N-1-naphthylaniline

Endpoint summary

Description of key information

A subchronic oral repeated dose toxicity study in rat (90 -day key study) was conducted. Although the assigned study director considered the findings at the lowest dose as incidental and non-adverse, the evaluating MSCA considers the lowest tested dose of 5 mg/kg bw/day in this study 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.In addition, the observed effects are considered to be dose-dependent starting at the lowest tested dose of 5 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

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

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

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.

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

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

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

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

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.

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).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 Oct 2000 to 17 Jan 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan winkelmann GmbH, Borchen, Germany
- Age at study initiation: 5 weeks for male and 6 weeks for female based on animal weights at the beginning of the study
- Weight at study initiation: approximately 140 g
- Housing: polycarbonate type III-cages (5 animals per cage, separated by sex) during acclimatization; during the treatment period the animals were housed individually in type II A-cages on low-dust wood granulate supplied by Ssniff Spezialdiäten GmbH / Soest, Germany.
- Diet (ad libitum): fixed-formula standard diet (NAFAG® 9439 25 W10, pelletized Diet for Rats and Mice) supplied by Eberle Nafag AG, Gossau.
- Water (ad libitum): tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 5 %
- Air changes (per hr): approx. 15 - 20 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial lighting

IN-LIFE DATES: From: 2000-10-09 To: 2000-11-09 (main group)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): test article was completely soluble in the vehicle used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data on stability of the test compound in the administration vehicle covering the concentration range used were obtained before start of this study. Since the test item was completely soluble in the vehicle used, investigations on homogeneity were not performed. During the study period, the test substance content in the vehicle was checked two times. For this purpose, samples were taken from the formulations used in the study. The analytical data verified that the test compound was stable in the concentration range used and that the content agreed with the target concentrations within defined limits.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were selected on the basis of a pilot tolerability study involving administration of the test compound (250 and 500 mg/kg bw/day for 7 days).
- Rationale for animal assignment: species recommended in guidelines for subacute toxicity studies
- Rationale for selecting satellite groups: possibility of recovery and occurrence of delayed toxic effects
- Post-exposure recovery period in satellite groups: 2 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On working days the animals were inspected once a day for morbidity and mortality. general examinations: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the individual experimental animals were determined before the beginning of the study and weekly thereafter up to scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The feed intake of each individual rat was determined once per week. These primary data were then used to calculate the following means for each feeding period of 7 days: feed consumption per animal and day (calculated from weekly determinations), feed consumption per kilogram body weight per day, mean feed consumption per animal per day, mean feed consumption per kg body weight per day

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not The water intake of each individual rat was determined once per week. These primary data were then used to calculate the following means for each watering period of 7 days: water consumption per animal and day (calculated from weekly determinations), water consumption per kilogram body weight per day, mean water consumption per animal per day, mean water consumption per kg body weight per day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood was withdrawn from animals by cardiac puncture at the time of necropsy.
- Anaesthetic used for blood collection: Yes (diethyl ether anesthesia)
- How many animals: 40
- Parameters checked:
Blood: erythrocytes, hemoglobin, hematocrit, thrombocytes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, leucocytes, differential blood count, hepato-quick, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Determination of glucose concentrations were taken in the morning from one of the caudal veins of non-anesthetized animals.
- Animals fasted: Yes
- How many animals: 40
- Parameters checked:
Enzyme activity: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, glutamate dehydrogenase
Substrates/Electrolytes: cholestereol, triglycerides, glucose, creatinine, urea, bilirubin (total), protein (total), albumin, sodium, potassium, calcium, chloride and inorganic phosphate, Hormones: triiodothyronine, thyroxine, thyroid stimulating hormone

