Registration Dossier

Administrative data

Description of key information

Toxicity of phenol, heptyl derivatives administered orally (gavage) to Crl:CD(SD) rats for 28 days was mainly observed at a dosage level of 450 mg/kg/day as evidenced by lethality, clinical observations (decreased defecation, dermal atonia, hypothermia), lower body weights, serum chemistry changes and several histologic changes (tubular nephropathy in the kidneys, fatty change of the liver, stratified squamous hyperplasia of the non-glandular stomach, thymic lymphoid depletion and hemorrhage and depletion of secretion of seminal vesicles). Therefore, the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of phenol, heptyl derivatives to Crl:CD(SD) rats for 28 consecutive days was 150 mg/kg/day as none of the aforementioned effects occurred at that dosage level.
No classification for repeat dose effects is required as the effect is limited to a local irritant effect on the forestomach, which is not relevant to humans. The irritation effects observed at the 150 mg/kg/day dose level were not considered to be adverse. The systemic toxic effects observed at 450 mg/kg/day were considered to be secondary to the lcoal irritant effects in the stomach.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-10-24 to 2006-08-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 217 - 264 g, females: 151 - 199 g
- Fasting period before study: No
- Housing: individually in clean stainless steel wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C (19.9°C - 23.2°C)
- Humidity (%): 50±20% (35.0% - 50.5%)
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2005-11-07 To: 2005-12-05
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: weight dependant per dose group
- Amount of vehicle (if gavage): 0, 10, 30, 90 mg/mL to give dose of 5 mL/kg
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for homogeneity and stability

GC with flame ionisation detection:
Instrument: Hewlett Packard 6890 GC
Column: Rtx-1, 30 m x 0.25 mm ID, 0.025 µm film thickness
Temperature: Initial temperature 75°C, hold 0.5 minutes
Ramp A at 25°C/minute to 250°C, hold 0.5 minutes
Carrier gas: Helium flow of 1µL/minute
Pressure: 13.3 psi at 75°C (EPC constant flow on)
Injector temperature: 150°C
Injection volume: 1.0 µL split (10:1)
Detector: FID at 250°C
Retention time: Ca. 6.5 - 6.8 minutes
Run time: 8.0 minutes

Quantitation:
Range: 62.5 - 500 µg/mL

Homogeneity (10 and 90 mg/mL refrigerated for 10 days):
Variability (RSD): =< 10%
Relative error (% RE): within ± 15%

Stability (10 and 90 mg/mL refrigerated for 10 days):
Post storage mean analyte concentration not < 90% of initial concentration

Test article concentrations:
Range: 95.0% - 98.1% of target values
Analyses concentrations within 15% of target concentrations.
Duration of treatment / exposure:
28 days
Following 4 weeks of dose administration, 5 rats/sex/group were euthanized; the remaining animals in the control and high dose groups were euthanized following a 14-day nondosing (recovery) period.
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Control and 450 mg/kg bw/day: 10 per sex/dose
50 and 150 mg/kg bw/day: 5 per sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day range finder
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pretest and then weekly physical examination
Detailed physical examinations were conducted on all animals weekly, beginning approximately 1 week prior to test article administration. Observations at the time of dosing were not performed on days when detailed physical examinations were conducted, provided the detailed physical examination was conducted prior to dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pretest and twice daily during exposure
Clinical examinations were performed twice daily, prior to dose administration and approximately 1 to 2 hours following dose administration (designated as 1 hour post-dosing for report presentation purposes). All significant findings were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Pretest and weeky during exposure

FOOD CONSUMPTION : Yes
- Time schedule for examinations: Pretest and weekly during exposure, calculated as g/animal/day

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of necrospy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked
Total leukocyte count (White Cells)
Erythrocyte count (Red Cells)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (Platelet)
Prothrombin time (ProTime)
Activated partial thromboplastin time (APTT)
Reticulocyte count
Percent (Reticulocyte)
Absolute (Retic Absolute)
Differential leukocyte count -
Percent and absolute
-Neutrophil
-Lymphocyte
-Monocyte
-Eosinophil
-Basophil
-Large unstained cell

