2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-05-08-1995-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 411, 1981; EPA Series 82-3, 1984; FIFRA Guideline 83-1, 1991; EEC Directive 87/302 Sub-Chronic Dermal Toxicity Study; MAFF, Subchronic Dermal Toxicity Study, 1985

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

EC Number:

500-033-5

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Cas Number:

25068-38-6

Molecular formula:

(C15 H16 O2 . C3 H5 Cl O)x

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Constituent 2

Reference substance name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

216-823-5

EC Name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Cas Number:

1675-54-3

Molecular formula:

C21H24O4

IUPAC Name:

2,2'-[propane-2,2-diylbis(4,1-phenyleneoxymethylene)]dioxirane

Details on test material:

Diglycidyl ether of bisphenol A (DGEBPA), lot number TB94060150389, was obtained from The Dow Chemical Company, Freeport, Texas. The identity of the test material was confirmed through the use of gas chromatography (GC)/mass spectrometry (MS) using the electron impact (EI) mode. The purity of DGEBPA was determined to be 99.65%  0.04% by GC using area percent quantitation (Ghaoui et al., 1995). A subsequent re-analysis (May 31, 1995) confirmed stability of the test material (Kiefer et al., 1995).

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Approximately 8-week-old male and nulliparous and nonpregnant female Fischer-344 rats (calculated birth date was March 6, 1995) were purchased from the Charles River Laboratories, Kingston, New York. This strain of rat was chosen because of its general acceptance in neurotoxicity testing and the availability of historical data. The laboratory veterinarian conducted eye examinations and examined the rats for health status shortly after arrival. All rats were determined to be in good health. The animals were acclimated to laboratory conditions for at least 1 week.

The rats were housed one per cage in suspended stainless steel cages which had wire-mesh floors. A 12-hour photoperiod was employed throughout the study with lights on at 7:00 am. Certified Purina Chow #5002 (Purina Mills, Inc., St. Louis, Missouri) and municipal drinking water were available ad libitum. Analysis of the Purina Certified Chow was performed by Purina Mills Inc. to confirm that the diet provided adequate nutrition, and to quantify the levels of selected contaminants. Analysis of the drinking water was performed by the City of Midland, Michigan and an independent laboratory in accordance with Laboratory SOPs. Contaminant levels were considered to be within normal limits, i.e., at levels that would not confound interpretation of the study.

Administration / exposure

Route of administration:

other: dermal- open

Vehicle:

acetone

Details on exposure:

Groups of 12 Fischer 344 rats/sex/dose level were dosed dermally with DGEBPA in acetone solutions at concentrations of 0%, 0.9%, 9%, or 90% (w/v) for male rats or 0%, 0.6%, 6%, or 60% (w/v) for female rats, respectively. The dosing volume was 300 ul per application which corresponded to approximate doses of 0, 10, 100, or 1000 mg DGEBPA/kg body weight/application. Each dose group followed a 5 applications/week (Monday through Friday) dermal dosing regimen for approximately 13 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the DGEBPA in acetone was determined in a previously conducted 13-week subchronic mouse study (Redmond et al., 1995c). Homogeneity of the test material in the low and high dose level solutions was determined concurrently with the conduct of the study. Analysis of dosing solutions by high performance liquid chromatography (HPLC) to verify the concentration of the test material was conducted on all dose levels at the beginning of the dosing period. Additional concentration analyses were conducted at week 6 and week 12 of the dosing period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100, or 1000 mg DGEBPA/kg body weight/application or 0%, 0.9%, 9%, or 90% (w/v) for male rats or 0%, 0.6%, 6%, or 60% (w/v) for female rats, respectively
Basis:
nominal conc.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 12 Fischer 344 rats/sex/dose level were dosed dermally with DGEBPA in acetone solutions at concentrations of 0%, 0.9%, 9%, or 90% (w/v) for male rats or 0%, 0.6%, 6%, or 60% (w/v) for female rats, respectively. The dosing volume was 300 ul per application which corresponded to approximate doses of 0, 10, 100, or 1000 mg DGEBPA/kg body weight/application. Each dose group followed a 5 applications/week (Monday through Friday) dermal dosing regimen for approximately 13 weeks. The rats were weighed and clinically examined weekly. Motor activity and a functional observational battery (FOB), including grip performance and hindlimb landing foot splay testing, were conducted before exposure and monthly thereafter. A battery of electrodiagnostic tests was conducted after 13 weeks of exposure on approximately half of the 12 rats/sex/exposure group. Electrodiagnostic tests, using evoked potential technology were used to screen for dysfunction of peripheral nerves, spinal cord, brainstem, cerebellum and cerebrum by evaluating evoked potentials from several modalities. FOB testing was conducted on all rats in a random order, whereas motor activity and electrodiagnostic testing were counterbalanced across dose groups.

