg/kg/day group.

2

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

in olive oil

Sex:

male/female

Basis for effect level:

other: Absence of clinicopahological evidence of hepatic injury associated with the vacuolative changes in 10 mg/kg/day group..

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP study conducted according to OECD Guideline 407 test substances (trade name Clearlink 1000) potential toxicity of repeated administration was evaluated. 6 Crj:CD (SD) IGS rats (male/female) were dosed daily by gavage with dose levels of 30, 10 and 3 mg/kg/day for 28 days. A recovery group was organized in 10 and 30 mg/kg/day groups and control group.

No death occured during the study. No signs of test substance related toxicity were observed in autopsy or in urinanalysis. Bode weight gain and food uptake were low in both sexes in the highest (30 mg/kg/day) dose level groups during the administration. Clinical signs (lowering of spontaneous motion, staining of nasal and peroral area, cyclopean eye and lacrimation in females, staining of lower abdomen and rough hair in males) were predominantly observed in the highest dose (30 mg/kg/day) group rats. Also increase in organ weights (relative weight of liver and kidneys, and absolute and relative weight of adrenals ) were observed in the highest dose group (30 mg/kg/day) rats.

In female rats in dose groups 10 mg/kg and 30 mg/kg/day, increase of monocytes in differentiation of leucocytes was noted. In 30 mg/kg/day group (male and female rats) increase of platelets and neutrophils and decrease of lymphocytes in ditterentation of leucocytes was observed. Female rats total cholesterol decreased in 10 mg/kg/day and 30 mg/kg/day groups. In the highes dose level (30 mg/kg/day) both males and females total cholesterol decreased, GOT, GPT, blood urea and blood nitrogen increased and alkaline phoshatase decreased. Inorganic phosphorus increased and total protein, creatine and total bilirubin decreased in the highes dose (30 mg/kg/day) group males.

Following histopathological findings were made in male and female rats in 10 mg/kg and 30 mg/kg dose groups: Foamy changes of bile duct epihelium of liver in males, foam cells in lungs, diffuse hyperplasia of transitional epithelium and fine vacuolation of transitional upithelium in urinary bladder in females. Fine vacuolation of hepatocytes and microgranuloma in liver in males and females in 30mg/kg/day group, foamy changes of bronchial epithelium of lungs, foam cells in sinusoids of liver, fine vacuolation of transitioinal epithelium of kidney, foamy changes of high epithelial cells of axillary lympnodes, foam cells in lymphatic sinus and foamy changes of high epitheliala cells in mesentric lymph nodes, foamy changes of high epithelial cells in agmen peyerianum, foam cells in red splenic pulp of spleen and fine vacuolation and cortical hypertrophy of adrenal cord bands in males, compex keratinization of proventriculus, foam cells in tunica mucosa propria of jejunum, foam cells in tunica mucosa propria of ileum, fine vacuolation of hepatocytes in liver and localized myocarditis of hear in females, foam cells of lungs, diffuse hyperplasia of transitional epithelium and fine vacuolation of transitional epithelium in urinary bladder.

Under the test conditions, the NOEL was indicated to be 3 mg/kg/day based on the presence of vacuolative changes in various organs of rats from the 10 mg/kg dose group. A review of the original study report was made at 2011 by George A. Parker. In the review summary report it is speculated that even the histopathological findings support the estimation of the NOEL, it should be noted that vacuolative changes of the type described in the report do not necessary indicate an adverse effect. Similar vacuolative changes have been observed in safety assessment studies of a number of merketed pharmaceutical products, and were determined to represent phospholipidosis.

In addition, clinical pathology indications of hepatic injury were observed only in rats at the 30 mg/kg/day group. The absence of clinical pathology indications of hepatocellular injury in the 10 mg/kg/day group posibly indicates, that the vacuolative changes were not adverse in that group. Based on this, the NOAEL is considered 10 mg/kg/day.

The results of this study would not lead to the classification for repeated dose toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.