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of necrospy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total Bili)
Urea nitrogen
Creatinine
Alkaline phosphatase (AlkalinePhos’tse)
Alanine aminotransferase (Alanine Transfer)
Aspartate aminotransferase (AspartatTransfer)
Gamma glutamyltransferase (GlutamylTransfer)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Triglycerides (Triglyceride)

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necrospy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked
Specific gravity (SG)
pH
Urobilinogen (URO)
Total volume (TVOL)
Color (COL)
Clarity (CLA)
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Bilirubin (BIL)
Occult blood (BLD)
Leukocytes (LEU)
Nitrites (NIT)
Microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Study week 3 prior to dosing
- Dose groups that were examined: All
- Battery of functions tested:
HOME CAGE OBSERVATIONS
Posture
Convulsions/tremors
Feces consistency
Biting
Palpebral (eyelid) closure
HANDLING OBSERVATIONS
Ease of removal from cage
Lacrimation/chromodacryorrhea
Piloerection
Palpebral closure
Eye prominence
Red/crusty deposits
Ease of handling animal in hand
Salivation
Fur appearance
Respiratory rate/character
Mucous membranes/eye/skin color
Muscle tone
OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE OBSERVATION PERIOD)
Mobility
Rearing
Convulsions/tremors
Grooming
Bizarre/stereotypic behavior
Time to first step (seconds)
Gait
Arousal
Urination/defecation
Gait score
Backing
SENSORY OBSERVATIONS
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation
NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength
Hindlimb foot splay
Grip strength-hind and forelimb
Rotarod performance
PHYSIOLOGICAL OBSERVATIONS
Catalepsy
Body temperature
Body weight
LOCOMOTOR ACTIVITY

Sacrifice and pathology:
GROSS NECROPSY: Yes
The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. The following tissues and organs were collected:
Adrenal glands (2)
Bone with marrow
Femur
Sternum
Bone marrow smear (from femur)
Brain
Cerebrum level 1
Cerebrum level 2
Cerebellum with medulla/pons
Cervix
Epididymides (2)
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes
Mesenteric
Mandibular
Ovaries with oviducts (2)
Peripheral nerve (sciatic)
Pituitary
Prostate
Seminal vesicles (2)
Spinal cord
Cervical
Midthoracic
Lumbar
Spleen
Testes (2)
Thymus
Thyroid [with parathyroids, if present (2)]d
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions (when possible)

