2,6-dibromo-4-cyanophenyl octanoate

Neurotoxicity

Description of key information

Key, M-458465-01-1; neurotoxicity (acute oral, single administration by gavage, rat, OECD 424, GLP):

NOAEL (systemic and neurotoxicity) = 100 mg/kg bw (males and females)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Sep 2012 - 11 Feb 2013

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:

adopted 1997

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: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 310 - 311 g (males), 215 - 218 g (females)
- Fasting period before study: no
- Housing: individually in suspended steel wire mash cages
- Diet: A04C-10 from SAFE (Augy, France), ad libitum
- Water: filtered and softened tap water, ad libitum
- Acclimation period: at least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 05 Sep 2012 To: 12 Oct 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended into corn oil (Sigma, France) to provide the required concentration. There was one preparation for each concentration for the study. When not in use, the formulations were stored at room temperature.

VEHICLE
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in corn oil has been demonstrated in a previous study at concentrations of 0.5 and 10 g/L for up to 4 days under similar conditions of usage and storage. The homogeneity of the test item in the formulation was verified on the preparation at the lowest and highest concentrations to demonstrate adequate formulation procedures. The concentration of the test item in the dosing formulation was verified for each dose level. The mean values obtained from the homogeneity check were taken as measured concentration. The results were 102 - 110% of nominal concentration for both the homogeneity and the concentration analysis and are therefore within the in-house target range of 90 - 110% of nominal concentration and were considered acceptable for use on the current study.

Duration of treatment / exposure:

Single treatment with 14 days of post-treatment observation period.

Frequency of treatment:

Only one treatment was performed.

Dose / conc.:

10 mg/kg bw (total dose)

Dose / conc.:

30 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The dose levels were set after evaluation of the results of acute oral rat toxicity studies with this substance and a preliminary study to this one, designed to determine the time-of-peak effect after a single oral administration of the test item.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for moribundity and mortality twice daily (once daily on weekends and public holidays), and were observed for clinical signs at least once daily. Cages and cage trays were inspected for evidence of ill health such as blood or loose feces.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on study Day 1 prior to dosing and then weekly during the study period as part of the FOB, and before scheduled necropsy.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization phase
- Dose groups that were examined: all animals

Specific biochemical examinations:

None

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Home cage observations: posture, piloerection, involuntary motor movements, gait abnormalities, vocalization, abnormal behavior
- Observations during handling: ease to remove from cage, reaction to being handled, muscle tone, eyelid, lacrimation, salivation, nasal discharge, staining, alopecia, emaciation, temperature upon touch
- Open-field observation: each animal was individually observed in an open field for 2 minutes for piloerection, respiration, arousal, gait abnormalities, posture, involuntary motor movements, stereotypic movements, vocalizations and number of rearings, urine and feces spots
- Reflex/physiologic observations and measurements: pupil size, pupillary reflex, surface righting reflex, corneal reflex, flexor reflex, auditory startle response, tail pinch response, grip strenght, landing foot splay, body weight, rectal temperature
- Minimization of bias: adequacy of training of technical personnel and inter-observer reliability has been verified
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: During pre-study phase, on Day 1 of the study (approximately 6 h after dosing), and on Day 7 and 14.

OTHER:
-Exploratory motor activity: Animals were tested individually using an automated photocell recording apparatus (Interonic, Bordeaux, France) designed to measure quantitatively spontaneous exploratory activity in a novel environment. Exploratory activity was recorded during the first 60 min, with data collected at 10-min intervals throughout the session.

Sacrifice and (histo)pathology:

- Time point of sacrifice: Day 15, 16, 17 or 18
- Number of animals sacrificed: all animals (6 per dose with perfusion, the remaining animals without)
- Parameters measured: macroscopic examination of external surfaces, all orifices and major body cavities
- Brain weight: Yes
- Length and width of brain: No

- Procedures for perfusion: Animals were deeply anesthetized by inhalation of Isoflurane and then euthanized by exsanguination during intravascular perfusion with a fixative solution. Prior to anesthesia, the animals were injected with an intraperitoneal dose of heparin sodium (60 mg/kg bw). The perfusion via the left ventricle consisted of a flush with phosphate buffer, followed by the fixative solution (4% formaldehyde and 1% glutaraldehyde in phosphate buffer).
- Number of animals perfused: 6 per dose level
- Tissues evaluated: Brain, dorsal root ganglia and their spinal nerve roots, eyes, gasserian ganglia, gastrocnemius muscle, optic nerves, peripheral nerves, spinal cord, macroscopic lesions in neural tissue or skeletal muscle

H&E staining:
- Type of staining: Hematoxylin and Eosin (H&E)
- Embedding media: paraffin wax
- Tissues: brain, spinal cord (cervical, thoracic, lumbar), eyes, optic nerves and gastrocnemius muscle
- Thickness: 4 µm
- Number of sections: not reported

Lee's staining
- Type of staining: Lee's
- Embedding media: glycol methacrylate (GMA)
- Tissues: dorsal root ganglia (including dorsal and ventral root fibers) from the cervical and lumbar swellings, gasserian ganglion, and peripheral nerves (sciatic, tibial and sural)
- Thickness: 2 - 3 μm
- Number of sections: not reported