Electrodiagnostic tests were divided into principal and ancillary. Principal tests included flash evoked potential (medium flash intensity) recorded from the visual cortex (FEP-V), somatosensory evoked potential recorded from the somatosensory cortex (SEP-S), auditory brainstem response to clicks (ABRc), and caudal nerve action potentials (CNAP). Ancillary tests included: Low intensity FEPs, cerebellar (FEP-C and SEP-C), and tone pip ABRs. Ten rats (5 males and 5 females) per exposure level were necropsied after electrophysiological testing.

Examinations

Observations and clinical examinations performed and frequency:

Prior to the start of the study, the eyes of all rats were examined using indirect ophthalmoscopy by a licensed veterinarian. A cageside visual evaluation for moribundity, mortality, the availability of feed and water, and treatment-related effects was conducted twice a day. Clinical observations are comparable to the hand-held portion of the FOB. Clinical observations were conducted once a week. The dermal test site was subjectively evaluated daily the first week of study and on the last day of each dosing week, using an acute dermal irritation scoring system similar to the one recommended by the Organization for Economic Co-Operation and Development (OECD, 1981). All rats were weighed during the predosing period and at weekly intervals during the dosing periods.

Specific biochemical examinations:

no biochemical examinations conducted

Neurobehavioural examinations performed and frequency:

Motor activity and a functional observational battery (FOB), including grip performance and hindlimb landing foot splay testing, were conducted before exposure and monthly thereafter. A battery of electrodiagnostic tests was conducted after 13 weeks of exposure on approximately half of the 12 rats/sex/exposure group. Electrodiagnostic tests, using evoked potential technology were used to screen for dysfunction of peripheral nerves, spinal cord, brainstem, cerebellum and cerebrum by evaluating evoked potentials from several modalities. FOB testing was conducted on all rats in a random order, whereas motor activity and electrodiagnostic testing were counterbalanced across dose groups.

Electrodiagnostic tests were divided into principal and ancillary. Principal tests included flash evoked potential (medium flash intensity) recorded from the visual cortex (FEP-V), somatosensory evoked potential recorded from the somatosensory cortex (SEP-S), auditory brainstem response to clicks (ABRc), and caudal nerve action potentials (CNAP). Ancillary tests included: Low intensity FEPs, cerebellar (FEP-C and SEP-C), and tone pip ABRs. Ten rats (5 males and 5 females) per exposure level were necropsied after electrophysiological testing (August 7-11, 1995).

Sacrifice and (histo)pathology:

Complete gross examination of tissues was conducted on all animals by a veterinary pathologist. An extensive list of tissues was collected. Tissues for the neuropathologic evaluation, were prepared from all perfusion-fixed rats in control and high-dose groups. Nine transverse sections of the brain were prepared from the: olfactory lobe, cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, cerebellum, and medulla oblongata. The following tissues were also prepared: trigeminal ganglia and nerve, pituitary gland, eyes with optic nerves, spinal cord (cervical and lumbar), nasal tissues with olfactory epithelium and skeletal muscles (gastrocnemius and anterior tibial). Tissues from the central nervous system and sections of skeletal muscle were embedded in paraffin, sectioned approximately 6 um thick, and stained with hematoxylin and eosin. Peripheral nerves (sciatic, tibial and sural) and dorsal root ganglia with roots (cervical and lumbar) were osmicated, embedded in plastic, sectioned approximately 2 um thick, and stained with toluidine blue. All tissues were examined by a veterinary pathologist using a light microscope.

