N-1-naphthylaniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Dec 2014 to 25 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Test guideline 5

Qualifier:

according to guideline

Guideline:

EU Method B.43 (Neurotoxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

N-1-naphthylaniline

EC Number:

201-983-0

EC Name:

N-1-naphthylaniline

Cas Number:

90-30-2

Molecular formula:

C16H13N

IUPAC Name:

N-phenylnaphthalen-1-amine

Details on test material:

- Stability: The stability under storage conditions over the test period was guaranteed
- Physical state/appearance: solid/white to light red
- Storage conditions: ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 49 ± days
- Weight at study initiation: mean males: 193.3 – 196.7 g, mean females: 147.5 – 150.9 g
- Housing: 5 animals per cage, in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dustfree wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria and Play tunnel large (Art. 14153), PLEXX B.V., Elst, Netherlands were added for environmental enrichment.
- Diet: ad libitum, Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, in water bottles
- Acclimation period: 20 – 25 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently mixed by a magnetic stirrer with a hotplate at 40°C, for 1h. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in corn oil. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90-110% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of the test item.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

In order to balance the groups for functional observational batteries and motor activity, the study was conducted with several subsets (Section A males and Section A females = first 5 animals of each test group; Section B males and Section B females = second 5 animals of each test group; Section C males and Section C females = third 5 animals of each test group). For functional observational batteries and measurements of motor activity, the animals were tested in randomized order. Thus, it was ensured, that:
- all animals were examined within the same day after start of test substance administration. Time of testing was therefore identical for all animals;
- for each examination day animals from all test groups (including controls) could be used;
- the examinations for all subsets could be performed on the same time of the day, thus potential diurnal effects could be negated

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50×37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals. Body weight was also determined on the days when functional observational batteries were carried out. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.

FOOD CONSUMPTION
Individual food consumption was determined weekly over a period of 3 days and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION
Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION
The eyes of all animals of Section B and C were examined prior to the start of the administration period. At the end of the administration period, i.e. study days 85 for male animals and study day 84 for female animals, the eyes of animals in test groups 0 (control) and 4 (125 mg/kg bw/d) of Section B and C were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, Heinz bodies. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). The prothrombin time was determined.

CLINICAL CHEMISTRY
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS
For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Gerrmany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Gerrmany). The following parameters were determined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume.

INTERFERENCE TEST
A stock solution of the compound of 1 g/L in 95% ethanol was established. This solution was diluted in two rat urine samples and two rat serum samples obtaining compound concentrations of 100, 10, 3.3, 1 and 0.1 mg/L in urine and 10, 3.3, 1 and 0.1 mg/L in serum. In each concentration sample the following parameters were measured: serum: total bilirubin; urine: protein, glucose, ketone bodies, urobilinogen, bilirubin and blood.

NEUROBEHAVIOURAL EXAMINATION: FUNCTIONAL OBSERVATIONAL BATTERY
Functional observational batteries (FOBs) were performed in all animals of Section A and B before the administration period (day -7) and on study days 1, 22, 50 and 85. The FOBs were performed each time from about 10.00 h onwards, starting with passive observations without disturbing the animals, followed by removal from the home cage and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. findings were ranked according to the degree of severity, if applicable.

- Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings
- Open field observations:
The animals were transferred to a standard arena (50×50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes
- Sensorimotor tests/reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision (“visual replacing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

NEUROBEHAVIOURAL EXAMINATION: MOTOR ACTIVITY ASSESSMENT
The motor activity assessment (MA) was carried out in all animals of Section A and B at the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed single in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Sacrifice and pathology:

GROSS PATHOLOGY (animals of section A)
- The first five surviving animals per sex and test group were sacrificed by perfusion fixation. SOERENSEN's phosphate buffer served as rinsing solution and the fixation solution according to KARNOVSKY served as fixative. The animals fixed by perfusion were necropsied with regard to the question of neuropathology and the visible organs or organ sections were assessed by gross pathology as accurately as it is possible after a perfusion fixation.
- The following organs/tissues were preserved in neutral buffered 4% formaldehyde: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, larynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina, brain (remaining material after trimming), spinal cord (parts of cervical and lumbar cord). The remaining organ material and the animal body were stored.

