N-butylbenzenesulphonamide

Neurotoxicity

Description of key information

Adult male Adult Sprague-Dawley male rats (Harlan) received corn oil vehicle or BBSA (100, 200, or 300 mg/kg/ d) via oral gavage (5 ml/kg bwt) daily/5 d/week for 27 days. Neurobehavioral examinations and gait were monitored. As well as hindlimb dysfunction. On day 27 animals were euthanized and examined for astrocyte/microglia morphology and hippocampus.

No evidence of peripheral nerve lesions or gliosis in the hippocampus or cerebellum was observed in the rat following 27-day oral exposure. Furthermore no alterations were observed in gait, locomotor activity, and rearing behavior. Also mRMA levels for glial fibrillary acidic protein, interferon gamma,CXCR-3, intercellular adhesion molecule-1,and CD11b were not altered in the hippocampus while Iba-1 levels were decreased. No neurotoxicity was reported for BBSA with-in a 4 week exposure regime in the male Rat. Also no histological lesions were observed in the testis seminal vesicles, coagulating glands, epididymis and prostate. In the liver, minimal centrilobular hypertrophy was evident in all rats in the HD group.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to US NTP quidelins ( OECD)

Principles of method if other than guideline:

Adult Sprague-Dawley male rats (Harlan) received corn oil vehicle or NBBS (100, 200, or 400 mg/kg/d) via oral gavage (5 ml/kg bwt) daily/5 d/week for 27 d. Deaths were observed in the 400 mg/kg/d dose group in the first 5 d and dosing was decreased to 300 mg/kg/d. Adult male spargue-Darwley rats were examined for hindlimb dysfunction, sciatic nerve histopathology, gliosis in the hippocampus and cerebellum, male reproductive organ weights and histopathology as well as pathological changes in the liver.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Thirty-day-old male Spraque-Dawley rats ( Haralan labs, dublin, VA) were housed 4 per cage and allowed to acclimate to the animal facility. Foos( NIH31) and distilled deionized water were available at libitum. At 50 d of age, rats were ramdomly assigned to dose groups and received a daily oral dose ( 5 d a week ) of either corn oil vehicle (n=8) or NBBS (Frinton Labs) (100(n=8), 200 (n=8) or 400mg/kg/d (n=11)) in a gavage volume of 5ml/kg body wt. The 400 mg/kg/d dose resulted in lethality 3 rats after 1-week of dosing and the dose was lowerd to 300 mg/kg/d with no further incidence of morbidity or death. Animals received 5 daily doses per week over a 27-day period. All procedures were conducted according to an animal protocol approved by the NIEHS animal care and use committee.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Oral gavage. Dose volume 5ml/kg body wt, one dose a day (5 day a week).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

27-day oral exposure

Frequency of treatment:

one dose a day (5 day a week)

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

Remarks:

The 400 mg/kg/d dose resulted in lethality, and the dose was lowered to 300 mg/kg/d after 1 week dosing , with no further incidence of morbidity or death

No. of animals per sex per dose:

100(n=8),200 (n=8), 400 (n=11), The 400 mg/kg/d dose resulted in lethality 3 rats after 1-week of dosing and the dose was lowerd to 300 mg/kg/d with no further incidence of morbidity or death.

Control animals:

yes, concurrent vehicle

Details on study design:

Adult male spargue-Darwley rats were examined for hindlimb dysfunction, sciatic nerve histopathology, gliosis in the hippocampus and cerebellum, male reproductive organ weights and histopathology as well as pathological changes in the liver.