ORGAN WEIGHTS:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Prostate
Seminal vesicles
Spleen
Testes
Thymus
Thyroid with parathyroids*
Uterus
Paired organs were weighed together.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less.
Body weight, body weight change, food consumption, continuous functional observational battery (FOB), locomotor activity, clinical pathology and organ weight data were subjected to a parametric 1-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
See Details on results
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Details on results
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL OBSERVATIONS AND MORTALITIES
Test article-related deaths were noted in the 450 mg/kg/day group. One male (no. 99925) and one female (no. 99952) in the 450 mg/kg/day group died on study day 27. Toxicologically relevant clinical observations in these animals prior to death included decreased defecation, dermal atonia, hypothermia (body cool to touch) and thinness. A specific cause of death for these animals could not be determined but the deaths were most likely attributed to test article administration. There were no other test article-related deaths. One male (no. 99922) in the 150 mg/kg/day group died of an accidental death on study day 28, and one female (no.99941) in the 450 mg/kg/day group died on study day 4, presumably due to some aspect of experimental manipulation, but this could not be confirmed based on the gross and histopathology report.
All other animals survived to the scheduled necropsies. Test article-related clinical observations in the surviving animals during the dosing period consisted of clear material around the mouth and/or ventral neck and forelimbs, and signs of unkempt appearance (yellow material on various body surfaces including the urogenital area) in the 450 mg/kg/day group. Dermal atonia and thinness were noted occasionally in the 450 mg/kg/day group. These clinical signs were not observed during the recovery period. There were no other test article-related clinical observations. All clinical findings in the test article-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.
BODY WEIGHTS
Test article-related lower body weights were noted in the 450 mg/kg/day group males. Lower mean body weights gains were noted in this group during the entire dosing period (study weeks 0 to 1 through 3 to 4) when compared to the control group. As a result, mean body weights were also lower (up to 14%) during the entire dosing period when compared to the control group. Mean body weights in the 450 mg/kg/day group males were still lower (8%) than the control group by the end of the recovery period; although some mean body weight gains were higher during study weeks 4 to 5 and 5 to 6.
FOOD CONSUMPTION
There were no test article-related effects on food consumption. A slight (5 gram), statistically significant reduction in food consumption was noted in the 450 mg/kg/day group males during the first week of dosing when compared to the control group. However, this transient reduction in food consumption was likely not attributed to test article administration because a sustained effect was not observed to correlate with the lower body weights noted throughout the dosing period in the 450 mg/kg/day group males.
FUNCTIONAL OBSERVATIONAL BATTERY (NEUROBEHAVIOUR)
Home cage observations: There were no test article-related effects on home cage observations. Several statistically significant changes were noted when compared to the control group. However, these findings were not noted in a dose-related manner or were not considered toxicologically significant without other effects on functional observational battery parameters.
Handling observations: The 450 mg/kg/day group males and females were noted with statistically significantly
lower number of animals with normal fur appearance (clean and groomed) when compared to the control group. These changes correlated with the clinical signs of unkempt appearance in this group and were considered test article-related. There were no other statistically significant changes in handling observations.
Open Field observations: Open field observations were unaffected by test article administration. There were no statistically significant differences when the test article-treated males and females were compared to the control group at the study week 3 evaluation.
Sensory observations: Sensory observations were unaffected by test article administration. There were no statistically significant differences when the test article-treated males and females were compared to the control group at the study week 3 evaluation.
Neuromuscular observations: There were no test article-related effects on neuromuscular observations. Mean rotarod performance was higher in the 50 and 450 mg/kg/day group females (statistically significant in the 50 mg/kg/day group) when compared to the control group. However, because decreased rotarod performance is typically indicative of toxicity, the difference was most likely the result of low control group values and was not considered the result
of test article administration. In addition, mean hindlimb footsplay in the 450 mg/kg/day group females was statistically significant higher in the 450 mg/kg/day group females. This finding was not considered test article-related because the 450 mg/kg/day group males were comparable to control group males and the female control value for footsplay was unusually low relative to historical control data. There were no other statistically significant differences in neuromuscular observations.
Physiological observations: There were no test article-related effects on physiological observations. Mean body weight in the 450 mg/kg/day group males was statistically significantly lower than the control group as noted previously (Section 6.3.). Mean catalepsy time in the 150 mg/kg/day group males was statistically significantly higher than the control group. As this slight difference from the control group was not noted in a dose-related manner, it was not considered the result of test article administration.