ORGAN WEIGHTS (animals of section A)
The following weights were determined: brain (without olfactory bulb)

HISTOPATHOLOGY (animals of section A)
- The following organ samples of the brain were processed histotechnically: frontal lobe, parietal lobe with diencephalon and hippocampus, midbrain with occipital and temporal lobe, pons, cerebellum, medulla oblongata. Samples obtained from test group 0 (control) and 3 (125 mg/kg bw/d) were stained with hematoxylin-eosin (HE) stain. Samples obtained from test group 1 (5 mg/kg bw/d) and 2 (25 mg/kg bw/d) were embedded in paraplast.
- The following other organs samples were processed histotechinically: eyes with retina and optic nerve, cervical cord (C3-C6), lumbar cord (L1-L4), trigeminus ganglia with nerve, gastrocnemius muscle, all gross lesions. Samples obtained from test group 0 (control) and 3 (125 mg/kg bw/d) were stained with hematoxylin-eosin (HE) stain. Samples obtained from test group 1 (5 mg/kg bw/d) and 2 (25 mg/kg bw/d) were preserved in 4% formaldehyde.
- The following organ specimens were removed after perfusion: dorsal root ganglion (3 of C3-C6), dorsal root fiber (C3-C6), ventral root fiber (C3-C6), dorsal root ganglion (3 of L1-L4), dorsal root fiber (L1-L4), ventral root fiber (L1-L4), proximal sciatic nerve (cross and longitudinal sections), proximal tibial nerve (at knee, cross and longitudinal sections), distal tibial nerve (at lower leg, cross and longitudinal sections). Samples obtained from test group 0 (control) and 3 (125 mg/kg bw/d) were embedded in an epoxy resin, sectioned with semithin and stained with Azure II-Methylene blue-basic Fuchsin (AMbf). Samples obtained from test group 1 (5 mg/kg bw/d) and 2 (25 mg/kg bw/d) were fixed and stored in the buffer solution.

GROSS PATHOLOGY according to OECD 408 (animals of sections B and C)
- Animals were sacrificed by decapitation.
- The following organs/tissues were preserved in neutral buffered 4% formaldehyde: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, larynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

ORGAN WEIGHTS (animals of sections B and C)
The following weights were determined in all animals sacrificed: anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, spleen, testes, thymus, thyroid glands, uterus with cervix

HISTOPATHOLOGY (animals of sections B and C)
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings was performed as follows:
- The following organs were assessed in all test groups: all gross lesions, kidneys (in low and mid dose group only males), liver, spleen
- The following organs were assessed only in the high dose group and the control group: adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum (with Peyer’s patches), larynx, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, larynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
For further determination of the pigment stored in the spleen, the spleens of animals No 38, 39, 106 and 125 were stained with Perl’s Prussian blue stain and Turnbull stain (for detection of iron).

Statistics:

Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed as follows: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means for body weight and body weight change. Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided) was used for rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, blood parameters, urine pH, volume, specific gravity and weight parameters. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians was used for Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related, adverse findings were observed in animals of test groups 1 to 3 (5, 25 and 125 mg/kg bw/d). Salivation after treatment was observed in all male and female animals of test group 3 (125 mg/kg bw/d) on several study days. The finding occurred for the first time on study day 12 in male animals and on study day 11 in female animals. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by the bad taste of the test substance or local affection of the upper digestive tract. One left-sided semi-closed eyelid was observed in male animal No. 16 of test group 3 (125 mg/kg bw/d) from study day 48 onwards until sacrifice. This single finding was assessed to be spontaneous in nature and not related to treatment. Reddish discolored urine was detected in all male and female animals of test group 3 (125 mg/kg bw/d), i.e. in male animals of Section A from study day 60 onwards, in male animals of Section B and C from study day 58 onwards as well as in female animals of Section A from study 57 onwards and in female animals of Section B and C from study day 55 onwards. The finding was assessed to be related to treatment but not adverse.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related effects on body weight development, i.e. mean body weight and body weight change values, were observed for male and female animals of test groups 1-3 (5, 25 and 125 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Increased food consumption values were observed for male and female animals of test groups 1 to 3 over the entire application period when compared to the control values. Significant deviations were observed in male animals of test group 3 (125 mg/kg bw/d) on study days 21 to 49, 63, 70 and 84 with a maximum of +25% on study day 42 and in female animals on study days 28 and 35 with a maximum of +21% on study day 28. In addition, a significantly increased value was observed in female animals of test group 1 (5 mg/kg bw/d) on study day 77. Although significant changes mainly occurred in the high-dose group, the findings were assessed not to be of toxicological relevance as no effects on body weight parameters became obvious. The reason was most likely spilling of food or a reaction to the bad taste of the applied test substance preparation.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

