2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Neurotoxicity

Description of key information

Similar to OECD 424, oral, rat, 90-days: NOAEL (neurotoxicity) = 996 mg/kg bw/day (highest dose tested)

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:

20 Jul - 6 Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF Guideline for a repeated oral neurotoxicity test (JMAFF 12 Noshan No. 8147, 24 November 2000)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: approximately 5 - 6 weeks
- Weight at study initiation: 129 - 162 g (males), 121 - 160 g (females)
- Housing: in groups of up to 4 per sex in polypropylene grid-floor cages, environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels
- Diet: Rat and Mouse SQC Ground Diet No. 1 (Special Diet Services Limited, Witham, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Dietary admixtures were prepared prior to treatment and then twice during the three month study period (at approximately monthly intervals).
- Storage temperature of food: The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique. The results indicate that the mean dietary admixture concentrations were within acceptable limits for the purpose of this study. To verify the homogeneity of the test substance in the diet, dietary admixtures were sampled from the middle and two opposite sides in triplicate and analysed prior to use. To determine the stability, dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for seven weeks. The dietary admixtures were considered to be stable for a period of at least seven weeks.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously (via diet)

Dose / conc.:

150 ppm

Remarks:

equivalent to 12 mg/kg bw/day

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 117 mg/kg bw/day

Dose / conc.:

12 000 ppm

Remarks:

equivalent to 996 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Observations and clinical examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 0 (the day before the start of treatment) and at weekly intervals thereafter. Body weights were also recorded at terminal sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and during final week of the study
- Dose groups that were examined:The eyes of all control and high dose group animals were examined. Since treatment-related effects were detected at the high dose during the final
week of dosing, examinations were extended to all low and intermediate dose animals.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: motor activity, forelimb/hindlimb grip strength, grasp response, touch escape, vocalization, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex, finger approach, Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behavior, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation, lachrymation
- Description of procedures: Detailed individual clinical observations were performed for each animal using a purpose built arena. An automated grip strength meter was used for measuring forelimb/hindlimb grip strength.
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: No data
- Time schedule for examinations: prior to start of treatment and during Weeks 2, 4, 8 and 12
- Duration of observation period for open field observations: not specified
- Description of equipment where required:

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: twenty purpose built 44 infra-red beam automated activity monitors
- Length of session, number and length of subsessions: 1 hour per day
- Parameters measured: percentage of time each animal was active and mobile
- Total activity: active: all motion including static movement (movement undertaken by the animal when it is stationary e.g. grooming, feeding, rearing up, head movement etc.); mobile: when the animal is actually moving from one place to another place

Sacrifice and (histo)pathology:

- Time point of sacrifice: after 90 days of treatment
- Number of animals sacrificed: 10 per sex per group
- Brain weight: yes
- Length and width of brain: no
- Procedures for perfusion: perfused with gluteraldehyde: paraformaldehyde via the heart, following initial perfusion with heparinised saline.
- Number of animals perfused: 5 per sex per group
- Tissues evaluated: brain (olfactory bulb, forebrain centre of cerebrum (including hippocampus), midbrain, cerebellum, pons and medulla oblongata), dorsal root ganglia (cervical and lumbar regions), dorsal and ventral root fibres (longitudinal cervical and lumbar sections), eyes (longitudinal sections), optic nerve (longitudinal sections), sciatic nerve (proximal - longitudinal and transverse sections), tibial nerve (proximal (at knee) and calf muscle branches - longitudinal and transverse sections), skeletal (calf) muscle (transverse sections), spinal cordn (longitudinal and transverse cervical and lumbar sections)
- Type of staining: haematoxylin and eosin
- Methodology of preparation of sections: not specified
- Thickness: 5 µm
- Embedding media: paraffin
- Number of sections: not specified
- Number of animals evaluated from each sex and treatment group: 5/ sex from control and 12000 ppm group

Positive control:

A neurotoxicity study in rats was conducted between 12 February and 14 April 2003 in the same laboratory to investigate the possible neurotoxicity of acrylamide and trimethyltin chloride. Acrylamide and trimethyltin chloride were administered by oral gavage for a period of up to 28 days at dose levels of 15 and 1 mg/kg bw/day, respectively. Histopathological examination of animals treated with acrylamide revealed mild treatment-related changes in the peripheral nervous system. Lesions of neuronal necrosis, karyorrhexis, astrocytic mitotic figures, gliosis and meningeal hypercellularity in various areas in the central nervous system were also observed. Animals treated with trimethyltin chloride showed lesions in the central nervous system, namely in the olfactory bulb, forebrain, cerebrum, hippocampus and midbrain. Based on the results of this study, the previously known effects of acrylamide and trimethyltin chloride were confirmed. Therefore, the laboratory showed the proficiency to identify and characterize neurotoxic substances.