Locomotor activity: Locomotor activity patterns (total and ambulatory activity counts) were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 evaluation. Values obtained from the 4 epochs evaluated (0-15 minutes, 16-30 minutes, 31-45 minutes and 46-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test article-treated groups when the animals were
evaluated during study week 3.
HAEMATOLOGY
There were no test article-related effects on hematology parameters. All statistically significant differences from the control group were not observed consistently in both sexes and/or did not occur in a dose-dependent manner and therefore, were not considered to be test article-related. While the statistically significant lymphopenia in the 450 mg/kg/day group males at study week 6 was consistent with a stress response, there was no such finding at the end of the dosing period at study week 4; therefore, the lymphopenia was considered to be unrelated to test article administration. Mean percent neutrophil counts were higher in the 450 mg/kg/day group males at the study weeks 4 and 6. The elevation as study week 4 appeared to be the result of individual outlying values. Therefore, although percent neutrophil count was statistically significant at study week 6, the values were comparable to the previous interval.
CLINICAL TOXICITY
See Table 1 in attached tables for statistically significant parameters.
The increases in urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were compatible with the test-article related histopathologic lesions involving parenchymal damage in the kidneys and liver. These abnormalities were present at the primary euthanasia. The week 6 recovery evaluation revealed that these values returned to normal, except for ALT and AST in males, which remained statistically significantly elevated compared to the control group. This continuous elevation of ALT and AST at study week 6 in males only was consistent with the increased severity of the liver lesion in the males, as compared to females, and suggested that the liver lesion in the males was not reversed. All remaining statistically significant changes in serum chemistry parameters were not observed consistently in both sexes and/or did not occur in a dose-dependent manner and
therefore, not considered to be test article-related.
URINALYSIS
See Table 2 in attached tables for statistically significant parameters.
MACROSCOPIC EXAMINATION
See Table 3 in attached tables for statistically significant parameters.
ORGAN WEIGHTS
See Tables 4 and 5 in attached tables for statistically significant parameters.
Test article-associated organ weight changes were associated with the histologic alterations shown in Table 5. The histologic changes in the liver and kidney were considered to be directly related to administration of the test article. The histologic changes in the seminal vesicles and the thymus were considered to be associated with stress, and thus were indirectly associated with administration of the test article.
MICROSCOPIC EXAMINATION
All males and females in the 450 mg/kg/day group, whether they were study week 4 primary euthanasia animals or found dead animals, had a renal lesion compatible with tubular nephropathy. This finding included 6 males and 6 females at 450 mg/kg/day. This lesion was also seen in the 150 mg/kg/day group males (3/4 animals). In females, the renal lesion was associated with gross findings of enlarged kidneys with a roughened surface and correlated to higher kidney weights in the 450 mg/kg/day group.
The renal lesion encompassed a variety of degenerative and inflammatory changes in the proximal and distal tubules within the cortex and occasionally within segments of the loop of Henle residing in the medulla. Most commonly, there was mild to moderate formation of basophilic tubules. These tubules typically contained protein casts. Occasionally, individualized tubules were lined by necrotic epithelium, flattened epithelium, or plump regenerative epithelium. Broad segments of basophilic tubules were typically associated with a mild degree of interstitial fibrosis and an infiltration of lymphocytes and, to a lesser extent, neutrophils. The combination of fibrosis and inflammatory cell infiltration was recorded as chronic active inflammation or chronic inflammation, depending on the presence or absence of neutrophils, respectively. The presence of basophilic tubules along with inflammation correlates to the gross finding of roughened surface of kidneys in the females.
In addition to the basophilic tubules and inflammation, several proximal tubules had minimal to moderate hyaline protein droplet accumulation. This change was more commonly seen in the males than in the females.
Tubular dilation was mild to severe in the affected kidneys and more pronounced in females. The tubular dilation typically involved only the basophilic tubules and imparted a spongy moth-eaten appearance to the kidney at sub-gross examination. This tubular dilation explains the gross findings of enlarged kidneys in females.
Mineralization of the tubules at the corticomedullary junctions and within the medulla were commonly seen to a mild to moderate degree in the 450 mg/kg/day group animals.
The lesions involving the kidneys may explain, and are consistent with, the elevation of urea nitrogen (BUN) and creatinine (Cr) in the 450 mg/kg/day group females at study week 4 and the elevated urea nitrogen in the 450 mg/kg/day group males at study week 4. The BUN and Cr values returned to normal by study week 6, suggesting, in part, that the renal lesion was non-progressive and possibly reversible.
The male and female recovery animals had a low incidence of basophilic tubules, dilation of tubules and hyaline droplets after 14 days recovery. The incidence and severity of the renal lesion were diminished, though still present at 14 days after dosing.
See Tables 6 and 7 in attached tables for statistically significant parameters.