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of necropsy
- Parameters checked:
Quantitative Determinations: urine volume, osmolality, protein
Semiquantitative Determinations: pH-value, protein, glucose, blood, bilirubine, ketone bodies, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
A Functional Observational Battery (FOB) was repetedly performed in main groups and in recovery groups, starting each time with the male groups. Motor Activity (MA) assessments were conducted during study week 4 (main and recovery groups) and study week 6 (recovery groups), starting each time with the male groups.
The following observations/examinations were performed approximately 1 hour after application of the test compound: home cage observations (posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations), observations during handling (ease of removing, reaction to being handled, muscle tone, palpebral closure, pupil size, lacrimation, salivation, nasal discharge, stains), open field observations (piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalizations, arousal, rearing, defecation, urination) and manipulative test (pupil response, approach response, touch response, auditory response, tail pinch response, righting reflex and forelimb and hindlimb grip strength (which was only performed at week 4 and 6).
Motor activity assessments were conducted in the fourth and sixth exposure week following the FOB examination. Animals were tested individually for 70 minutes in one of twelve figure-eight mazes. Motor activity (MA) and locomotor activity (LMA) were examined as activity for the
70-minute session (Summary Session MA and LMA) and activity during each of the seven 10-minute intervals (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occur during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Therefore a distinction can be made between changes in true locomotor (ambulatory) activity and changes in general motor activity (ambulatory and nonambulatory movements). Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were necropsied after exsanguination under deep ether anesthesia. Animals that died spontaneously or were killed in a moribund state during the study were necropsied at the earliest opportunity.

ORGAN WEIGHTS:
Following organs were weighed at necropsy: brain, adrenal glands, heart, liver, spleen, thymus, kidneys, testes, epididymides, prostate, seminal vesicle (calculated from sum of prostate+seminal vesicle weight substracted by prostate weight), ovaries and uterusCAGE SIDE ed.

HISTOPATHOLOGY: Yes
List of fixed tissues: Adrenal glands, Brain (Cerebrum, Cerebellum, Pons/Medulla), Epididymides, Esophagus, Femur, Heart, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum and remaining intestine), Larynx, Liver, Lungs, Lymph nodes (mandibular and mesenteric), Ovaries, Oviducts, Prostate, Sciatic nerve, Seminal vesicles (incl. Coagulating glands), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum, Stomach (Forestomach and Glandular stomach), Thymus, Thyroid gland with Parathyroid gland, Trachea, Urinary bladder, Uterus with uterine cervix, Vagina, and all organs or tissues with macroscopic findings.Kidneys and Testes were fixed in Davidson's solution. Prior to fixation, the lungs and the urinary bladders were instilled with 4 % formaldehyde solution.
Osseous tissues (femur, sternum, and vertebrae with spinal cord) were first decalcified and then like all other organs, embedded in Paraplast. Sections approximately 5 μm thick were prepared from the organs listed above (except remaining intestinal tissues) from all animals of the control and the high dose groups. All slides were stained with hematoxylin and eosin (H&E). From all main group animals, cryocuts obtained from the liver were stained with Oil Red O (ORO). Histopathological examination of the animals of the recovery groups was not performed.

Other examinations:

no data

Statistics:

The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups thatreceived the test substance were compared with those for the control group and significant differences were indicated by + for p ≤ 0.05 and ++ for p≤ 0.01. Statistical tests employed are descriptive analysis, continuous random variables, Dunnett test, adjusted Welch test, Kruskal-Wallis test followed by adjusted U tests (Mann-Whitney-Wilcoxon) and discrete random variables.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were detected during treatment and recovery period. Gait incoordination was observed during detailed clinical examinations on day 28 in main group animals of both genders with the following incidence rates in ascending order of dose level (males: 0-0-1-1; females: 0-2-2-3). Gait incoordination was also observed in one male (day 28 and 35) and in one female (day 28) animal of the recovery high-dose group. A tilted head was observed during the study in one male rat each belonging to the main control and the recovery control group, respectively and in one female rat of the 80 mg/kg group. Reddish discoloured urine was detected in main group animals of both genders treated with 20 and 80 mg/kg body weight between day 29 and day 31 during daily general clinical examinations.