Observations and clinical examinations performed and frequency:

BODY WEIGHT: Yes at termination
ORGAN WEIGHT: Yes
FOOD CONSUMPTION : No data
WATER CONSUMPTION: no data
TISSUE COLLECTION: Yes
Seminal vesicles, epididymides, testes, ventral prostate, liver, sciatic nerve, brain, hippocampus.
OPHTHALMOSCOPIC EXAMINATION : No
HAEMATOLOGY: no data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
Neurological examination : Yes
Neurobehavioral observations
Rats where monitored in the home cage twice weekly, prior to dosing and at 4h post-dosing, for changes in general activity level and rearing behavior. Rearing in the home cage was recorded as absent or present for rearing aided and for rearing unaided by the cage wall.
24h after dosing , gait was assessed using the following rating scale
(1) Normal straight line gait with nosplay
(2) Minor splay(turn out of the toes) with hindlimbs maintained under the body
(3) Splay with hindlimbs extended out from the body
(4) Similar to 3 with addition of a wobble to the gait
(5) Full spayed hindlimbs with the inability to get/keep hindquarters under the body.
Within the observation chamber ,the animals were scored for gait over the first 2 min. The observer was blind to dosing conditions.
On dosing day 26, 3 h following dosing, individual animals were observationally evaluated for changes in locomotor activity (# grid crossing), and rearing (# rears unassisted by chamber wall) within an open field area ( 70 cm diameter x46 cm high Plexiglas cylinder) over a 10 min period.

Neurobehavioural examinations performed and frequency:

1 ) qRT-PCR of glia related genes in the hipocampus.
mRNA levels were evaluated for GFAP,IFN-gamma,CXCR-3, and CD11b.

2) Astrocyte and microglia morphology
Examination of cellular changes in hippocampus and cerebellum/brain stem of rat

3)Neurobehavioral observations
Rats where monitored in the home cage twice weekly, prior to dosing and at 4h post-dosing, for changes in general activity level and rearing behavior. Rearing in the home cage was recorded as absent or present for rearing aided and for rearing unaided by the cage wall.
24h after dosing , gait was assessed using the following rating scale
(1) Normal straight line gait with no splay
(2) Minor splay(turn out of the toes) with hindlimbs maintained under the body
(3) Splay with hindlimbs extended out from the body
(4) Similar to 3 with addition of a wobble to the gait
(5) Full spayed hindlimbs with the inability to get/keep hindquarters under the body.
Within the observation chamber ,the animals were scored for gait over the first 2 min. The observer was blind to dosing conditions.
On dosing day 26, 3 h following dosing, individual animals were observationally evaluated for changes in locomotor activity (# grid crossing), and rearing (# rears unassisted by chamber wall) within an open field area ( 70 cm diameter x46 cm high Plexiglas cylinder) over a 10 min period.

Sacrifice and (histo)pathology:

On day 27 24 h following last dosing, animals were euthanized by CO2 and tissues were rapidly collected for histological evaluation. Seminal vesicles with coagulating gland, epididymides,, testes, and ventral prostate were excised, weighed, and immersed in Davidson's fixative. The liver was collected and immersion fixed in 10% neutral buffered formalin. The sciatic nerve was excised, gently attached to an index card, maintaining proximal/distal orientation and immersion fixed in either 4 % paraformaldehyde /0.1 M phosphtate buffer (PB;pH 7,2) for paraffin embedding or 3
% glutaraldehyde/PB for embedding in Spurrs resin. The brain was rapidly excised from the cranium, bisected in the mid-sagittal plane,and one hemisphere was immersion fixed in 4 % paraformaldehyde/PB. The hippocampus was dissected from the contralateral hemisphere and rapidly frozen on dry ice and stored at -80 °C

Positive control:

not present

Statistics:

Terminal body weight and organ weight were analyzed by one way ANOVA.
Gait was assessed by a rating scale and analysed using Mann-whitney U test. Subsequent independent group mean comparisons to control were conducted by Dunnett's test.mRNA levels for each transcript were expressed as a ratio of target gene to GAPDH for each aniaml and analyzed by a one-way ANOVA.Subsequent independent group mean comparisons to controls were conducted by Dunnett's test. All statistical tests were conducted using Prism software and significance levels were set at p<0.05.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