Other examinations:

None

Positive control:

None

Statistics:

All statistical analyses were carried out separately for males and females. Group means were compared at least at the 5% level of significance. Statistical analyses were carried out using Pristima, version 6.3.2 build 17, Xybion Corp. Variables analyzed were body weight and body weight change parameters, absolute and relative organ weights, exploratory motor activity, grip strength, landing foot splay and rectal temperature. A schematic overview of the statistical analysis procedure is provided in Attachment 01.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A slightly higher incidence of piloerection was observed in the 100 mg/kg bw males when compared to controls. See behavior (functional findings) for details.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

not applicable

Body weight and weight changes:

no effects observed

Description (incidence and severity):

not applicable

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no adverse treatment-related neurobehavioral findings from the home cage and open-field observations. A slightly higher incidence of piloerection (8/12 vs 5/12) at the time of peak effect (1 h) in males of the highest dose group (100 mg/kg bw) was observed in the home cage observation as well as in the open field observation (10/12 vs 3/12), but since this effect was in isolation and not observed at any other time point it was regarded to be probably a general reaction to treatment. Furthermore, it was considered not to be adverse as it was an isolated finding which was rapidly reversible.
There were no treatment-related changes during handling observations or in sensory reactivity, grip strength, landing foot splay, rectal temperature, body weights and exploratory motor activity. A statistically significant slightly lower mean landing foot splay value observed in males at 30 mg/kg bw during the last session was considered not to be treatment-related since it was observed without any dose-relationship, at the last session only and not at the time of peak effect on Day 1 (see Attachment 03).

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 mg/kg bw mean absolute brain weight was statistically significantly higher in females when compared to controls. However, the values for this group were within the normal range of historical control data (see Attachment 02). Therefore, in the absence of any other correlated finding, this difference was considered to be incidental and not treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The few macroscopic findings observed were considered to be incidental in origin.

Neuropathological findings:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Critical effects observed:

no

Conclusions:

The study at hand was conducted under GLP conditions and is in accordance with OECD guideline 424. The study is considered reliable and valid. Single oral administration of the test item to male and female Wistar rats did not result in any evidence of neurotoxicity up to the highest dose of 100 mg/kg bw. An isolated occurrence of piloerection in males of this group at time of peak effect during the FOB was rapidly reversible and was considered not to be adverse. Slightly increased brain weight in females at 100 mg/kg bw was within the historical control range and had no histopathological correlate. It was considered to be unrelated to treatment. Thus, under the conditions tested the NOAEL was set to be > 100 mg/kg bw due to the absence of any adverse effect induced by the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rat

Quality of whole database:

The quality of the database is good. The NOAEL is based on an acute neurotoxicity study due to the absence of any adverse effect.

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

Mode of Action Analysis / Human Relevance Framework

To characterize the neurotoxic potential of the test substance, an acute oral neurotoxicity study in rats is available. This key study (M-458465-01-1) was performed under GLP conditions and in accordance with OECD guideline 424. It is considered reliable and valid. Male and female Wistar rats were dosed once with the test item via gavage at dose levels of 0, 10, 30 and 100 mg/kg bw. Clinical observations, mortality, body weight, automated measurements of exploratory activity, a functional observation battery (FOB), brain weight, gross pathology and microscopic examination of the brain, dorsal root ganglia and their spinal nerve roots, eyes, gasserian ganglia, gastrocnemius muscle, optic nerves, peripheral nerves, spinal cord and macroscopic lesions in neural tissue or skeletal muscle were included in the study. During the study, no deaths occurred at any dose level prior to scheduled terminal sacrifice. There was also no effect on body weight or body weight gain noted. The neurobehavioral assessment showed no treatment-related effect for the home cage and open field observations, except for a slightly higher incidence of piloerection in male rats of the 100 mg/kg bw dose group on Day 1 of the study. However, since this effect was not observed at any other time point and since it was rapidly reversible it was considered a general reaction to treatment and not to be adverse. During the FOB, also a statistically significant lower mean landing foot splay value was observed in male rats of the 30 mg/kg bw group when compared to controls. This findings, however, was limited to the last session, showed no dose-relationship and did not occur at the time of peak effect on Day 1. It can therefore be regarded incidental and not treatment-related. No other effects were noted during the FOB. No treatment related macroscopic or microscopic findings were noted. The mean absolute brain weight of females at 100 mg/kg bw was statistically significantly higher than that of the control group. However, it was only 7% higher than the control and was within the range of historical control values. There were also no other, associated histopathological findings. Overall the test item was well tolerated by the animals up to and including the top dose of 100 mg/kg bw and treatment did not result in any evidence of neurotoxicity. On the basis of the results obtained in this study the test item is considered to not be neurotoxic following a single oral dose to male and female rats. The NOAEL for general and neurotoxicity was set to be 100 mg/kg bw based on the absence of any adverse effect under the conditions tested.

 

Justification for classification or non-classification

The available data on neurotoxicity of the test substance do not meet the criteria for classification and labelling according to the CLP Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.