Vacuolation of the hepatocytes was seen in 6/6 males and 2/7 females in the 450 mg/kg/day group for unscheduled and primary euthanasias. Vacuolation of the hepatocytes was characterized by individualized to coalescing, variable-sized, cytoplasmic vacuoles within the hepatocytes typical of fatty change. The centrilobular and mid-zonal hepatocytes were more commonly affected than the periportal hepatocytes. The fatty change was mild to severe in the males and mild to moderate in the females. One found dead male in the 150 mg/kg/day group had minimal fatty change in the liver.
The fatty change correlated to the higher liver weights in males and females in the 450 mg/kg/day group. The fatty change also correlated to the higher ALT and AST in 450 mg/kg/day males at study weeks 4 and 6 and the higher ALT in 450 mg/kg/day females at study week 4. There was fatty change in the liver after 14 days of recovery in 4/4 males in the 450 mg/kg/day group. This finding, along with still elevated ALT and AST values at study week 6 in males, suggested that the liver lesion in males, albeit decreasing in severity, was not reversed following a 14-day recovery period.
There was no indication of any fatty change in the liver of females after the 14-day recovery period. This, along with the normal ALT and AST liver enzymes by study week 6 in females, suggested that the liver lesion was reversed following the 14-day recovery period in the females.
See Tables 8 and 9 in attached tables for statistically significant parameters.
Squamous hyperplasia of the non-glandular stomach was present in 2/6 males in the 450 mg/kg/day group. This lesion was characterized by mild to moderate diffuse thickening of the keratinized stratified squamous epithelium with retention of normal cellular differentiation. There was focal erosion and focal subacute inflammation of the submucosa in each of these 2 affected animals, and the hyperplasia was likely secondary. Erosion was characterized by an incomplete loss of superficial epithelium with retention of the basement membrane and basilar layer of epithelium and its basement membrane, covered by a few degenerative epithelial cells from the stratum germinativum, within which there were clusters of neutrophils and necrotic inflammatory cells. This hyperplasia, in conjunction with the focal erosion and inflammation, was compatible with, though not specific for, direct irritation by the test article.
Squamous hyperplasia, erosion or inflammation was not seen in test article-treated females.
Squamous hyperplasia of the non-glandular stomach was not present in any of the recovery animals.
Lymphoid depletion of the thymus in 450 mg/kg/day group males and females and hemorrhage of the thymus in the 50 and 450 mg/kg/day group males were seen in a test article-related manner. Lymphoid depletion was mild to severe in the males and moderate in the females. This change was characterized by a decrease in thickness of the thymic cortex and an overall reduction in the density of the thymic lymphocytes. Rarely, pyknotic lymphocytic nuclei were present. Lymphoid depletion correlated to a decrease in thymic weight in the 450 mg/kg/day group males at study week 4. These changes in the thymus were non-specific and associated with stress. These findings were compatible with the stress-related findings in the seminal vesicles and with the stress-related clinical pathology findings (i.e., lymphopenia at study week 6 in 450 mg/kg/day group males).
Thymic lymphoid depletion decreased in incidence after a 14-day recovery period; however, the change was not completely reversed.
See Tables 10 and 11 in attached tables for statistically significant parameters
Depletion of secretion of the seminal vesicles was seen to a mild to moderate degree in 4/5 males in the 450 mg/kg/day group. It was seen to a mild degree in 1/5 males in each of the control and 150 mg/kg/day groups. This change was associated with a decreased organ weight at study week 4 and with a gross finding of small seminal vesicles. Aside from a diminished amount of eosinophilic secretion within the seminal vesicles, there was no associated glandular epithelial change.
Depletion of secretion is a non-specific change that has been associated with stress in laboratory animals (Almeida et al., 1998; Harvey et al., 1992). In this study, there was other histologic evidence of stress, including the lymphoid depletion in the thymus and the lymphopenia in the 450 mg/kg/day group rats at study week 6.
Mild depletion of secretion was seen in 1/4 males at 450 mg/kg/day after the 14-day recovery period, suggesting that the lesion persisted, though did not progress.
There was a dose-related and treatment-related increase in the incidence of medullary plasmacytosis and lymphoid hyperplasia of the mandibular lymph nodes in males. There were no such patterns in the females.
See Table 12 in attached tables for statistically significant parameters
Medullary plasmacytosis was characterized by an expansion of the medullary cords with well differentiated plasma cells. Lymphoid hyperplasia was typified by prominent lymphoid follicles with active germinal centers and an expansion of the paracortex by well differentiated small lymphocytes. In males, lymphoid hyperplasia and medullary plasmacytosis was mild to moderate. In the control and/or 450 mg/kg/day group females, these changes were also seen as mild to moderate.
There was no associated gross finding involving the mandibular lymph nodes in the 450 mg/kg/day group males, nor were there any other enlarged or reactive lymph nodes identified at gross or microscopic evaluation. Therefore, the lymph node changes in the males were considered to have no biologic significance and most likely represented normal biologic variation.
Remaining histologic changes were considered to be incidental findings, manifestations of spontaneous diseases, or related to some aspect of experimental manipulation other than administration of the test article. There was no test article-related alteration in the incidence, severity, or histologic character of those incidental and spontaneous tissue alterations


Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, toxicity of phenol, heptyl derivatives administered orally (gavage) to Crl:CD(SD) rats for 28 days was mainly observed at a dosage level of 450 mg/kg/day as evidenced by lethality, clinical observations (decreased defecation, dermal atonia, hypothermia), lower body weights, serum chemistry changes and several histologic changes (tubular nephropathy in the kidneys, fatty change of the liver, stratified squamous hyperplasia of the non-glandular stomach, thymic lymphoid depletion and hemorrhage and depletion of secretion of seminal vesicles). Therefore, the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of phenol, heptyl derivatives to Crl:CD(SD) rats for 28 consecutive days was 150 mg/kg/day as none of the aforementioned effects occurred at that dosage level. The systemic toxic effects observed at 450 mg/kg/day were considered to be secondary to the local irritant effects in the stomach.
Executive summary:

Test Guidance

OECD Guideline 407

Method and material

Phenol, heptyl derivatives in the vehicle, corn oil, were administered orally by gavage once daily for 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 50, 150 and 450 mg/kg/day. A concurrent control group (Group 1) received

the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups.

Groups 1 and 4 each consisted of 10 animals/sex and Groups 2 and 3 each consisted of 5 animals/sex. Following 4 weeks of dose administration, 5 rats/sex/group were euthanized; the remaining animals in the control and high dose groups were euthanized following a 14-day nondosing (recovery) period.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly.

Functional observational battery and locomotor activity data were recorded for all animals during study week 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed on up to 5 animals/sex/group on the days of the scheduled

necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in Groups 1 and 4. Kidneys, liver and gross lesions were examined from animals in Groups 2 and 3 at the primary necropsy and all animals at the recovery necropsy; the stomach (non-glandular), seminal vesicles, thymus were examined from all males in Groups 2 and 3 at the primary necropsy.

Results

There were no test article-related effects on home cage, open field, sensory, neuromuscular and physiological observations. Locomotor activity patterns were unaffected by test article administration. There were no test article-related effects on food consumption or hematology parameters. One male and one female in the 450 mg/kg/day died due to test article administration. Clinical observations in these animals prior to death included decreased defecation, dermal atonia, hypothermia and thinness. There were no other test article-related deaths. Clinical observations in the surviving animals consisted of clear material around the mouth and signs of unkempt appearance in the 450 mg/kg/day group. These signs of unkempt appearance correlated with a lower number of animals with normal fur appearance during the functional observational battery. Dermal atonia and thinness were occasionally noted in the 450 mg/kg/day group. These clinical observations were not observed during the recovery period.

Body weights were lower in the 450 mg/kg/day group males throughout the dosing period (study weeks 1 through 4) but recovered slightly following the termination of dosing. Higher urea nitrogen, creatinine (females only) and alanine aminotransferase were noted in 450 mg/kg/day group males and females and higher aspartate aminotransferase (AST) was noted in 450 mg/kg/day group males. Higher urine volume was noted in 450 mg/kg/day group males.

Review of the gross necropsy revealed suspected test article-related alterations of small seminal vesicles in 450 mg/kg/day group males and enlarged/rough surface kidney and small thymus in 450 mg/kg/day group females.

Organ weight changes attributed to test article administration in the 450 mg/kg/day groups included increases in liver (males and females) and kidney (females) weights along with decreases in seminal vesicle (males) and thymus (males) weights.

Histopathologic findings included vacuolation of the hepatocytes (450 mg/kg/day group males and females), squamous hyperplasia of the non-glandular stomach (450 mg/kg/day group males), lymphoid depletion of the thymus (450 mg/kg/day group males and females) and hemorrhage of the thymus (50 and 450 mg/kg/day group males). After the 14-day recovery period, total recovery from vacuolation of the hepatocytes (females only) and squamous hyperplasia was observed in the female and male groups. All other mentioned histopathologic findings were observed with partial recovery after the 14-day recovery period.

Conclusions

Based on the results of this study, toxicity of phenol, heptyl derivatives administered orally (gavage) to Crl:CD(SD) rats for 28 days was mainly observed at a dosage level of 450 mg/kg/day as evidenced by lethality, clinical observations (decreased defecation, dermal atonia, hypothermia), lower body weights, serum chemistry changes and several histologic changes (tubular nephropathy in the kidneys, fatty change of the liver, stratified squamous hyperplasia of the non-glandular stomach, thymic lymphoid

depletion and hemorrhage and depletion of secretion of seminal vesicles). Therefore, the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of phenol, heptyl derivatives to Crl:CD(SD) rats for 28 consecutive days was 150 mg/kg/day as none of the aforementioned effects occurred at that dosage level. The systemic toxic effects observed at 450 mg/kg/day were considered to be secondary to the local irritant toxicity to the stomach.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Satisfactory

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

There is one key sub-acute 28-day repeat oral dose study in the rat performed in accordance with OECD Guideline 407

Phenol, heptyl derivatives in the vehicle, corn oil, were administered orally by gavage once daily for 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 50, 150 and 450 mg/kg/day. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups.