Samples of the right dorsal caudal nerve from male rats were also evaluated by a teased nerve fiber technique. Approximately 0.6 cm sections of caudal nerve for this procedure were taken just distal to the primary section (approximately 4 cm from the base of the tail). The nerves were stained with 1% osmium tetroxide, dehydrated in graded ethanols, and infiltrated with Quetal 651. The fibers were then teased apart in a drop of resin on a glass slide using a needle and fine forceps until approximately 50 fibers were separated. The slides were then cured in a 60 degree oven overnight, cover slipped with additional resin and again cured prior to examination

Other examinations:

see other sections

Positive control:

no positive control

Statistics:

see below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):not applicable (migrated information)

Details on results:

Dosing Solution Stability, Homogeneity and Concentration: The stability of DGEBPA in acetone was determined to be approximately two weeks (Redmond et al., 1995a). Homogeneity of DGEBPA in acetone was determined once prior to the start of the study. The male high-dose solution was shown to be homogenous, with a standard deviation (S.D.) of 1.8 and percent relative standard deviation (% R.S.D.) of 1.94%. The female low-dose solution was also shown to be homogenous, with a S.D. of 0.01 and % R.D.S. of 1.76%.

To further verify the mixing method and test material delivery, samples of dose solutions and control (100%) acetone were assayed for concentrations of DGEBPA prior to study start and during weeks 6 and 12 of the dosing period. Results indicated an acceptable agreement between actual and target levels, with mean percent of target for all dose solutions ranging from 89% to 103%.

Feed and Water Analyses: Feed was determined to be nutritionally adequate for rats, and the feed or water did not contain unacceptable levels of contaminants.

Environmental Conditions: During the study, the average (±SD) holding room temperature was 22.01°C (±0.31) and the average (±SD) relative humidity was 53.39% (±5.83). A 12 hour light/dark cycle was maintained with light on at 7:00 am and off at 7:00 p.m. Airflow was maintained at 10 to 12 changes per hour.

Ophthalmology: Prior to the start of the study eye examinations were conducted via indirect ophthalmoscopy, and animals with eye anomalies were excluded from the population prior to randomization and assignment to study.

Sample Size: The goal of the study design was to have a sample size of 10 rats/sex/group. Two additional rats/sex/group were placed on study to ensure that this sample size was maintained in case of surgical death. Animals which died during surgery were removed from the study. Because of surgical deaths the sample size for all parameters of the study excluding the electrodiagnostic were as follows:

Control Low Mid High
Males 11 11 10 11
Females 12 12 11 12

Evaluation of Dermal Test Site: No treatment-related irritation was observed at dosages up to 1000 mg/kg/application in male or female rats throughout the duration of the study.

Cageside and Clinical Observations: No treatment-related effects were seen in cageside or clinical observations at any time during the study.

Body Weights: Treatment-related changes in body weights were observed in both sexes at the high-dose level. Body weights of male and female rats responded differently to treatment (a significant Treatment X Sex X Month p = 0.0141). Because of this significant interaction with sex, each sex was analyzed separately. Both sexes had significant changes in body weights (Treatment X Month for males, p = 0.003, and females, p = 0.004). Analysis of each treatment level versus control showed significant differences in males at the low- and high-dose levels (low dose p = 0.0063; mid-dose p = 0.0566; high dose p = 0.001). The lack of dose response in males (a non-significant mid-dose effect), coupled with statistical significance only in high-dose rats in the companion study of Redmond and Crissman (1995b), led to a conclusion that only the high-dose males had a treatment-related effect. The male low-dose statistical significance was regarded as spurious. In females, only the high-dose rats were significantly lighter than controls (female high dose p = 0.0001). High-dose female changes in body weight were attributed to treatment.

