Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Neurotoxicity

Description of key information

Acute and subchronic (90 day) neurotoxicity studies were performed to assess the neurotoxic potential of the test substance.

An acute neurotoxicity study at a limit dose (2000 mg/kg bw) did not reveal any signs of neurotoxicity.

There was no indication of clinical (general clinical observation, FOB and motor activity) or neurohistopathological neurotoxicity in a subchronic neurotoxicity study. Systemic signs of toxicity, namely body weight reduction in the 90-d study, were consistent with those seen in repeated dose studies with the test substance.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: March 11, 2014 To: January 06, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Health Effects Test Guidelines; OPPTS 870.6200; Neurotaxicity Screening Battery;

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.43 (Neurotoxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Lot number: 080722
Physical state/appearance: solid/yellow
Expirydate of the lot/batch: November 01, 2017

Species:

rat

Strain:

other: Crl:WI(Han)

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 ± 1 days at dosing
Weight at dosing: 174-219 g (males) and 135-164 g (females)
Housing: Housed together (5 animals per cage) in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2) with dust free bedding enriched with wooden gnawing blocks (TYP NGM E-022; Abedd® Lab. and Vet. Service GmbH, Vienna, Austria). During the motor activity measurements the animals were housed individually in Polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 800 cm2).
Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
Water: Tap water in bottles, ad libitum
Acclimation period: 13 –16 days

ENVIRONMENTAL CONDITIONS
Temperature: 20–24 °C
Humidity: 30–70 %
Air changes: 10 air changes per hour
Photoperiod: Alternating 12-hour light and dark cycles, artificial light from 6:00 am to 6:00 pm

IN-LIFE DATES: From: March 11, 2014 To: July 17, 2015

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DIET
For each dose level, a specified amount of test substance was mixed with a small part of basal feed and this pre-mix was mixed with the remaining part of the basal feed to obtain test diets with the target concentrations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean values of the test substance in Ground Kliba maintenance diet mouse/rat „GLP“ meal were found to be in the range of 90 – 110 % of the nominal concentrations.

The stability of the test substance in the diet over a period of up to 34 days at room temperature was proven before the start of the study. Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control analyses. In samples of the mid concentration only concentration control analyses were performed.

Duration of treatment / exposure:

At least 90 days.

Frequency of treatment:

Daily in the diet.

Dose / conc.:

0 ppm (nominal)

Remarks:

plain diet

Dose / conc.:

300 ppm (nominal)

Remarks:

corresponding to 20 mg/kg bw/d in males, 24 mg/kg bw/d in females

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 73 mg/kg bw/d in males, 92 mg/kg bw/d in females

Dose / conc.:

4 000 ppm (nominal)

Remarks:

corresponding to 396 mg/kg bw/d in males, 438 mg/kg bw/d in females

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, plain diet

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- mortality and moribundity: at least twice a day (except for Saturdays, Sundays, and public holidays when animals were observed at least once a day).
- general clinical observations: at least once a day.

DETAILED CLINICAL OBSERVATIONS
- all animals once prior to initiation of treatment and once weekly during the treatment period.
- 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
- on the day of initiation of treatment (test day 0), weekly thereafter and when functional observational batteries were carried out.
- The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION
- Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per animal and day.
- The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and food consumption.
FCx * C/BWx = test substance intake for study day x

BWx= body weight on study day x [g]
FCx = mean daily food consumption on study day x [g]
C = Concentration of test substance in the food on study day x [mg/kg]

WATER COMSUMPTION
Water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume but no water consumption data were recorded

Specific biochemical examinations:

Not performed

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATION BATTERY (FOB)
- all animals before the administration period (day -7) and on study days 1, 22, 50 and 85.
- The FOB started with passive observations without disturbing the animals (home cage observations), followed by removal from the home cage and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The findings were ranked according to the degree of severity, if applicable. Passive observations without disturbing the animals were performed in the home cage. Attention was paid to: posture tremor, convulsions, abnormal movements, gait abnormalities and other relevant findings.

Open field observation:
a) Environmental conditions: Any disturbing activities (touching the cage or rack, noise) were avoided during home cage observation. Animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) during open field observation.
b) Duration of observation period for open field observations: ≥ 2 minutes.
c) The following parameters were assessed: 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 (no. fecal pellets/appearance/consistency) within 2 minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.

The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision ("visual placing 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 hind limbs, landing foot-splay test, other findings.