Statistics:

Brain weight (absolute and relative to terminal bodyweight), weekly body weight gain and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinically observable signs detected during the study that could be attributed to neurotoxicity.
Clinically observable signs were detected for the three males which died at 12000 ppm. Broken teeth were reported for one male on Day 39, together with lethargy and pallor of the extremities. The animal was found dead later the same day. A further male was killed in extremis on Day 63 due to an open wound on the tail (evident from Day 35) which showed no signs of healing. On Day 82 a male showed clinically observable signs of hunched posture, pilo-erection, lethargy, pallor of the extremities, hypothermia, decreased respiratory rate and labored respiration. The animal was subsequently killed in extremis on the same day.
At 12000 ppm, fur loss developed from Day 4, becoming excessive in a number of animals as the study progressed. Hunched posture was evident in females from Day 54 onwards together with incidents of tiptoe gait and fur staining. One male and one female treated with 12000 ppm also showed corneal opacity by the end of the study. This condition was more severe at 150 and 1500 ppm. Opacity was evident for 150 and 1500 ppm females from Day 54 followed by the males on Day 68. However, these observations were considered not to be indicative of neurotoxicity. In addition, exophthalmia was also evident from Day 20 for two females and up to four males treated with the high dose. Full recovery was detected from Day 31 onwards.
See Attachments for tabulated data.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males died at 12000 ppm. One male was found dead on Day 39, one male was killed in extremis on Day 63 and a further male was terminated on Day 82.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in body weight gain was detected for animals of either sex treated with 12000 and 1500 ppm during the first week of the study, extending to the 150 ppm females. Body weight gain remained reduced for 12000 ppm animals until Week 6, recovering thereafter. This was probably attributable to the unpalatable nature of the dietary admixture and was not a direct effect of toxicity. At study termination, body weights were significantly reduced in males (-12%) at 1500 ppm and in males and females (-21 and -18%, respectively) at 12000 ppm.
See Attachments for tabulated data.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced dietary intake was evident for animals of either sex treated with 12000 ppm during the first two weeks of the study, recovering in females, but still evident in the males (-8%) throughout the study. Food consumption was also reduced at 1500 ppm.
See Attachments for tabulated data.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency was reduced for animals of either sex treated with 12000 ppm. This reduction however, was confined to the first week of the study only and complete recovery was detected thereafter. The reduction in Week 1 was therefore considered to be isolated and of no toxicological significance.
See Attachments for tabulated data.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated at all dietary concentrations showed corneal opacity and neovascularization of the cornea, with effects more severe at the 150 and 1500 ppm dose levels. These findings were not considered to be a neurotoxic effect of treatment.
One female treated with 12000 ppm showed an abnormal blood vessel in the right eye, during the pre-test observations. This was considered to be congenital and unrelated to treatment.

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, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects detected that could be considered attributable to neurotoxicity.
Open-field observations confirmed the clinical signs of hunched posture and exophthalmia detected at 12000 ppm. Hunched posture, pilo-erection, pallor, respiratory pattern changes and hypothermia were also reported during the Week 12 assessments, for the animal that was subsequently killed in extremis on Day 82.
All remaining inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

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):

