Phenol

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The Pesticide Assessment Guidelines, Subdivision F, Series 82-7 (PB91-154617) were followed; this guideline is comparable with OECD Guideline 424. Minor restriction: no ophthalmological examination.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Pesticide Assessment Guidelines, Subdivision F, Series 82-7 (PB91-15461J7); comparable to OECD Guideline 424

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phenol

EC Number:

203-632-7

EC Name:

Phenol

Cas Number:

108-95-2

Molecular formula:

C6H6O

IUPAC Name:

phenol

Details on test material:

- Physical state: white cristalline powder
- Analytical purity: 100% (determined by GC/TCD area%)
- Impurities (identity and concentrations): impurities >0.1% were not detected; water content 0.02%
- Purity test date: 1997-01-17
- Lot/batch No.: 112796/102596
- Stability under test conditions: stability and homogeneity of drinking water solutions were analysed; acceptance criteria (± 10% of target concentration) fullfilled (dose formulations prepared for administration to the animals during weeks 1, 7 and 13)
- Storage condition of test material: glass container (nitrogen blanket) at RT in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Crl:CD®(SD)BR
- Source: Charles River Canada Inc., St. Constant, Quebec
- Age at study initiation: appr. 7 weeks
- Weight at study initiation: 210 to 292 g (males) or 155 to 199 g (fema!es)
- Fasting period before study: no
- Housing: individually
- Diet (ad libitum except during behavioral evaluations): certified diet, analysed for contaminations
- Water (ad libitum except during behavioral evaluations): Municipal tap water which had been further treated by reverse osmosis and ultraviolet sterilization
- Acclimation period: 2 we


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Randomization approximately one week before treatment initiation (all animals weighed and 15 males and 15 females assigned to each of 4 groups using a computer-based selection procedure). Five rats/sex/group for the following phases of the study: perfusion after 13 weeks of treatment, perfusion at the end of the recovery period of 4 weeks, and necropsy at the end of the recovery period of 4 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of drinking water solutions were analysed; acceptance criteria (± 10% of target concentration) fullfilled (dose formulations prepared for administration to the animals during weeks 1, 7 and 13).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily ad libitum

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 200, 1000, 5000 ppm
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
18, 83, 308 mg/kg bw/day for males
Basis:
actual ingested
mean values

Doses / concentrations 3

Remarks:

Doses / Concentrations:
25, 107, 360 mg/kg bw/day for females
Basis:
actual ingested
mean values

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Post exposure observation period in the recovery group: 4 weeks

Examinations

Observations and clinical examinations performed and frequency:

Rats examined twice daily; a complete detailed examination was performed weekly.
Body weight: weighed weekly including days of behavioral testing as well as prior to terminal sacrifice.
Food consumption: Individual food consumption measured weekly including one week prior to treatment start.
Water consumption: Individual water consumption measured daily including the week prior to treatment start.
Body temperature measured once prior to initiation and at treatment week 4, 8, 13, and week 4 of recovery period

Specific biochemical examinations:

no data

Neurobehavioural examinations performed and frequency:

Functional Observational Battery (FOB)
All animals tested prior to the initiation and once during the 4th, 8th and 13th weeks of treatment. In addition, recovery phase animals (1O/sex/group) were tested during the 4th week of the recovery period.

Qualitative
Observations in home cage: body position, tremors, twitches, convulsions, bizarre/stereotypic behavior
Removal from home cage: ease of removal, vocalization, observations in arena, rearing, ataxie, hypotonic and impaired gait, overall gait incapacity, bizarre/stereotypic behavior, palpebra1 closure, tremors, twitches, convulsions, piloerection, respiratory rate/pattern, locomotor activity level, arousal, grooming, defecation, urination, olfactory response
Handling observations: lacrimation, pupil size, salivation, urinary staining, diarrhea, body tone, extensor thrust, corneal reflex, pinna reflex, toe and tail pinch, visual placing,
On Surface: auricular startle, air righting reflex, positional passivity.
Quantitative: hindlimb and forelimb grip testing, hindlimb splay, motor activity (sessions of 1-hour duration and activity counts recorded).

Sacrifice and (histo)pathology:

After 13 weeks of treatment, 5 rats/sex/dose were deeply anesthetized and perfused with lactated Ringer's solution followed by 3% glutaraldehyde & 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3 to 7.5). Same procedures were performed on 5 rats/sex/group euthanized after a 4-week recovery period.
Tissues from the high dose and control animals were processed for neuropathological evaluation. The nervous system tissues of animals in all groups were grossly examined at the time of sampling and any pathology observed was recorded.
Tissues for paraffin embedding and light microscopy:
Brain (6 levels) - forebrain (through the septum), center of the cerebrum (through the hypothalamus), midbrain, cerebellum and pons, midcerebellum and medulla oblongata, and medulla oblongata. Spinal cord - cervical and lumbar (cross-section). Brain weight (excluding olfactory bulbs), length and maximum coronal width were recorded prior to trimming.
Tissues for Epoxy Embedding:
Sciatic nerve (mid-thigh region) (cross-section)
Sciatic nerve (at sciatic notch) (longitudinal and cross-sections)
Sural nerve (at knee) (cross-section)
Tibial nerve (at knee) (longitudinal and cross-sections)
Gasserian ganglion - left (cross-section)
Lumbar dorsal root ganglion (L4) (cross-section)
Lumbar dorsal root (L4) (cross-section)
Lumbar ventral root (L4) (longitudinal and cross-sections)
Cervical dorsal root ganglion (C5) (cross-section)
Cervical dorsal root (C5) (cross-section)
Cervical ventral root (C5) (cross-section)
Grossly abnormal central or peripheral nervous system tissues.