Mortality:

no mortality observed

Description (incidence):

No animal died throughout the entire treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight development was not impaired by treatment. The statistically significantly higher mean body weights in female rats of the recovery high-dose group beginning with the 2nd week were considered as chance variation, since corresponding data in main high-dose groups in females as well as in male high-dose groups were inconspicuous.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption intake by main group and by recovery animals per day and per kg body weight per day was not affected by treatment. Significantly lower daily feed intakes per kg body weight per day in mid-dose females in the 2nd week of treatment as well as in male recovery high-dose animals in the 4th week of treatment were considered to be incidental findings since the changes were only small and were poorly correlated with time and dose.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Mean daily water intake by main group animals was unaffected by treatment. Singularily, female and male recovery high-dose animals showed a higher (females) or lower (males) mean daily water intake per kg body weight during the treatment period, which became statistically significant in the first and/or the fourth week of treatment. These changes were considered not treatment-related since they did not occur in the main groups, were only slight and ranged in opposite directions.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The examination of the red blood cells, showed a mild but dose-correlated reduction in erythrocyte count, hemoglobin and hematocrit concentration in female rats at 20 mg/kg and above, but with no changes in mean corpuscular hemoglobin. Male animals showed no changes in erythrocyte counts and in hemoglobin concentrations. The statistically significant increase in mean corpuscular hemoglobin concentration in low- and mid-dose males of the main group is considered not to be treatment-related since no dose correlation exists. Furthermore, all individual hemoglobin and hematocrit levels of these animals ranged within the 2s-limits of historical controls. No deviations from control were observed in recovery animals previously treated with the test item. The statistically significant increase in MCV in high-dose recovery females (p≤0.05) is considered to be of no biologic significance. Thrombocyte counts and clotting times showed no toxicologically relevant changes at the end of treatment and at the end of the recovery period. At term of treatment the investigations on white blood cells showed a trend to increased total leucocyte and lymphocyte counts in females at 20 mg/kg and above and in males at 80 mg/kg (p≤0.05 in 20 mg/kg females only).
At the end of the recovery period no relevant differences in total leucocyte counts and in lymphocytes were noted in animals previously treated with the test item when compared to concurrent controls. Recovery high-dose male animals only showed a slight increase in neutrophils (p≤0.05) which is considered to be not related to treatment since the difference was very small and four of five individual values ranged within the 2s-limits of historical controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period bilirubin concentrations were dose-dependently elevated in male animals starting at 20 mg/kg and - to a lesser extent - in females at 80 mg/kg. Male animals dosed at 80 mg/kg had also slightly but statistically significantly elevated albumin concentrations (p≤0.05). At the end of the recovery period the above mentioned changes were not any longer noted in animals previously treated with the test item when compared to concurrent controls. An interference test with concentrations of 3 mg, 10 mg and 30mg test material/l rat plasma revealed bilirubin level elevations of 3.4, 10.6, and 35.0 μmol/l, in ascending order of dose levels, whereas 1 mg test item/l rat plasma showed no influence on the determination of bilirubine in rat plasma. At term of treatment cholesterol and triglyceride concentrations were slightly but statistically significantly lower in males receiving 80 mg/kg. Since all individual cholesterol and triglyceride values of the high-dose group ranged within the 2s limits of historical controls and the changes occurred only in one sex, a relationship to treatment is questionable. At the end of the recovery period no differences in cholesterol and triglyceride concentrations were noted in animals previously treated with the test item when compared to concurrent controls. The determination of blood electrolyte concentrations revealed slightly but statistically significantly higher chloride concentrations in main group female rats at 80 mg/kg. The statistically significant decrease in mean sodium concentration in low and mid-dose males of the main group is considered to be of no biologic significance since it is not correlated with dose. The determination of enzyme activities in plasma (ALAT, ASAT, APh, GLDH) did not yield relevant test compound-related differences from control values. The determination of hormones in blood (T3, T4, TSH) at the end of the treatment period and at the end of the recovery period did not yield relevant test compound related differences from control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At term of treatment urine volumes and osmolality showed no relevant test compound related differences from control. Protein concentration tended to be slightly elevated (p>0.05) in both genders whereas protein excretion was significantly elevated in high-dose males of the main groups. The semi-quantitatively determined bilirubin was increased in the urine at the end of treatment in male arid female groups in a dose correlated manner starting at 5 mg/kg. No differences did exist in recovery animals. An interference test with urine concentrations of 100 and 1,000 mg/I test item showed a strong reaction with the urine stix (= bilirubin intensity step 3) whereas 1 and 10 mg/I showed no reaction. A trend to an increase in ketone bodies was observed in animals of the main groups treated with 80 mg/kg. Glucose, blood and urobilinogen concentrations were unremarkable in the main group and recovery animals. Dipstick measurements of urinary pH revealed no differences to controls in main groups and in recovery groups.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The functional observations exhibited gait incoordination on day 25 in two females at 20 mg/kg and two females at 80 mg/kg. One female at 5 mg/kg and two females at 80 mg/kg showed a stilted gait. Male animals of the main groups showed no gait abnormalities during the weekly open field observations. Animals of both genders of the high-dose recovery groups showed no gait abnormalities neither at the end of the study (day 24/25) nor at term of the recovery period (day 38/39). Grip strength measurements showed no differences attributable to the administration of the test substance at term of treatment as well as at the end of the recovery period. The pooled means of the high-dose groups and of the controls as well as the means of the low- and mid-dose groups ranged within the maximum historical pretreatment variability for males and females, respectively, which were 11 and 13% in forelimbs and 15 and 13% in hindlimbs (M. Dreist and A. Popp 1998).
Summary interval locomotor activitiy and motor activity measurements revealed no obvious differences between treatment groups and controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At term of treatment a trend towards higher relative liver weights was noticed in female animals receiving 80 mg/kg. A trend towards an increase in mean absolute liver weights was noted at the end of treatment in male (significant at p≤0.05) and female rats (p > 0.05) receiving 80 mg/kg. In the recovery groups previously treated with 80 mg/kg a slight increase in relative spleen weights was observed in males (significant at p≤0.05) and in females (p > 0.05), together with an increase in absolute spleen weights. Singularily, isolated absolute organ weight increases were detected in recovery high dose female rats (e.g. kidney-, liver- and ovary-weight) which were mainly due to slight differences in body weight means between recovery groups and are therefore considered not to be related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At term of treatment necropsy showed no test substance induced findings