No overt changes with respect to water consumption were observed visually for animals treated with the test substance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings. All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in rats of both sexes of test group 3 (125 mg/kg bw/d) a regenerative anemia was present: red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased and relative reticulocyte counts were increased. Additionally, in males of the mentioned test group mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were increased. In males of test group 3 (125 mg/kg bw/d) absolute basophil counts were higher compared to controls. The median was marginally above the historical control range (absolute basophils 0.00-0.02 Giga/L) and this was the only altered differential blood cell parameter. Therefore, this change was regarded as treatment-related but not adverse. In males of test group 1 (5 mg/kg bw/d) RBC counts were higher compared to controls. This change was not dose-dependent and, therefore, it was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in rats of both sexes of test group 3 (125 mg/kg bw/d) creatinine values were decreased and total bilirubin values were increased. Both mentioned parameters were also changed in the same way in females of test group 2 (25 mg/kg bw/d). In males of test group 1 and 2 (5 and 25 mg/kg bw/d) total bilirubin values were also higher compared to controls. The mentioned changes in test groups 1 and 2 were within historical control ranges (females: creatinine 28.7-40.7 μmol/L, total bilirubin 1.29-3.12 μmol/L; males, total bilirubin 0.56-2.76 μmol/L), however taking into account the dose-dependent increase, this may indicate a treatment-related effect already at 5 mg/mg bw/d. However, the assigned study director considered these changes in test groups 1 and 2 to be incidental and not treatment-related. In males of test group 3 (125 mg/kg bw/d) urea values were increased and cholesterol values were decreased. The urea mean was within the historical control range (urea 4.38-5.88 mmol/L) and, therefore, this alteration was regarded as incidental and not treatment-related. In females of test groups 1 and 3 (5 and 125 mg/kg bw/d) total protein and albumin levels were higher compared to controls, but the values were within historical control ranges (total protein 63.21-68.55 g/L, albumin 37.14-43.65 g/L). Therefore, these changes were regarded as incidental and not treatment-related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Because of the dark-brown colored urine of the rats in test group 3 (125 mg/kg bw/d), the dipsticks evaluating endogen analytes in the urine with dry chemistry reactions could not be used apart from one male sample. Urine sediment microscopy could be performed from all samples including test group 3. In rats of both sexes of test group 2 (25 mg/kg bw/d) bilirubin excretion was higher compared to controls. This was also true for females of test group 1 (5 mg/kg bw/d) and in the male sample of test group 3 (125 mg/kg bw/d). In males of test groups 2 and 3 (25 and 125 mg/kg bw/d, test group 3 one sample) urobilinogen levels in the urine were higher. Additionally, blood was found in the male sample of test group 3. In rats of both sexes of test groups 1 and 2 (5 and 25 mg/kg bw/d) as well as in the one male sample of test group 3 (125 mg/kg bw/d) glucose was found in the urine. In females of test group 2 (25 mg/kg bw/d) urine pH values were higher compared to controls. This finding is not related to the other mentioned changes in the urine and it is per se not an adverse effect. Regarding urine sediment microscopy, only slightly higher incidences of transitional epithelial cells in males of test group 3 (125 mg/kg bw/d) were observed. This amount of transitional epithelial cells can also be found in controls and, therefore, this observation was regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals of test groups 1-3 (5, 25 and 125 mg/kg bw/d) when compared to the control animals.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (set to 100%), the following mean absolute weights were significantly changed: the kidneys and livers of both males and females of test groups 3 (125 mg/kg bw/d); the liver of males in test group 2 (25 mg/kg bw/d).
When compared with control group 0 (set to 100%), the following mean relative organ weights were significantly changed: the kidneys and livers of both males and females of test groups 3 (125 mg/kg bw/d); the liver of males in test group 2 (25 mg/kg bw/d) and females in test group 1 (5 mg/kg bw/d).
The kidney weight increase in males and females of test group 3 (125 mg/kg bw/d) might be treatment-related but not adverse due to missing histopathologic findings (for females) or no histopathologic finding that could cause such a weight increase (for males). The liver weight increase in males of test groups 2 and 3 (25 and 125 mg/kg bw/d) and females of test group 3 (125 mg/kg bw/d) were regarded to be treatment-related.
Applying a linear mixed model approach (‘sex’ as random factor) a significant increase in spleen weight was observed at 5 mg/kg bw/d (p = 0.034).

