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EC number: 216-823-5
CAS number: 1675-54-3
There were no treatment-related effects on clinical observations, FOB (functional observations, grip performance, rectal temperature and landing foot splay), motor activity, sensory evoked potentials (FEP, SEP and ABR), and neuropathology (Text Table3). Differences from controls were observed in the caudal nerve action potentials (CNAP) of male 10, and male and female 1000 mg/kg/day groups, but, were considered not to be an adverse effect of treatment. The only clear treatment-related effect was a decrease in body weights at the high-dose group in males and females. This difference in body weight persisted 1 month post-treatment in 1000 mg/kg male rats; females were not held post treatment.
Due to implant loss, approximately half of the animals scheduled to be tested for electrophysiology could not be tested. The impact of this to the overall study is believed to be minimal for several reasons. First, a complete sample size was attained for all of the neurotoxicity screening battery parameters (clinical observations, FOBs, MA, and neuropathology). Therefore, only the sample size of the electrodiagnostic portion of this study was impacted by the loss of implants. Second, the data was analyzed by a factorial ANOVA which uses data from both sexes. In spite of smaller sample sizes, the statistical power exceeds that attained in a standard analyses where each sex is analyzed separately (i.e. 10/sex/dose). Figure 40 illustrates the degree of power that is attained with the factorial ANOVA versus ANOVA by sex. Finally, animals which lost implants underwent surgery to repair the scalp defect. These rats were retained on study for measures other than cerebral evoked potentials.
Electrodiagnostics, using evoked potentials technology, are techniques that can be use to evaluate multiple nervous system pathways (Mattssonet al., 1992). The flash evoked potential (FEP) was used to functionally evaluate the visual pathway from the retina to visual cortex, and cortical-subcortical circuits. The somatosensory evoked potential (SEP) evaluates sensory pathways from the caudal nerve, through the spinal cord and brainstem to the thalamus and then the sensory cortex. The auditory brainstem response (ABR) evaluates the integrity of the lower and upper brainstem, and tone-pip ABRs evaluate areas of the cochlea for evidence of ototoxicity. No treatment related effects were observed in the FEP, SEP or the ABRs. The CNAP was used to test peripheral nerve function.
Text Table 3. Summary of Results
Parameter 0 10 100 1000
Body weight - +(m) - +(m&f)
Recovery - -/+(m) - +(m)
nociception - - - -
touch - - - -
Grip performance - - - -
Landing foot splay - - - -
Motor activity - - - -
Tail temperature + + + +
latency - + - ++
shape - - - -
power - - - -
peak N1 analyses (males only)
latency ratio* - nd nd -
amplitude ratio* - nd nd -
Recovery - nd nd -/+
Somatosensory (SEP) - - - -
Flash (FEP) - - - -
Response (ABR) - - - -
Caudal nerve + ++(m) ++(m) +
Tibial - - - -
Sciatic - - - -
Sural - - - -
+ = increased severity or incidence of effect; - = negative
m = males only; f = females only; nd = not done
*Latency to peak N1 for the paired-stimulus response compared to peak N1 for the single-stimulus response. Similarly, the amplitude ratios were calculated.
Visual inspection of the waveforms and statistical evaluation of the CNAP data indicated slowing in 1000 mg/kg/day group, and visually the male 10 mg/kg/day group was slower than controls. The 100 mg/kg/day group was not different from the control. Shapes of the CNAPs composites from treated rats were robust and correlation to the control template was high. Although there was no treatment-by-sex interaction statistically, it was readily apparent from the composite waveforms that the 1000 mg/kg/day females were not slow compared to the controls. At the high dose, the males were 9% slower than controls whereas the high dose females were only 2% slower than control. The amount of slowing was calculated by determining the msec shift required to achieve an optimal correlation (best fit or similarity of shape) of the individual waveform to the template. The technique is especially useful for screening, but can be somewhat imprecise since the CNAP is a compound action potential which has a dominant peak (N1). The female 1000 mg/kg/day composite clearly shows no difference for peak N1but is slightly slower for N2which may account for the slight slowing that was calculated using the screening analysis. Since the male 1000 and 10 mg/kg/day composites were clearly slower than the controls, further analyses focused on the male CNAP data, specifically N1latency and amplitude.