Locomotor activity:
Type of equipment used: Motor activity examinations were performed in a darkened room using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. For the measurements animals were placed in new clean polycarbonate cages with absorbent material. Motor activity measurements started at 14:00 h. Because of the staggered measurement procedure, the starting time varied according to the time needed to place the animals in the cages. The numbers of beam interrupts were counted over 12 intervals of 5 minutes each. Measurement started individually for each animal when the 1st beam was interrupted and lasted exactly 1 hour. No food or water was offered during the measurements.

Auditory startle reflex habituation: Yes

Sacrifice and (histo)pathology:

a) Number of animals evaluated from each sex and treatment group: 5 per sex per treatment group for Neuropathology; the remaining 5 were subject to gross pathology only.
b) Neuropathology: Five animals per sex and test group were selected for neuropathology evaluation. These animals were sacrificed by perfusion fixation under deep Isoflurane anesthesia. SOERENSEN's phosphate buffer was used as the rinsing solution and fixation was performed with a solution according to KARNOVSKY. The sacrificed animals were necropsied and the visible organs or organ sections were assessed by gross pathology as accurately as it is possible for perfused animals. The weight of the brain (without olfactory bulb) was determined in all perfused animals after removal of the brain but before any other preparation. Additionally to organ/ tissues listed in paragraphs below, the following organs/tissues were preserved in neutral buffered 4% formaldehyde: Brain, spinal cord, adrenal glands

Various peripheral nerves, parts of the brain and brain-associated organs, parts of the spinal cord and muscles were embedded and histologically examined. The remaining organ material and the animal bodies were stored in neutrally buffered 4% formaldehyde solution.

The following organ samples were embedded in paraffin, sectioned and stained with hematoxylin-eosin (H&E) and assessed by light microscopy (only control and high dose - low and mid dose organs were stored in 4% formaldehyde):
- Brain (cross sections): Frontal lobe, Parietal lobe with diencephalon and hippocampus, Midbrain with occipital and temporal lobe, Pons, Cerebellum, Medulla oblongata.
- Brain-associated organs/tissues: Eyes with retina and optical nerve.
- Spinal cord (cross and longitudinal sections): Cervical swelling (C3-C6), Lumbar swelling (L1-L4).
- Peripheral nervous system: Gasserian ganglia with nerve, Gastrocnemius muscle.

The following nerves were embedded in an epoxy resin, semi thin sectioned and stained with Azure II - Methylene blue basic Fuchsin (AMbf) and assessed by light microscopy (only control and high dose, - low and mid dose organs/tissues were stored in buffer solution):
- 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, Proximal tibial nerve (at knee), Distal tibial nerve (at lower leg).

c) Pathology:
Animals not selected for perfusion fixation were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Positive control:

No concurrent positive control was employed in this study. However, positive control studies are periodically performed at BASF SE with various neurotoxicants. These studies demonstrate the ability to detect signs of neurotoxicity. The summaries of relevant studies are attached to the study report.

Statistics:

Means and standard deviations of each test group were calculated for several parameters.
- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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.
- Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related clinical signs of toxicity were observed throughout the study.
No test-substance effects were observed during the weekly detailed clinical observations. One female (group 1) was observed with blood in the right eyeball. This finding was considered to be spontaneous in nature.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight changes in male animals of test group 3 (4000 ppm) were significantly decreased over the whole study. The study report considered this observation to be treatment-related, but not adverse. There was no effect on female bodyweight.

No treatment-related changes in body weight parameters were observed in male and female animals of test groups 1 and 2 (300 and 1000 ppm) and in female animals of test group 3 (4000 ppm). The significant increase of body weight changes in females of test group 2 between study day 0 and 14 was considered to be incidental.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Due to the unusually high spilling of food in groups 2 and 3 (1000 ppm and 4000 ppm) food consumption could not be accurately assessed.

An approximate mean daily test substance intake in mg/kg bw/d over the entire study period was calculated based on the 95% lower confidence interval limit of historical food consumption data, to ensure there was no overestimation of test article exposure.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations:
No treatment-related findings were observed.

Open-field observations:
No test substance-related findings were observed. Only one female animal (300 ppm) showed blood in the right eyeball and no eyelid closure reflex in the right eye on study day 85.
Rearing was significantly increased in male animals of all test groups (1-3; 300, 1000, 4000 ppm) on study day 85 and in female animals of test group 1 (300 ppm) on study day 22. The finding was assessed to be incidental because no dose-response relationship occurred.