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histopathological examination of the animals revealed findings which were evenly distributed among the groups. These findings are known from control animals of previous studies to be spontaneous alterations.

Histopathological findings: neoplastic:

no effects observed

Details on results:

RECOVERY GROUP
Delayed occurence of toxic effects was not observed - except for slightly increased relative spleen weights- and all changes were completely reversible during a recovery period of about 2 weeks.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Overall there are signs indicative of a mild normochromic anemia in female animals at 20 mg/kg and above with a slight hyperbilirubinaemia. The reason for the reduction in the number of circulating blood cells has not been detected but might be related to hemolytic anemia. The effects were not accompanied by a regenerative hyperplasia of the bone marrow and/or increased circulating reticulocytes. The increase in leucocyte and lymphocyte counts cannot be explained but might be secondary to the changes in red blood. This slight increase in relative spleen weight in recovery animals previously treated with test material might be secondary to hemolytic anemia resulting in an increased extramedullary hematopoiesis.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 Aug to 16 Aug 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The systemic tolerance of male and female rats to Additin 30/Vulkanox PAN was examined in a pilot tolerability study involving administration of test compound by gavage for 7 days. The study methodology folllowed no specific guideline.

GLP compliance:

not specified

Species:

rat

Strain:

other: Wistar; HsdCpb:WU

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

2

Control animals:

yes, concurrent vehicle

Critical effects observed:

not specified

Mortality:

One female animal receiving 500 mg/kg died after 2 days of treatment.

Clinical signs:

Clinical signs occured in both genders at a dose of 250 mg/kg and above. Signs included reduced motility, decreased reactivity, uncoordinated gait, laboured breathing, discoloured urine, piloerection, and -in one case salivation.

Gait abnormalities appeared early during treatment (day 1) in all dose groups. A discoloured urine was also seen from day 1 or 2 until end of treatment in all dose gorups, except in males receiving 250 mg/kg. In that dose group, only one animal showed discoloured urine on day 7.

Towards the end of treatment (day 6 to 7) laboured breathing was noted in male and female animals of all dose groups and reduced motility was observed in 500 mg/kg males and in females receiving 250 and 500 mg/kg.

There was no body weight gain in one female animal receiving 250 mg/kg. Body weight development was not affected in the second female rat of that dose group as well as in the surviving animal of the 500 mg/kg group, when compared to historical control data of animals of the same age group. Body weight development in males receiving 500 mg/kg was slightly retarded, whereas both animals of the 250 mg/kg group seemed not be affected by treatment.