A sensitive method to evaluate peripheral nerve toxicity is to examine the ability of the nerve to respond to a second stimulus which is presented very soon after an initial stimulus (recoverability). The interval (3 msec) between the first and second stimuli of the paired stimuli was selected so that a normal caudal nerve could not fully recover from the first stimulus by the time the second stimulus was presented, i.e. even control nerves were challenged. A ratio was calculated for latency and amplitude (latency to N1paired/latency to N1single; amplitude of N1paired/amplitude of N1single. This ratio tends to control for between animal variability in latency and amplitude which makes it ideally suited to evaluating compromised peripheral nerve function. To support a hypothesis of treatment-related neurotoxicity of the caudal nerve, one would expect the second response from paired stimuli to recover more slowly in an affected nerve than a non-affected nerve in amplitude and/or in latency. Peak N1recovery function of the 1000 mg/kg male group was virtually the same as the controls for latency, and was better than the controls for amplitude. The latency to N1of the second of the pair for the controls was 1.11 of the single-stimulus response and was 1.12 for the 1000 mg/kg group. Amplitude for the control second of the pair was 0.54 of the single stimulus response and was 0.71 for the 1000 mg/kg group. Clearly, recoverability of nerve function was not affected by treatment.
A conclusion of caudal nerve toxicity is further weakened by a lack of consistent neuropathology of the caudal nerves from 1000 mg/kg males compared to controls. To support a conclusion of an adverse effect on the CNAP one would have to argue for a central-peripheral distal axonopathy (C-PDA). However, histopathological examination of the caudal nerves revealed a high incidence of spontaneous lesions in controls as well as the treated groups. There were no changes that could be correlated with a treatment-related increase in latency and the pattern of histopathologic effects was not consistent with the pattern of effects seen electrophysiologically. The most severe histopathology was in the 10 mg/kg/day and 100 mg/kg/day groups while animals in the control and 1000 mg/kg/day group were similarly affected but to a lesser degree than at 10 and 100 mg/kg/day. It should be noted that the animals randomly selected for histopathology overlapped only slightly with electrodiagnostic rats.
Since the caudal nerve is similar to other peripheral nerves, one would expect pathologic changes in peripheral nerves other than the caudal nerve (e.g. sciatic, sural and distal tibial nerves) if C-PDA were present. There were, however, no pathologic changes in the sural or the tibial nerves. Further, if DGEBPA caused a C-PDA, neuropathology of the ascending end of the dorsal column (entry to the brain stem) would be expected. No lesions were observed in the dorsal column nor were there an changes in the far-field portion of the SEP (activity from the dorsal column and nucleus gracilis). Additionally, the tail touch response and the test for nociception (pinch response), conducted during the FOB, designed to assess diminished sensitivity in the tail, were not different between treatment and control groups.
In addition to the lack of correlation of histopathology with the effects seen electrophysiologically, similarly there was no correlation with grip performance or landing foot splay. Grip performance and landing foot splay are techniques that were included in EPA’s neurotoxicity guideline to screen for potential peripheral neuropathy. Animals with the slowest CNAP responses did not have smallest grip performance nor did they have the greatest hind limb splay. Conversely, animals with the fastest CNAP responses did not have the largest grip performance or smallest hind limb splay.
Finally, there appears to be an association between the body weight differences in the low- and high-dose male rats, and the changes in CNAP conduction times. It seems unlikely that by coincidence the same groups that had lower body weight (male 10 and 1000 mg/kg/day and female 1000 mg/kg/day) also had varying degrees of slow CNAP responses. Although the nature of the effect is obscure, the overwhelming preponderance of data indicates the slowing of the caudal nerve was not due to peripheral neuropathy.
Mattsson, J. L., Boyes, W. K., and Ross, J. F. (1992). In
Neurotoxicology, edited by Hugh Tilson. Chapter 7. Incorporating evoked
potentials into neurotoxicity test schemes. Raven Press, Ltd., New York
Male and female Fischer 344 rats were
dermally exposed to Diglycidyl Ether of Bisphenol A (DGEBPA) at
approximately 10, 100 or 1000 mg/kg, 5 dayslwk, for 13 weeks. The rats were
weighed and clinically examined weekly. A functional observational
battery (FOB) and motor activity (MA) were conducted preexposure and
monthly. In addition to the FOB and MA evaluations, the postexposure
neurotoxicity evaluation focused on evoked potential testing of the
visual (FEP), auditory (ABR) and somatosensory systems (SEP), conduction velocity
evaluation of caudal nerves (CNAP), and a comprehensive
neuropathological examination. The only effect clearly related to
treatment was a decrease in body weight at the 1000 mg/kg/day dose group
for both sexes. Slower caudal nerve conduction times observed in the
low- and high-dose males was not attributed to dermal treatment with
DGEBPA because of the lack of dose response, the absence of
toxicological patterns among the parameters evaluated, the absence of
any consistent histopathological findings in multiple peripheral nerves,
including the caudal nerve, and the lack of an effect on caudal nerve recoverability.
The No-Observed-Effect-Level (NOEL) for dermal exposure to DGEBPA was
100 mg/kg/day for both males and females based on a decrease in body
weights at 1000 mg/kg/day.
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