All other animals: necropsy performed, tissue preserved in 10% formalin

Other examinations:

no

Positive control:

no

Statistics:

Group variances Bartlett's test; when the differences between group variances were not significant (P>0.001), a one-way analysis of variance (ANOVA) was performed. If significant differences (P<0.05) were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups. When the differences between group variances were significant (P<0.001) by Bartlett's test, the Kruskal-Wallis test was then performed. Where significant differences (P<0.05) between the groups were indicated by the Kruskal-Wallis test, the values for the control and treated groups were compared using Dunn's test (or WilcoxonIMann Whitney "U" test).
Qualitative FOB data analyzed by Fisher's exact probability test.
Level of significance declared at 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

Mortality and clinical signs
5000 ppm: One female was euthanized on day 14 due to poor condition (dehydratation, hunched posture/prominent backbone/thin/weak appearance, uncoordinated movements, tremors, reduced activity, pallor, partly closed eyes, reduced body temperature). One male and 1 female showed dedydration, hunched posture/prominent backbone/thin appearance, reduced activity, decreased feces and fur staining. Nine females and seven males showed a dehydrated appearance commencing as early as the end of week 1; one male and six females showed a prominent backbone/thin appearance.
1000 ppm: two males and two females showed a dehydrated appearance commencing as early as the end of week 1.
200 ppm: no treatment related effects.

Body weight
200 & 1000 ppm: no significant effects
5000: mean body weights in males and females significantly reduced during treatment period but rats showed marked gains in body weight during the recovery period (in males no significant difference compared with control).

Food consumption
No significant differences in mean food consumption were noted between the control and 200 or 1000 ppm groups during the treatment period.
5000 ppm: significant decrease in males and females during exposure period and significant increase in males during recovery period.

Water Consumption
5000 ppm: mean daily water consumption significantly (P1000 ppm: On occasion, significant reductions in mean daily water intake in males and females; at other times, the water intake of the 1000 ppm rats was generally lower than control values.
200 ppm: no significant differences detected.

Body temperature
A significant decrease in body temperature was noted for males in the 1000 and 5000 ppm groups at the week 13. However, as these values fell within the historical control range (37.1 to 38.4°C) together with the absence of FOB findings, this was considered not to be of toxicological significance.

Functional Observational Battery (FOB) and Motor Activity
No behavioral changes were observed for animals treated with phenol.
Exception: At the week 4 a significant (P< 0.01) reduction in total counts in motor activity was observed for 5000 ppm females. Correlation of the total counts of individual animals with dehydration shows that while the control group had an average increase of > than 20% at week 4 compared to the prestudy evaluation, females in the 5000 ppm group with dehydration had an average decrease of 17%; however, females without dehydration had an average increase of 2% over prestudy values suggesting that the decrease in motor activity was secondary to markedly lower (40-50% of control) water intake.
Other changes at 1000 and 5000 ppm were also considered not to be of neurotoxicological significance.

Brain weight
No effects on organ weight detected in brains of perfused animals.

Gross pathological evaluation
There were no necropsy findings considered treatment-related

Neuropathology
There were no gross or histopathological findings following treatment and no gross pathological findings following recovery in nervous tissue for males or females that were considered treatment related.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

200 ppm

Sex:

male/female

Basis for effect level:

other: effects secondary to reduced water intake; dose corresponding to 18 mg/kg bw/day in males and 25 mg/kg bw/day in females

Remarks on result:

other:

2

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Sex:

male/female

Basis for effect level:

other: neurotoxicity; corresponding to 360 mg/kg bw/day for females and 308 mg/kg bw/day for males

Remarks on result:

other:

Applicant's summary and conclusion

Conclusions:

In male and female rats the NOAEL for neurotoxic effects was 5000 ppm in a 13 week drinking water study. The NOEL concerning overall effects was 200 ppm.

Executive summary:

The Pesticide Assessment Guidelines, Subdivision F, Series 82 -7 (PB91 -154617) were followed; this guideline is comparable with OECD Guideline 424. Minor restriction: no ophthalmological examination.

In a drinking water study 15 rats per dose per sex were exposed to 0, 200, 1000, or 5000 ppm for 13 weeks. Five rats/sex/group were used for the following phases of the study: perfusion after 13 weeks of treatment, perfusion at the end of the recovery period of 4 weeks, and necropsy at the end of the recovery period of 4 weeks.

At a concentration of 5000 ppm lower body weight, reduced food and water consumption and abnormal clinical signs including dehydrated appearance was reported; one female was sacrificed due to poor condition. At 1000 ppm, decreased water intake and on occasion dehydrated appearance were seen. Marked improvements were noted following recovery. No effects were noted for any parameters at 200 ppm. Neurobehavioral evaluations (functional observation battery) did not reveal any findings of neurotoxicological significance at any concentration following treatment or recovery. Observations of altered motor activity (females only) were noted, however, these findings were considered to be secondary to the reduction in water and/or food intake. No gross or histopathological lesions in nervous tissue attributed to treatment with phenol were detected. Based on these findings, a NOAEL for neurotoxicity under the conditions of this study was 5000 ppm (308 and 360 mg/kg bw/day for males and females, respectively). The overall study NOEL under the conditions of this study was 200 ppm (18 and 25 mg/kg bw/day for males and females, respectively).

Conclusion: In male and female rats the NOAEL for neurotoxic effects was 5000 ppm in a 13 week drinking water study. The NOEL concerning overall effects was 200 ppm.