Food consumption was not affected by treatment in male and female animals of both dose groups.

Mean daily water intake was significantly higher in male and female animals of both dose groups, when compared with historical controls. There was no evidence of a dose correlation.

Gross necropsy revealed enlarged kidneys in males rats receiving 500 mg/kg. The kidneys of female rats of both dose groups showed a rough and discoloured surface. Adrenals were enlarged in 500 mg/kg female rats and in one 250 mg/kg female rat.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

haematopoietic

Organ:

blood

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

The systemic tolerance of male and female rats to N-phenyl-1-naphtylamine was examined in a pilot tolerability study involving administration of the test compound by gavage (Temerowski, 2000). The 7-day treatment with the test compound using oral doses of 250 and 500 mg/kg bw revealed toxic effects, mainly on the kidneys with severe clinical symptoms in all dose groups of both genders. Female rats seemed to be the more sensitive sex.

Based on the outcome of the pilot study, a subacute repeated dose toxicity study following OECD guideline 407 was performed with dose levels of 5, 20 and 80 mg/kg bw/day (Temerowski, 2002). The test item was administered orally via gavage to (main) groups of 5 male and 5 female Wistar rats using target doses of 0, 5, 20 and 80 mg/kg body weight once a day for approximately 4 weeks. In parallel, recovery groups of 5 male and 5 female rats were treated with 80 mg test substance/kg body weight or the vehicle for 28 days and were observed for reversibility, continuance or delayed occurrence of toxic effects during a recovery period of about 2 weeks. Regarding survival rate there was no difference between treated and untreated animals. Mean daily food and water intake per kg body weight as well as mean body weight development was not affected by treatment. Detailed clinical examinations and/or functional observations revealed gait abnormalities towards the end of the treatment period in some female rats throughout all treatment groups and in few male rats at 20 and 80 mg/kg. Grip strength measurements conducted in the fourth week of treatment and at term of recovery showed no differences attributable to the administration of the test substance. Summary interval locomotor activity (LMA) and motor activity (MA) measurements revealed no obvious differences between treatment groups and controls at the end of treatment. A dose-correlated reduction in erythrocytes, hemoglobin and hematocrit concentration was noted in female rats at 20 mg/kg and above. White blood ccunt revealed an increase in total leucocyte and lymphocyte counts in females at 20 mg/kg and above and in males at 80 mg/kg. Thrombocyte counts and clotting times were not affected by treatment with the test compound. At the end of the treatment period bilirubin concentrations in blood were dosedependently elevated in male animals starting at 20 mg/kg and - to a lesser extent - in females at 80 mg/kg. The semi-quantitatively determined urinary bilirubin was also increased in the urine in male and female groups in a dose correlated manner starting at 5 mg/kg. Most of these changes in bilirubin concentrations were considered to be mainly due to an interference of the test compound with the test kits. Male animals receiving 80 mg/kg showed slightly elevated albumin and slightly lower cholesterol and triglyceride concentrations. The determination of blood electrolyte concentrations revealed slightly but statistically significantly higher chloride concentrations in female rats at 80 mg/kg. At the end of the recovery period the above mentioned changes were no longer noted in animals previously treated with the test item when compared to concurrent controls. The determination of liver enzyme activities in plasma as well as the hormones in blood (T3, T4, TSH) did not yield relevant test compound-related differences from control values. Urinary protein concentration arid excretion was slightly elevated in animals receiving 80 mg/kg. Glucose, blood, ketone body and urobilinogen concentrations were unremarkable as well as urinary pH, urine volumes and osmolality. At the end of treatment a trend towards higher relative liver weights was noticed in female animals receiving 80 mg/kg with no microscopic correlates. The recovery groups of both genders previously treated with the test item showed a slight increase in absolute and. relative spleen weights when compared with the concurrent controls. The histopathological examinations revealed no treatment-related findings in the spleen as well as in other organs and tissues at term of treatment and after 2 weeks of recovery. In conclusion, administration of the test item to male and female Wistar rats for approximately 4 weeks resulted in a systemic no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight mainly with effects on blood cells and on liver function. With regard to local effects a NOAEL was not established due to unspecific gait abnormalities occurring in two female rats at 5 mg/kg body weight. Delayed occurrence of toxic effects was not observed -except for slightly increased relative spleen weights- and all changes were completely reversible during a recovery period of about 2 weeks.