No toxicologically significant effects of treatment were detected.
A slight but statistically significant increase in relative brain weight was detected in males at 12000 ppm. The minimal significance was considered attributable to the lower body weight in this group. Statistically significant reductions in absolute brain weight were detected for animals of either sex treated at all dietary concentrations, in comparison to controls (excluding males treated with 150 ppm). These reductions were not reflected in the relative brain weights and in the absence of any histopathological correlates, were considered to be of no toxicological significance.
See Attachments for tabulated data.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 12000 ppm showed thickening of the glandular gastric epithelium at terminal kill whilst all females showed varying degrees of fur loss. No such effects were detected at the other dose levels, however, corneal opacity was evident in the majority of animals treated with 1500 and 150 ppm; one male and one female treated with 12000 ppm were also affected.
The interim deaths were all associated with minor physical injuries. One male showed broken teeth and dark stomach contents. The male sacrificed on Day 63 showed an open wound on the tail and red lungs. Following exsanguination of this animal, the blood was reported to be pale and tail and red lungs. Following exsanguination of this animal, the blood was reported to be pale and thin. The remaining interim death showed pallor of most internal organs. Staining around the mouth was evident and was probably attributable to the tongue injury reported at necropsy.
See Attachments for tabulated data.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The statistically significant reductions in total percentage activity detected for males treated at 12000 and 150 ppm during the pre-test assessments were minimal (p<0.05) and in the absence of the administration of test material, were considered to have arisen fortuitously.
During the Week 8 assessments, the eyes of females at 150 and 1500 ppm were recorded as opaque but this was unrelated to neurotoxicity.
All remaining inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes observed in the tissues examined.
The few lesions present were insignificant and commonly observed in laboratory maintained rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

> 12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: not determinable due to absence of adverse toxic effects

Remarks on result:

other: equivalent to 996 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 117 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Remarks on result:

other: equivalent to 996 mg/kg bw/day

Critical effects observed:

no

Conclusions:

The study was performed under GLP conditions and according to Japanese MAFF Guideline for a repeated oral neurotoxicity test (JMAFF 12 Noshan No. 8147, 24 November 2000). Dietary administration of the test substance to rats for a period of up to ninety consecutive days at concentrations of 150, 1500 and 12000 ppm (equivalent to a mean achieved dosage for males and females of 12, 117 and 996 mg/kg bw/day, respectively) resulted in no evidence of neurotoxicity, therefore, the NOAEL was considered to be 12000 ppm for neurotoxicity (equivalent to 996 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

996 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

The study was designed to investigate the possible neurotoxicity of the test material and was conducted similar to OECD TG 424. The test material was administered by dietary admixture to three groups, each with ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to 90 consecutive days (13 weeks), at dietary concentrations of 150, 1500 and 12000 ppm (equivalent to a mean achieved dosage for males and females of 12, 117 and 996 mg/kg bw/day, respectively). A further group of ten males and ten females was exposed to basal laboratory diet to serve as a control.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Ophthalmoscopic examination was performed on control group and high dose animals before the start of treatment and on all animals during the final week of the study.

Five animals per sex from each dose group were subjected to whole body perfusion and histopathological examination of neural tissue was performed on all perfused animals from high dose and control treatment groups.

There were three unscheduled deaths during the study but these were not attributed to neurotoxicity. The three interim deaths of male rats at 12000 ppm were attributed to physical injury (an open wound to the tail, broken teeth and tongue injury). Normally, such injuries would not have resulted in death, however, the blood was reported as thin and pale for one of these animals, suggesting an adverse effect on clotting potential. However, this was a systemic effect of treatment and was not indicative of neurotoxicity. Clinically observable signs were confined to opacity of the eyes and varying degrees of fur loss and scab formation. A treatment-related reduction in bodyweight for animals of both sexes treated with 12000 ppm was considered to be attributable to the effects of test item administration, due to a reduction in food efficiency in the same group. Temporary reduction in bodyweight gain at the early stages for groups treated with 1500 ppm was probably attributable to the unpalatable nature of the dietary admixture and was not a direct effect of toxicity. Ophthalmoscopic examination revealed opacity and neovascularisation of the cornea, throughout the treatment groups, with effects more severe at 150 and 1500 ppm. A reduction in absolute brain weight was detected at all treatment levels. This was most probably due to the lower bodyweight gains seen during the first week of the study and not a neurotoxic effect of treatment. In addition, necropsy findings from 12000 ppm animals showed thickening of the glandular gastric epithelium. Histopathological examination showed no evidence of neurotoxicologically significant ocular or gastric change and neural tissue of 12000 ppm animals was similar to that of controls. No toxicologically significant microscopic changes were detected. 

Conclusion: Since no neurotoxic effects were observed up to the highest dose level, a 'No Observed Adverse Effect Level' (NOAEL) of 12000 ppm (equivalent to a mean achieved dosage of 996 mg/kg bw/day) for neurotoxicity was derived.

Justification for classification or non-classification

The available data on neurotoxicity with 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (CAS 473278-76-1) does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.