Groups 1 and 4 each consisted of 10 animals/sex and Groups 2 and 3 each consisted of 5 animals/sex. Following 4 weeks of dose administration, 5 rats/sex/group were euthanized; the remaining animals in the control and high dose groups were euthanized following a 14-day non-dosing (recovery) period.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly.

Functional observational battery and locomotor activity data were recorded for all animals during study week 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed on up to 5 animals/sex/group on the days of the scheduled necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in Groups 1 and 4. Kidneys, liver and gross lesions were examined from animals in Groups 2 and 3 at the primary necropsy and all animals at the recovery necropsy; the stomach (non-glandular), seminal vesicles, thymus were examined from all males in Groups 2 and 3 at the primary necropsy.

There were no test article-related effects on home cage, open field, sensory, neuromuscular and physiological observations. Locomotor activity patterns were unaffected by test article administration. There were no test article-related effects on food consumption or hematology parameters. One male and one female in the 450 mg/kg/day died due to test article administration. Clinical observations in these animals prior to death included decreased defecation, dermal atonia, hypothermia and thinness. There were no other test article-related deaths. Clinical observations in the surviving animals consisted of clear material around the mouth and signs of unkempt appearance in the 450 mg/kg/day group. These signs of unkempt appearance correlated with a lower number of animals with normal fur appearance during the functional observational battery. Dermal atonia and thinness were occasionally noted in the 450 mg/kg/day group. These clinical observations were not observed during the recovery period.

Body weights were lower in the 450 mg/kg/day group males throughout the dosing period (study weeks 1 through 4) but recovered slightly following the termination of dosing. Higher urea nitrogen, creatinine (females only) and alanine aminotransferase were noted in 450 mg/kg/day group males and females and higher aspartate aminotransferase (AST) was noted in 450 mg/kg/day group males. Higher urine volume was noted in 450 mg/kg/day group males.

Review of the gross necropsy revealed suspected test article-related alterations of small seminal vesicles in 450 mg/kg/day group males and enlarged/rough surface kidney and small thymus in 450 mg/kg/day group females.

Organ weight changes attributed to test article administration in the 450 mg/kg/day groups included increases in liver (males and females) and kidney (females) weights along with decreases in seminal vesicle (males) and thymus (males) weights.

Histopathologic findings included vacuolation of the hepatocytes (450 mg/kg/day group males and females), squamous hyperplasia of the non-glandular stomach (450 mg/kg/day group males), lymphoid depletion of the thymus (450 mg/kg/day group males and females) and hemorrhage of the thymus (50 and 450 mg/kg/day group males). After the 14-day recovery period, total recovery from vacuolation of the hepatocytes (females only) and squamous hyperplasia was observed in the female and male groups. All other mentioned histopathologic findings were observed with partial recovery after the 14-day recovery period.

Based on the results of this study, systemic toxicity of phenol, heptyl derivatives administered orally (gavage) to Crl:CD(SD) rats for 28 days was mainly observed at a dosage level of 450 mg/kg/day as evidenced by lethality, clinical observations (decreased defecation, dermal atonia, hypothermia), lower body weights, serum chemistry changes and several histologic changes (tubular nephropathy in the kidneys, fatty change of the liver, stratified squamous hyperplasia of the non-glandular stomach, thymic lymphoid depletion and hemorrhage and depletion of secretion of seminal vesicles). Therefore, the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of phenol, heptyl derivatives to Crl:CD(SD) rats for 28 consecutive days was 150 mg/kg/day as none of the aforementioned effects occurred at that dosage level.

No classification for repeat dose effects is required as the effect is limited to a local irritant effect on the forestomach, which is not relevant to humans. The irritation effects observed at the 150 mg/kg/day dose level were not considered to be adverse.

The supporting data from the 14-day range finder support the assertion that irritatnt effects in the forestomach attributed to the toxicity observed in the main study.