Differences in body weight gains of treatment versus control groups were also examined. High dose males had a 20% lower rate of growth while females in the high dose group had a 19% lower rate of growth.

Functional Observational Battery:

Hand-held and Open field Observations: The mean scores for FOB ranked observations provided an overall summary of the average rank of the group for each ranked category. Only one was sufficiently disparate between groups as to merit further attention (Text Table 1). Females in the high-dose group at week 13 demonstrated and increase in urination of almost 1 rank over control females. While this pattern appears to be dose response this is not supported by the incidence table (Text Table 2) and is not present at any other time in females or at any time in males.

Text Table 1. Average rank FOB Observations of Subjective Interest

MALES FEMALES
Dose mg/kg/day 0 10 100 1000 0 10 100 1000
Number of rats 11 11 10 11 12 12 11 12
WEEK 13
Urination 2.0 1.8 1.6 2.1 1.8 2.0 2.2 2.7


Ranked FOB observations were evaluated statistically, treatment vs control, by the test of proportions. There were potentially 384 pairwise comparisons, only two of which was significant (alpha = 0.02; Text Table 2). An increase number of rats in the mid-dose group had a moderate response to tail pinch. This finding lacks any kind of dose-response relationship and supports the absence of a treatment-related effect.

Urination was increased in the high-dose females at week 13. This becomes more apparent by combining the ranks of moderate and pronounced. An increase in perineal soiling was not noted on the FOB at this time point and this difference was not present at any other time point in females or at anytime in males. For these reasons the increase in urination of the high-dose females is believed to spurious and unrelated to treatment.

Text Table 2. Summary of Significant FOB Observations
(Test of Proportions; p< 0.02)

MALES FEMALES
Dose mg/kg/day 0 10 100 1000 0 10 100 1000
Number of rats 11 11 10 11 12 12 11 12
WEEK 4
Responsiveness to Tail Pinch
minimal 4 2 1 5 1 0 0 1
moderate 6 8 9 6 6* 11 12*(1) 7
pronounced 2 2 2 1 5 1 0 4

WEEK 13
Urination
none 2 3 6 3 4 4 3 1
minimal 7 7 2 5 7 4 5 4
moderate 2 1 2 2 1 4 1 5
pronounced 0 0 0 1 0 0 2 2
pronounced +
moderate 2 1 2 3 1* 4 3 7*(1)

*Significant pair , Test of proportions, treatment vs control: (1) z = 2.83, p < 0.02.

Rectal Temperature: No treatment-related effects were seen in rectal temperature at any time during the study.

Grip Performance: No treatment-related effects were seen in hindlimb or forelimb grip performance at any time during the study.

Landing Foot Splay: No treatment-related effects were seen in landing foot splay at any time during the study.

Motor Activity: No treatment-related effects were seen in motor activity at any time during the study.

Electrodiagnostics:

Surgery: A total of five animals were lost during surgery due to anesthetic complications. Additionally, complications with adherence of the implant to the skull of rats was encountered which resulted in a sample size of less than 10/sex/dose group for electrodiagnostic testing. In a factorial format combined male plus female sample sizes ranged from 11 to 16 per dose group.

Temperature: Body temperatures were recorded just before and just after collection of electrodiagnostic tests. Tail temperatures were recorded during the CNAP testing. There were no treatment-related differences in body temperature (treatment p=0.3582). Tail temperatures were cooler than what has been historically observed for other studies (tail temperatures approximately 22C for this study and approximately 25C for previous studies; Mattsson et al., 1992; Albee et al., 1992; Albee et al., 1993; Mattsson et al., 1993) for all animals, however, no exposure-related differences were identified (Treatment p=0.4370).


Flash Evoked Potentials (FEPs): Visual examination of FEPs did not reveal qualitative differences in either medium or low intensity FEPs recorded from the visual cortex or cerebellum. Quantitative evaluation of medium intensity FEPs recorded from the visual cortex did not reveal any exposure-related differences.