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Mean absolute and relative weight parameters did not show significant differences when compared to the control group 0. A single gross lesion finding occurred. This was considered to be incidental or spontaneous in origin and without any relation to treatment. No neuropathological lesions were noted during histopathlogy.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys of three and eight male animals of test group 2 and 3, respectively, degeneration/regeneration mainly of the proximal tubules was observed. Degeneration was characterized by increase of cellular eosinophilia, desquamated cells or necrosis/apoptosis whereas regeneration was represented by increase in basophilia and large vacuolar nuclei of tubular epithelial cells. The chronic progressive nephropathy (CPN) is a well known phenomenon in the male aging rat. Chemicals can exacerbate the lesion of CPN or cause similar findings compared to CPN. These findings were regarded to be treatment-related. In test group 1 animals (5 mg/kg bw/d) the CPN was not regarded to be treatment-related, as the incidence was comparable to control animals.
In the livers of females of test group 3 (125 mg/kg bw/d) and males of test group 2 and 3 (25 and 125 mg/kg bw/d) a centrilobular hypertrophy was observed. This finding was regarded to be treatment-related.
In the spleen in females of all test groups there was an increase in extramedullary hematopoiesis. This means an increase in blood cell production that in adult animals usually takes place in the bone marrow. Two animals of test group 1 showed a grade 1 or grade 2 response, respectively, while only a grade 1 response was found in 1 animal of test group 2. At 125 mg/kg bw/d, grade 2 or 3 responses were observed in a total of 3 animals, indicating increased severity and incidence at the highest dose level. In test group 3 animals (125 mg/kg bw/d) this finding was regarded to be treatment-related. Due to the missing dose-response relationship in test groups 1 and 2 (5 and 25 mg/kg bw/d) in these two test groups the assigned study director considered this finding to be incidental. Furthermore, males of test group 3 (125 mg/kg bw/d) and females of all treated groups revealed an increase in brownish pigment storage in the spleen. By special staining (Perl’s Prussian blue stain and Turnbull stain) it could be demonstrated that this pigment was a Fe-containing compound and, therefore, regarded to be hemosiderin, a degradation product of the red blood pigment. These findings were regarded to be treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Interference test: The parent compound interfered with the serum bilirubin measurement down to a concentration of 0.1 mg/L. In urine samples the bilirubin measurement interfered at compound concentration of 100 mg/L, blood (at least in one sample) and ketone body tests down to a concentration of 1.0 and 0.1 mg/L, respectively.

Effect levels

Dose descriptor:

LOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Organ weights

Absolute organ weights

When compared with control group 0 (set to 100%), the following mean absolute weights were significantly changed:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (5)	2 (25)	3 (125)	1 (5)	2 (25)	3 (125)
Kidneys	99%	109%	114%**	101%	101%	115%**
Liver	100%	117%**	128%**	108%	101%	131%**

* : p ≤ 0.05, **: p ≤ 0.01

Relative organ weights

When compared with control group 0 (set to 100%), the following mean relative organ weights were significantly changed:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (5)	2 (25)	3 (125)	1 (5)	2 (25)	3 (125)
Kidneys	98%	102%	114%**	101%	105%	116%**
Liver	99%	111%*	129%**	109%*	106%	132%**

* : p ≤ 0.05, **: p ≤ 0.01

Histopathology

Treatment-related findings were observed in males and females with incidences and grading according to the tables below:

Kidneys	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10
Degeneration/regeneration	0	0	3	8
·    Grade 1			3	2
·    Grade 2				2
·    Grade 3				4
Nephropathy, chronic	1	2	6	5
·    Grade 1	1	2	6	1
·    Grade 2				4

In the kidneys of male animals degeneration/regeneration mainly of the proximal tubules were observed. Degeneration was characterized by increase of cellular eosinophilia, desquamated cells or necrosis/apoptosis whereas regeneration was represented by increase in basophilia and large vacuolar nuclei of tubular epithelial cells. The chronic progressive nephropathy (CPN) is a well known phenomenon in the male aging rat. Chemicals can exacerbate the lesion of CPN or cause similar findings compared to CPN (Frazier et al., 2012). These findings were regarded to be treatment-related. In test group 1 animals (5 mg/kg bw/d) the CPN was not regarded to be treatment-related, as the incidence was comparable to control animals.

Liver	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (5)	2 (25)	3 (125)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10	10	10	10	10
Hypertrophy, centrilobular	0	0	5	9	0	0	0	10
·    Grade 1			5
·    Grade 2				9
·    Grade 3								10

In the livers of females of test group 3 (125 mg/kg bw/d) and males of test group 2 and 3 (25 and 125 mg/kg bw/d) a centrilobular hypertrophy was observed. This finding was regarded to be treatment-related and correlated to the observed liver weight increases. For males and females of test group 3 (125 mg/kg bw/d) this finding was regarded to be treatment related and adverse due to the magnitude of weight increase (+30%) and the severity of the hypertrophy (Grade 2-3). In males of test group 2 (25 mg/kg bw/d) the weight increase was only +11% and the hypertrophy Grade 1. As no other findings were observed this finding for the male animals of test group 2 (25 mg/kg bw/d) was regarded to be treatment-related but not adverse.

Spleen	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (5)	2 (25)	3 (125)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10	10	10	10	10
Hematopoiesis, extramedullary	0	0	0	0	0	2	1	3
·    Grade 1						1	1
·    Grade 2						1		2
·    Grade 3								1
Pigment storage	2	0	0	6	1	6	9	5
·    Grade 1						5	8
·    Grade 2	2			6	1	1	1	5

In the spleen in females of all test groups there was an increase in extramedullary hematopoiesis. This means an increase in blood cell production that in adult animals usually takes place in the bone marrow. In test group 3 animals (125 mg/kg bw/d) this finding was regarded to be treatment-related. Due to the missing dose-response relationship in test groups 1 and 2 (5 and 25 mg/kg bw/d) in these two test groups the assigned study director considered this finding to be incidental. Furthermore, males of test group 3 (125 mg/kg bw/d) and females of all treated groups revealed an increase in brownish pigment storage in the spleen. By special staining (Perl’s Prussian blue stain and Turnbull stain) it could be demonstrated that this pigment was a Fe-containing compound and, therefore, regarded to be hemosiderin, a degradation product of the red blood pigment. This finding matches the previously described anemia observed during clinical pathology and the anemia was regarded to be hemolytic in nature with deposition of hemosiderin in the spleen. These findings were regarded to be treatment-related. For test group 3 males and females (125 mg/kg bw/d) this finding was regarded to be treatment-related and adverse. In females of test group 3 (125 mg/kg bw/d), a slight increase in extramedullary hematopoiesis occurred. This was interpreted as a reaction to the anemia and, therefore, assessed to be adverse. Some females of test group 1 and 2 (5 and 25 mg/kg bw/d) showed extramedullary hematopoiesis and pigment storage as well.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test item to Wistar rats over a period of 3 months revealed signs of systemic toxicity in animals at a dose level of 5 mg/kg bw/d taking pathology and hematology findings into account. Although the assigned study director considered the effects at the lowest dose as incidental and non-adverse, the evaluating MSCA considers the lowest tested dose of 5 mg/kg bw/day as a LOAEL instead of a NOAEL, because at this level significantly increased bilirubin levels in plasma of exposed animals, extramedullary haematopoiesis and pigment storage in spleen, as well as bilirubin in urine and a significant increase in spleen weight was observed, indicative of a haemolytic anemia. All these findings are considered to be dose dependent starting at the lowest tested dose of 5 mg/kg bw/day. Under the conditions of this study, the lowest observed adverse effect level (LOAEL) for systemic toxicity was 5 mg/kg bw/d.