Phenol, heptyl derivatives in the vehicle, corn oil, were administered orally by gavage once daily for 14 consecutive days to 5 groups (Groups 2-6) of Crl:CD (SD) rats. Dosage levels were 50, 100, 200, 300 and 450 mg/kg/day. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. All groups consisted of 3 animals/sex. Following 14 days of dose administration, all animals were euthanized. For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy.

All animals survived to scheduled necropsy. There were no test article-related effects on body weight or food consumption. There were no test article-related macroscopic findings. Clinical observations (including excessive salivation and yellow material on various body surfaces) were noted in males and females at 300 and 450 mg/kg/day and to a lesser extent at 200 mg/kg/day males and females. Slight increases in kidney weights were noted in a probable test article-related manner. Increases of up to 22% in the 300 mg/kg/day group males and up to 18.8% in the 450 mg/kg/day group females were noted in liver weights compared to the control group. These changes were considered to be test-article related although no microscopic examination was performed.

Phenol, heptyl derivatives, administered orally by gavage, did not produce any severe signs of toxicity in Crl:CD(SD) rats at dosage levels up to 450 mg/kg/day for 14 consecutive days. However, clinical observations (excessive salivation and yellow material) and higher liver and/or kidney weights were noted in the 300 and 450 mg/kg/day group males and females. Therefore, the dosage level of 450 mg/kg/day was considered the maximum tolerated dose (MTD) and was recommended as the high dose for a subsequent 28-day study.

The corrosive/irritant nature of the test material at low concentrations also indicates that any effects observed in a 90-day study would also be attributable to the local irritant effect prior to observation of any systemic effect as noted in the existing studies via the oral route. It may be concluded that adverse systemic toxicity only occurs at or above dose levels that cause severe local irritation and therefore, a 90-day repeat dose study by the oral route is considered to be scientifically unjustified.

Second route of exposure

90-day repeat dose studies by the inhalation or dermal routes are scientifically unjustified. The substance is unreactive, insoluble and not inhalable and there is no evidence of dermal absorption.

EC 276-743-1 has a relatively low molecular weight of 192.3 gm/mol, the divalent form having a molecular weight of 290 gm/mol.The substance is slightly soluble in water (42.1 mg/L), with a relatively high octanol/water partition coefficient (log Kow = 4.5) and a low vapour pressure (0.26Pa @ 25oC).

Although the physical chemical properties suggest that EC 276-743-1 is of adequate molecular size to participate in endogenous absorption mechanisms within the mammalian gastrointestinal tract should that material be ingested, acute oral, a 28-day repeated-dose oral gavage toxicity and reproduction/developmental toxicity screening studies identified that systemic toxicity was secondary to the local irritation effects on the forestomach of rats.

EC 276-743-1 was also tested for acute toxicity following dermal application. The single-dose dermal application of the test material resulted in no manifestations of systemic toxicity that would suggest systemic absorption through cutaneous barriers. Dermal absorption studies on alkyl phenols and structural analogues have shown thatexposure of human skin to concentrations of up to 10% NPE result in minimal exposure. The percentage dose absorbed was concentration dependant (ca. 1% for a 0.1% solution, 0.1% for a 1% solution and 0.01% for a 10% solution). There were no large species differences for absorption seen in the study. Therefore systemic exposure via the dermal route would be secondary and minimal with respect to local effects.

The potential for inhalation toxicity was not measured. However, the EC 276-743-1 vapour pressure indicates a very low propensity to enter atmospheric air in a respirable form (predicted to be ca. 0.1 ppm under ambient conditions). Thus, respiratory absorption under normal use and handling of this material is expected to be inconsequential.

The corrosive/irritant nature of the test material at low concentrations also indicate that any effects observed in a 90-day study would be attributable to the local irritant effect prior to observation of any systemic effect as noted in the studies via the oral route.

In addition human health risk mitigation measures will be in place to minimise the corrosive/irritant and skin sensitisation properties of the substance, resulting in a reduction of potential human exposure.

Therefore, 90-day repeat dose studies by the inhalation or dermal routes are considered to be scientifically unjustified.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP Guideline study

Justification for classification or non-classification

No classification for repeat dose effects is required as the effect is limited to a local irritant effect on the forestomach, which is not relevant to humans.