In a follow-up GLP compliant subchronic neurotixicity study study (BASF SE, 2016) 1-Naphthalinamine, N-phenyl- 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. 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.

With regard to clinical examinations, no signs of systemic toxicity changes were observed in male and female animals at any dose level (up to 125 mg/kg bw/d). In addition, FOB and MA measurements did not reveal test substance-related neurobehavioral effects at any concentration on study days -7, 1, 22, 50 and 85. Salivation shortly after treatment was observed in all male and female animals of test group 3 (125 mg/kg bw/d) on several study days. From the temporary, short appearance immediately after dosing it was concluded that the finding 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 a single male animal of test group 3 (125 mg/kg bw/d) but 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). It was assumed that the reddish discoloration of the urine was related to the physical property of the test substance (white to light red color) and/or its metabolites as no endogenous product is known to change the urine color this way. Thus, the discoloration was treatment-related but assessed to be non-adverse.

Regarding clinical pathology, in rats of both sexes of test group 3 (125 mg/kg bw/d) a regenerative anemia was present (decreased red blood cell [RBC] counts, hematocrit and hemoglobin values and increased relative reticulocyte counts). Most probably haemolytic anemia was leading to increased serum total bilirubin levels. However, regarding bilirubin measurement (2,5-dichlorophenyl diazonium tetrafluoroborate (DPD) colorimetric method), an interference test performed with the parent compound at concentrations of 0.1; 1; 3.3 and 10 mg/L resulted in an interference at least down to a concentration of 1 mg/L. According to Sikka et al. (1981) one oral gavage administration of 160 mg/kg bw of the compound led to peak levels of about 3 mg/L serum after two hours with minimal residues of parent compound left after 24 hours. At the administered dose (test group 3: 125 mg/kg bw/d) the increased serum bilirubin values were due to an interference of the compound with the test method and therefore the change was regarded as artefact and not treatment-related. Looking at the urinalysis findings, conjugated bilirubin was excreted in the urine but this measurement did also interfere with parent compound excretion at 100 mg/L (method: DPD colorimetric method; interference test with 0.1; 1; 3; 10; 30 and 100 mg/L parent compound). Because no method interference with metabolites could be evaluated, the validity of positive urine bilirubin results in rats of both sexes of test group 2 (25 mg/kg bw/d) and in males of test group 3 (125 mg/kg bw/d) and females of test group 1 (5 mg/kg bw/d) should be questioned. However, in males of test groups 2 and 3 (25 and 125 mg/kg bw/d) urobilinogen was found in the urine and this measurement did not interfere with excreted parent compound. Urobilinogen was synthetized from bilirubin by bacteria in the gut and reabsorbed from the intestine into the blood and excreted in the urine. Therefore, a hemolytic anemia with increased urobilinogen excretion in the urine did probably happen in rats of both sexes of test group 3 (125 mg/kg bw/d; in females without urobilinogen excretion) and in males of test group 2 (25 mg/kg bw/d; urobilinogen excretion only). Regarding glucose excretion in the urine 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 of test group 3 (125 mg/kg bw/d), no changes in clinical chemistry serum parameters were found in these individuals reflecting an affection of the energy metabolism. In the anatomical pathology a degeneration/ regeneration of the proximal tubule cells in the kidney was found in males of test groups 2 and 3, but not in females. Therefore, a pathohistological correlate to the seemingly increased glucose excretion was not found. The glucose measurement was not affected by the parent compound excretion. However, the compound is primarily metabolized via hydroxylation with subsequent glucuronidation and sulfation followed by partial excretion via urine (Sikka et al., 1981). An interference of the glucose measurement in the urine (GOD/POD method) by metabolites of the compound coloring the urine red-brown which is not done by the parent compound can be assumed. Therefore, the glucose level increase in the urine was regarded as incidental and not treatment-related. In one male of test group 3 (125 mg/kg bw/d) hemoglobin was found in the urine, but again, this measurement was affected by the compound excretion (interference at least in one urine sample down to a compound concentration of 1 mg/L) and therefore was regarded as an artefact with no pathophysiological relevance. Lower serum creatinine values in rats of both sexes of test group 3 (125 mg/kg bw/d) were most probably due to a higher rate of glomerular filtration in the kidneys, because of higher urine volume. This is at least true for males with higher urine volume in test group 3. The higher filtration rate of creatinine was regarded as an adaptive rather than an adverse effect.