However, no adverse neurobehavioral effects in male and female Wistar rats were observed at any dose level. In addition, no test substance related effects were observed during the neurohistopathology investigation at any dose level. The NOAEL for neurotoxicity was 125 mg/kg bw/d in male and female animals (highest dose tested).

Executive summary:

The test item was administered orally by gavage to groups of 15 male and 15 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 5 mg/kg bw/d (test group 1), 25 mg/kg bw/d (test group 2) and 125 mg/kg bw/d (test group 3) over a period of 3 months. Corn oil served as vehicle. Every test group was divided into 3 sections, i.e. sections A, B and C, each containing 5 animals per sex, to cover the requirements of both study types, i.e. a repeated-dose 90-day oral toxicity study according to OECD 408 and a repeated-dose 90-day neurotoxicity study according to OECD 424.

For all animals of sections A, B and C, food consumption and body weights were determined once a week. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Functional observational batteries and motor activity measurements were carried out in 10 animals per sex and test group on study days -7, 1, 22, 50 and 85 (animals of sections A and B). Ophthalmological examinations were performed in 10 animals per sex and test group before the beginning and at the end of the administration (animals of sections B and C). Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period (animals of sections B and C). Five animals per sex and test group were fixed by in situ perfusion and subjected to neuropathological examinations (animals of section A). The remaining animals were sacrificed and assessed by gross pathology, followed by organ weight determinations and histopathological examinations (animals of sections B and C).

The following test substance-related, adverse findings were noted:

Test group 3: 125 mg/kg bw/d

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in both sexes

• Increased relative reticulocyte counts in both sexes

• Increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in males

• Decreased cholesterol values in males

• Increased urobilinogen levels in the urine sample of one male animal

• Increased mean absolute and relative liver weights in male and female animals, i.e. +28% (absolute) and +29% (relative) in males as well as +31% (absolute) and +32% (relative) in females, in correlation to a mild to moderate centrilobular hypertrophy

• Minimal to moderate degeneration/regeneration of proximal tubules in kidneys of eight males

• Minimal to slight chronic nephropathy in kidneys of five males

• Slight to moderate extramedullary hematopoiesis in the spleen of three females

• Minimal to slight pigment storage in the spleen (hemosiderosis) in six males and five females

Test group 2: 25 mg/kg bw/d

• Increased urobilinogen levels in the urine of males

• Minimal degeneration/regeneration of proximal tubules in kidneys of three male animals

• Minimal chronic nephropathy in kidneys of six males

Test group 1: 5 mg/kg bw/d

• No findings were considered test substance-related or adverse by the study director, however, the following findings were considered by the evaluating authorities:

• Significant increased bilirubin levels in plasma of exposed animals

• Onset of extramedullary haematopoisesis in females

• Pigment storage in spleen in females

• Bilirubin in urine in females

• Significant increase in spleen weight when applying a linear mixed model approach ('sex' as random factor) (p= 0.034)

All findings are considered indicative of a haemolytic anemia.

The oral administration of the test item to Wistar rats over a period of 3 months revealed signs of systemic toxicity in animals at a dose level of 5 mg/kg bw/d taking pathology and hematology findings into account. Although the assigned study director considered the effects at the lowest dose as incidental and non-adverse, the evaluating MSCA considers the lowest tested dose of 5 mg/kg bw/day as a LOAEL instead of a NOAEL, because at this level significantly increased bilirubin levels in plasma of exposed animals, extramedullary haematopoiesis and pigment storage in spleen, as well as bilirubin in urine and a significant increase in spleen weight was observed, indicative of a haemolytic anemia.

All these findings are considered to be dose-dependent starting at the lowest tested dose of 5 mg/kg bw/day. However, no adverse neurobehavioral effects in male and female Wistar rats were observed at any dose level. In addition, no test substance-related effects were observed during the neurohistopathology investigation at any dose level.

Under the conditions of this study, the lowest observed adverse effect level (LOAEL) for systemic toxicity was 5 mg/kg bw/d. The NOAEL for neurotoxicity was 125 mg/kg bw/d in male and female animals (highest dose tested).