Sensorimotor tests / reflexes:
No treatment-related findings were observed. Due to blood in eyeball of one female animal (300 ppm) no pupillary reflexwas observed.
Grip strength of forelimbs (GSF) was slightly significantly increased in female animals of test group 1 (300 ppm) on study day 1 and study day 50. Due to the isolated occurrence and the lack of dose-response relationship, this finding was assessed as spontaneous in nature and, therefore, not test substance-related.


Motor activity:
On study day -7 and 1, no changes were observed for male and female animals at any dose level. On study day 22, interval 4 was slightly significantly increased in male animals of test groups 3 (4000 ppm) and significantly decreased in interval 12 in male animals of test group 1 (300 ppm). On study day 50, interval 1 was significantly increased in male animals of test group 1 (300 ppm). In female animals of test group 2 (1000 ppm) interval 12 was increased. On study day 85, interval 5 was significantly increased in male animals of test group 1 (300 ppm). In female animals of test group 3 (4000 ppm) intervals 7 and 12 was increased.
All these findings were assessed as incidental because as no dose-response relationship and no changes in overall activity was observed at any day of examination.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute brain weights of females in group 1 and 3 (300 ppm and 4000 ppm) were significantly decreased. There was not a statistical reduction in relative weights in these groups and no dose response relationship, these changes were considered to be incidental and not related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related gross pathology findings were observed.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment related neurohistopathological findings were observed.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneaus in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

238 mg/kg bw/day

Sex:

male

Basis for effect level:

other: no adverse neurobehavioral or neurohistopathological effects

Remarks on result:

other:

Remarks:

corresponding to 4000 ppm

Key result

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

273 mg/kg bw/day

Sex:

female

Basis for effect level:

other: no adverse neurobehavioral or neurohistopahtological effects

Remarks on result:

other:

Remarks:

corresponding to 4000 ppm

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

238 mg/kg bw/day

Sex:

male

Basis for effect level:

other: no adverse findings

Remarks on result:

other:

Remarks:

corresponding to 4000 ppm

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

273 mg/kg bw/day

Sex:

female

Basis for effect level:

other: no adverse findings

Remarks on result:

other:

Remarks:

corresponding to 4000 ppm

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

238 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study: Neurotoxicity 90d oral 2015/1175552

The oral administration of the test substance at dose levels of 300, 1000 and 4000 ppm (20, 73 and 396 mg/kg bw (males) and 24, 92 and 438 mg/kg bw females respectively) to Wistar rats over a period of 3 months revealed neither adverse neurobehavioral effects nor test substance-related effects in the neurohistopathology investigation at any concentration. There was a significant reduction (-11.7%) in the final body weight of males at the highest dose tested (4000 ppm).

Under the conditions of this study the no observed effect level (NOEL) for neurotoxicity was 4000 ppm for male (238 mg/kg bw/d) and female animals (273 mg/kg bw/d).

The no observed effect level (NOEL) for general systemic toxicity was 4000 ppm for male (238 mg/kg bw/d) and female animals (273 mg/kg bw/d).

Supporting study: Neurotoxicity acute oral 2012/1219658

The test substance administered as a single oral gavage dose at 0, 200, 700 or 2000 mg/kg bw caused transient test substance-related adverse effects at 700 and 2000 mg/kg/bw. Test substance-related effects were observed on the day of test substance administration at dose levels of 700 and 2000 mg/kg bw, in a limited number of animals i.e. hypothermia and slight tremors in test group 3 (2000 mg/kg bw), unsteady gait and high-stepping gait in test groups 2 and 3 (700 and 2000 mg/kg bw), piloerection in test groups 2 and 3 (700 and 2000 mg/kg bw); and reduced motor activity in both sexes of test group 3 (2000 mg/kg bw) on study day 0. These findings were reversible and not observed on study days 7 and 14. All findings were assessed as being related to an impairment of the overall condition of the animals rather than being related to a neurotoxic mode of action.

Brain weight determination, necropsy and neuropathology examination by light microscopy did not reveal any neuropathological, treatment-related findings up to 2000 mg/kg bw. Therefore, the NOAEL for general effects was 200 mg/kg bw/d, the NOAEL for neurotoxicity was 2000 mg/kg bw/d.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No adverse neurobehavioral effects nor test substance-related effects in the neurohistopathology investigation at the highest concentration tested. As a result the substance is not considered to be classified for neurotoxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.