Regarding neuropathology, brain weight determination, necropsy and neuropathology examination by light microscopy did not reveal any neuropathological, treatment-related findings.

Regarding pathology (performed according to OECD 408), the male kidneys, the liver and the spleen were target organs. In the kidneys of male animals of test group 2 and 3 (25 and 125 mg/kg bw/d) three males and eight males, respectively, showed degeneration/regeneration of the proximal tubules. In addition, animals of these two test groups showed a higher incidence of basophilic tubules compared to control animals. These findings were regarded to be treatment-related and adverse. In the livers of males of test group 2 and 3 (25 and 125 mg/kg bw/d) and females of test group 3 (125 mg/kg bw/d) a centrilobular hypertrophy was observed. This also 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. In the spleen, there was a brownish pigment stored mainly within histiocytes that showed a positive reaction when stained with Perl’s Prussian blue stain and Turnbull stain. This led to the diagnosis of hemosiderin storage, 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. 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. All other findings occurred either individually or were biologically equally distributed over control and treatment groups.

Although the assigned study director considered the findings at the lowest dose as incidental and non-adverse, the evaluating MSCA considers the lowest tested dose of 5 mg/kg bw/day in this study 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.In addition, the observed effects are considered to be dose-dependent starting at the lowest tested dose of 5 mg/kg bw/day.

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).

 

Repeated dose toxicity: other routes

N-phenyl-1 -naphthylamine (219 mg/kg) was administered intraperitoneally to mice for 3 days (Nomura, 1977). 48 hours after the last administration methaemoglobin formation was measured. A slightly increased methaemoglobin level (1.6 % versus 0.4 % in controls) was noted. Examination of methaemoglobin formation after 9 consecutive days of intraperitoneal injection showed no alteration compared to the controls (0.6 % versus 0.4 % in controls) (Nomura, 1977).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

After repeated exposure of rats to the registered substance over a period of 3 months in a highly reliable GLP guideline study signs of systemic toxicity were revealed taking pathology and hematology findings into account.

The observed effects comprise 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 at 5 mg/kg bw/d and were considered indicative of haemolytic anemia.

Nevertheless, at this lowest tested dose the bilirubin levels in plasma were within the range of the historical control data. Moreover, the extramedullary hematopoiesis and pigment storage in the low and mid test group (5 and 25 mg/kg bw/d) showed no correlation to clinical pathology (no severe anemia). Thus, these effects as well as the statistically significant increase in spleen weight are not considered of biological significance at the low dose group.

Regarding the urinalysis, it is stated in the study report that the validity of the positive urine bilirubin results in the mid dose group (25 mg/kg bw/d), in males of the high dose group (125 mg/kg bw/d) and females of the low dose group (5 mg/kg bw/d) should be questioned due to interference with the parent compound in the corresponding measurement. Thus, reliable increased values are only observed for the mid and high dose with regard to urobilinogen.

Additionally, adverse effects were observed in kidney of male animals, indicating chronic nephropathy at the mid dose level of 25 mg/kg bw/d, which may also be alpha-2-μ globulin- mediated. As this is a specific mechanism unique to male rats, the relevance of the observed adverse effects in rat kidney for human health may be questionable.

Taken together, effects observed at the lowest dose level of 5 mg/kg bw/d might be indications of the onset of haemolytic anemia, however, since significant toxic effects are only observed at higher (moderate) dose levels, the criteria for classification in Category 2 for target organ toxicity after repeated exposure are met.

Therefore, classification of the registered substance for Specific Target Organ Toxicity - Repeated Exposure Category 2 according to Regulation (EC) No 1272/2008 is warranted.