Ancillary data (FEP-C medium intensity, low intensity FEPs: Qualitative evaluation of ancillary FEPs did not reveal any exposure-related differences.

Auditory Brainstem Response (ABR): Visual inspection of ABR clicks and tone-pips did not reveal qualitative differences between treatment and control groups in either males or females.

Click Auditory Brainstem Response (ABRc): Quantitative evaluation did not reveal any treatment-related differences for either males or females.

Tone-pip Auditory Brainstem Response (ABR10 kHz& ABR30 kHz) Visual inspection of tone-pip ABRs did not reveal any treatment-related differences for either males or females at 10 or 30 kHz.

Somatosensory Evoked Potentials (SEP): Visual inspection of SEPs recorded from the cerebellum and from the somatosensory cortex (SEP-S) showed robust and well shaped waveforms that had similar peak latencies across females in all groups. Males were well shapes with similar peak latencies across groups, however, male controls exhibited waveforms which were less robust than treated groups. Comparison of the SEP power of control males to historical values from past studies conducted in this laboratory revealed that control males were less robust than what has been historically observed (male control, SEP long timebase power approximately 39uV; historical SEP long timebase power approximately 60uV; Mattsson et al., 1991; Mattsson et al., 1992; Albee et al., 1992; Albee et al., 1993). Treated males demonstrated power values for SEPs which were consisted with those observed historically. No quantitative differences were statistically identified between treatment and control groups.

Caudal Nerve Action Potentials (CNAP): Visual inspection of the CNAPs recorded in response to single stimuli and to pairs of stimuli showed that waveforms were well developed but somewhat slower than controls in the male low-dose and high-dose groups.

Quantitatively, overall MANCOVAs for single and paired pulse CNAPs were not statistically significant . At the univariate level, there were not statistical differences for waveform shape or power . However, for single-pulse CNAPs, the overall ANCOVA for latency differences was statistically significant (Treatment p=0.0150), and both sexes were affected similarly (non-significant Treatment X Sex p = 0.2790). Contrasts of treatment groups versus control indicated that only CNAPs of high-dose rats were different from control ( low-dose p = 0.4845; mid-dose p = 0.5971; high-dose p = 0.0079). Male high-dose CNAPs were 0.49 msec slower than controls, and female high-dose CNAPs were 0.12 msec slower.

Since the actual stimulus-response latencies to the major negative peak of the CNAP were about 5.2 msec, male high-dose CNAPs were about 9% slower than control, and female high-dose CNAPs about 2% slower than control.

Paired-pulse CNAPs overall ANCOVA for latency differences was also statistically significant (Treatment p = 0.0023), and both sexes were affected similarly (nonsignificant Treatment X Sex p = 0.1422). Contrast of treatment groups versus control indicated that only paired-pulse CNAPs of high-dose rats were different from control (low-dose p = 0.4501; mid-dose p = 8107; high-dose p = 0.0005). Only the male high-dose CNAPs demonstrated increased slowing of the paired-pulse over the single-pulse suggesting a decrement in the recoverability of the caudal nerve.

Post Hoc Caudal Nerve Action Potentials (CNAP) ) Male rats which were retested by warming the tails and using needle electrodes under isoflurane anesthesia demonstrated the same pattern of effects as rats initially tested. Qualitatively the low dose and high dose males were slower than controls. Comparison of CNAPs collected at the base of the tail or 4.5 cm distal to the base of the tail did not indicate that the degree of slowing was increasing across the caudal nerve.

Post-exposure testing conducted a 2 weeks and 1 month post-exposure demonstrated. males in the high dose group were approximately 0.2 msec slower as opposed to 0.49 msec two weeks earlier. At 1 month post-treatment the high dose males were still 0.2 msec slower.