3 animals at 400 mg/kg/BW

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

There was no evidence of dose dependent or treatment–related peripheral nerve degeneration following BBSA exposure.
We observed no elevations in the hippocampus for mRNA levels of related genes. Levels of mRNA for GFAP, IFN-gamma, CXCR-3 and CD11b were not altered by BBSA exposure and remained within constitutive levels seen in the control hippocamous. Daily oral gavage for 5d/week for 4 weeks did not produce clinical signs of hindlimb dysfunction, altered gait and rearing behavior, and ambulatory motor activity. Upon examination of the H&E staining in the hippocampus and cerebellum, no increased of GFAP or morphological indicators of astrocyte or microglial reactivity in the hippocampus or cerebellum.
In this study there was no evidence of decreased male reproductive organ weights up to 400 mg/kg/BW. We observed no significant changes in the liver in the 100 and 200 mg/kg/d BBSA dose group as compared to the controls. However all rats of the high BBSA dose group displayed some indication of minimal centrilobular hepatocyte hypertrophy.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No evidence of peripheral nerve lesions or gliosis in the hippocampus or cerebellum was observed in the rat following 27-day oral exposure. Furthermore no alterations were observed in gait, locomotor activity, and rearing behavior. Also mRMA levels for glial fibrillary acidic protein, interferon gamma,CXCR-3, intercellular adhesion molecule-1,and CD11b were not altered in the hippocampus while Iba-1 levels were decreased. No neurotoxicity was reported for BBSA with-in a 4 week exposure regime in the male Rat.

Key result

Critical effects observed:

no

No alterations were observed in gait, locomotor activity, and rearing behavior. There was no evidence of peripheral nerve lesions or gliosis in the hippocampus or cerebellum. mRNA levels for glial fibrillary acidic acid protein, interferon gamma, CXCR-3, intracellular adhesion molecule-1, and CD11b were not altered in the hippocampus while Iba-1 levels were decreased. These data do not support neurotoxicity effects for NBBS within a 4-week exposure regimen.

Conclusions:

No evidence of peripheral nerve lesions or gliosis in the hipocampus or cerebellum was observed in the rat following 27-day oral exposure. Furthermore no alterations were observed in gait, locomotor activity, and rearing behavior. Also mRMA levels for glial fibrillary acidic protein, interferon gamma,CXCR-3, intercellular adhesion molecule-1,and CD11b were not altered in the hippocampus while Iba-1 levels were decreased. No neurotoxicity was reported for BBSA with-in a 4 week exposure regime in the male Rat. Also no histological lesions were observed in the testis seminal vesicles, coagulating glands, epididymis and prostate. In the liver, minimal centrilobular hypertrophy was evident in all rats in the HD group.

Executive summary:

At 400 mg/kg/Day : 3 rats died between day 3 and 5, with no clinical signs of toxicity prior to death. Therefore dosing was decreased. To 300 mg/kg/day.

At 300 mg/kg/Day: There was no evidence of dose dependent or treatment-related peripheral nerve degeneration following NBBS exposure. All indices of degenerative changes in sciatic nerves across dosed groups and control were considered minimal background changes. GFAP immunostainiong (DAB) in the suprapyramidal blade and hilus of the dentate gyrus of rats at day 27 demonstrate a normal fibrous astrocyte morphology within the blade and the hilus, as well as for the vehicle and control . Immunostaining for GFAP in the cerebellum showed normal asterocyte morphology between vehicle control and 300mg/kg/day BBSA rats. Cage-side observations did not detect any dose-related difference in the behavior or general health status of the animals with no evidence of altered activity and rearing behavior. All animals displayed rearing behavior both in the center and at the wall of the home cage. Analysis of activity level as detected by locomotor activity and rearing in an open field confirmed a lack of motor deficits with no differences detected between vehicle controls and NBBS dosed rats. Within the open field arena, assessment of gait by a rating scale detected no significant difference between groups, with all animals falling within the rating score of 1. No male reproductive organ toxicity was observed up to 300 mg/kg/d. Only in the 300mg/kg /d dose group minimal changes in the liver were observed.

At 200 mg/kg/day: There were no treatment related macro-or microscopic findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD 407 oral in rodents

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

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), N-butylbenzenesulphonamide is not classified for neurotoxicity.