Post Hoc Statistical Analyses of CNAPs: Analysis to optimized the evaluation of N1 of the caudal nerve action potential ( ratios of peak latency and amplitude of N1 of paired vs single pulse) for male control and high-dose rats did not support a hypothesis of reduced recoverability. Latency ratios were similar for both control and high-dose rats (control latency ratio = 1.11; high-dose latency ratio = 1.12). Amplitude ratios were better in high-dose rats than control rats suggesting that recoverability of the caudal nerve was better for the treated group (control amplitude ratio = 0.54; high-dose amplitude ratio = 0.71).

Analyses of CNAP by MANCOVA using body weight and tail temperature as a covariate suggested that the differences observed between treatment and control groups were associated with differences in body weights and tail temperature between groups (Latency ANCOVA, single-pulse p = 0.0866 with body weight, vs p = 0.0150 without bodyweight).

Overall CNAP results: Male high-dose and low-dose groups were qualitatively slower than control male rats in both cool and warm tails. Quantitatively, only the high-dose group was identified as significantly different than controls and this difference was identified in the latency component of the waveform. The caudal nerve conduction time was found to be equally slow across it’s entire length. Partial recovery of the caudal nerve action potential was observed at 2 weeks post-treatment and remained unchanged by 4 weeks post-treatment. The slowing in the caudal nerves of male low and high dose rats is associated with the differences in body weights and tail temperatures between controls and treatment groups. Analysis optimizing for N1 indicated that high-dose males had better recovery than control males.

Neuropathology:
There were no treatment-related gross lesions observed. There was no clear treatment effect visible by histopathologic examination. However, because of difficult to interpret electrophysiologic findings in the caudal nerves of male rats, their caudal nerves were examined in both methacrylate sections and teased nerve preparations. The observations in these nerves were tabulated under the general diagnostic category, "degeneration, myelin." The observations included a small increase in thinly myelinated fibers, and various proportions of fibers with myelin ellipsoids and balls. In nerves graded as "severe" virtually all nerve fibers appeared affected, although they were not severe in the sense of causing organ failure; i.e., flaccid tails were not observed, nor was any other visually apparent clinical neurologic deficit. The morphologic pattern of myelin degeneration appears to be most consistent with Wallerian degeneration or crush injury (Dyck et al., 1984), which indicates a primary effect on axons, not Schwann's cells; a pattern consistent with the effects of repeated local trauma, as would be predicted in the caudal nerves because of their anatomic vulnerability.

The incidence pattern of nerves with some degree of myelin degeneration was not related to test material concentration and was different than the pattern of nerve conduction velocity slowing observed in-life. Nerves with more severe anatomic lesions occurred more frequently in the middle-dose group males. However, nerve conduction velocities were not different from controls in this group. Additionally, no treatment effect was suggested by data in females -- either in-life or histopathologically. Also, the other peripheral nerves examined histopathologically were almost universally normal; so, clearly, there was no systemic effect on peripheral nerves.

Effect levels

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Based on a decrease in body weights at 1000 mg/kg/day.

Remarks on result:

other:

Any other information on results incl. tables

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                                                                                    Dose (mg/kg) Parameter                              0                    10                  100                1000 Body weight                          -                   +(m)                   -               +(m&f) Recovery                           -                 -/+(m)                  -                   +(m)        FOB (observations) nociception                         -                      -                      -                      - touch                                   -                      -                      -                      - Grip performance                  -                      -                      -                      - Landing foot splay                -                      -                      -                      - Motor activity                        -                      -                      -                      - Evoked Potentials Tail temperature                 +                     +                     +                     + Caudal nerve latency                             -                     +                     -                    ++ shape                                -                      -                      -                      - power                               -                      -                      -                      - peak N1 analyses (males only)      latency ratio*             -                    nd                   nd                    -      amplitude ratio*        -                    nd                   nd                    - Recovery                         -                    nd                   nd                   -/+ Somatosensory (SEP)         -                      -                      -                      - Flash (FEP)                         -                      -                      -                      - Auditory Brainstem Response (ABR)             -                      -                      -                      - Pathology 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

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

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.