4-ethylphenol

Endpoint summary

Description of key information

In a repeated dose 28-Day oral toxicity study equivalent to OECD Technical Guideline 407, doses of 0, 100, 300 and 1000 mg/kg bw/d 4-ethylphenol were administered by gavage to male and female rats. No mortality was observed in any group exposed to 4-ethylphenol. In the 300 mg/kg bw/d dose group an increased organ weight of the liver and a squamous cell hyperplasia in the forestomach was observed in male rats. An expression of toxic symptoms like a staggering gait, salivation was observed in the highest dose group (1000 mg/kg) for male and female rats. Also, low body weights, high relative organ weights for liver or kidney and mucosal and squamous cell hyperplasia in the forestomach were determined. At the end of the recovery period in both, males and females a recovery tendency of the body weight and a reversibility of the other changes was observed

According to the observations described, the No-Observed-Effect-Level (NOEL) for male rats was 100 mg/kg bw/d and was 300 mg/kg bw/d for female rats.

In a second, supporting repeated 28-Day oral toxicity study equivalent to OECD Technical Guideline 407 young rats were exposed to 4-ethylphenol at the same dose levels. Almost the same toxicological symptoms as salvation, staggering gait and high values of liver weight were observed in the higher dose groups. Based on lesions in the forestomach the NOAEL for rats in this study was derived to be 100 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

SPF Crj:CD(SD) IGS rats from the Atsugi Breeding Center of Charles River Laboratories Japan, Inc. were used for the testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
49 rats of each sex with an age of 4 weeks were purchased on 3 March 1999. The weight range of the animals at the time of arrival was 80 - 92 g for the males and 70 - 78 g for the females. After receiving, the general appearance of each individual was observed once a day for 6 days in case of males and for 7 days in case of females. In addition, their weight was determined twice during this period. For the duration of quarantine and acclimatization no abnormalities were observed in the animals.
After the end quarantine and acclimatization, 42 healthy animals of each sex in an age of 5 weeks were selected and provided for the tests. They were divided by the stratification random sampling method into groups with the same average weight based on the weight on the day of the end of quarantine and acclimatization (the second last day before administration). The weight range of the animals was 130 - 145 g for the males and 117 -131 g for the females, and was less than +/- 20 % of the average weight (males 138.5 g, females 122.2 g). The animals excluded from the selection were not used for the test.

Before separation into groups, the animals were marked with an oil-based marker on the tail for individual identification. After separation into groups the animal number was tattooed into the auricle for individual identification. Before separation into groups, the breeding cages were marked on the front with labels in a different color for each sex, on which the test number and the animal numbers had been specified. After separation into groups they were marked on the front with labels in different color for each sex, on which the test number, the test group and the animal number had been specified.

HOUSING
The animals were raised in an animal breeding room with a temperature of 23 +/- 3 °C (measured range 21 - 24 °C), a humidity of 55 +/- 10 % (measured range 37 - 61 %), an air change rate of 10 - 15 times per hour and an illumination time of 12 hours (artificial light switched on 8 a.m. and turned off 8 p.m.). The temperature and the humidity of the breeding room were monitored every day.
During the quarantine and acclimatization period, 4 to 5 animals separated by sex were accommodated in cages made from bracket-type metal with a metallic wire mesh bottom (260W x 380D x 180H, mm), after separation into groups 1 animal was accommodated. The cage and the feeder were cleaned once at the time of separation into groups and the tray was exchanged twice a week for sterilization. The automatic water supply equipment was drained once a week. The animal breeding room was cleaned and disinfected in a frequency of once a day. For disinfection chlorine-based and iodine-based disinfectants were used alternating weekly.

Solid food CRF-1, made by Oriental Yeast Co., Ltd., irradiated with gamma rays, was provided ad libitum in a metallic food supplier.
The presence or absence of contaminating material or the presence or absence of microorganisms, which may have an adverse effect on the test, was analyzed in the used lots of the food (981209, 990109, 990203). The analysis of contaminating material was carried out in the Japan Food Research Laboratories and the bacteriological testing was carried out by the food manufacturer. The items to be analyzed and the acceptable values were in compliance with the standard operating procedures of the Safety Research Institute for Chemical Compounds (Ltd.). As a result of the analysis no values exceeding the acceptable values were observed.

Sapporo tap water was provided ad libitum using automatic water supply equipment.
Samples were taken on 12 January 1999 and 6 April 1999 and analyzed for the presence or absence of contaminating material which may have an adverse effect on the test. The analysis was carried out by NIHONEISEI Co. Ltd. The items to be analyzed and the acceptable values were in compliance with the standard operating procedures of the Safety Research Institute for Chemical Compounds (Ltd.). As a result of the analysis no values exceeding the acceptable values were observed.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The test compound was administered according to the "test guidelines" with a stomach tube forcibly orally into the stomach once a day between 9:00 a.m. and 12:00 a.m. for 28 days.
The dosage capacity was set to 5 mL/kg, and the individual administration volume was calculated on the basis of the bodyweight on the measurement day next to the day of administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

It was confirmed as result of an analysis of the stability of 4-EP in the preparation liquid with a concentration of 0.2 mg/mL and 400 mg/mL, carried out prior to the dosage, that the test material is stable in the preparation liquid for 3 hours at room temperature and for 8 days in a cool and dark place (measured range 1 - 3 °C).
It was confirmed as a result of the analysis of the test material concentration in the preparation liquids of each concentration used for administration at the time of the first and last preparation that the rate of content was 98.5 - 102 % of the predetermined concentration and that the preparation liquids contained the predetermined concentration. The above mentioned analysis were carried out in the Hodogaya JRF Contract Laboratories Co., Ltd.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Details on study design:

The dosage of the test material was set on the basis of the results of a preliminary test (test number SR-9884). In the preliminary test, doses of 0, 250, 500, 1000 and 2000 mg/kg were administered orally to 1 group of each 5 male and female SD-rats for 14 days.
It was found that in the 2000 mg/kg group until 3 days of administration both males and females of all examples had died. In the 1000 mg/kg group the death of 1 female, low values of the body weight and reduced food consumption of males and females, and mucosal hyperplasia of the forestomach in males and females as autopsy findings were observed and, in addition, high values of the liver weight of males and females and low values of the spleen weight of females were found as change of organ weights. In the 500 mg/kg group high values of the liver weight of males and low values of the spleen weight of females were recognized. Further, in the blood chemical analysis, high values of GPT and total cholesterol were found in males and low values of alkaline phosphatase and high values of potassium in females of the 1000 mg/kg group. In males of the 500 mg/kg group, high values of GPT were observed. On the other hand, changes assumed to be due to the administration of the test material were not recognized in the 250 mg/kg group.

Accordingly, in the present 28 day repeated dose oral toxicity test, 1000 mg/kg/day, which is estimated to show certainly toxic effects in male and female rats, is taken as highest dose, and by dividing this dose as follows by a common factor of about 3 to 300 and 100 mg/kg/day, 3 dosage groups for both males and females were established. In addition, a control group was established to which only the solvent (olive oil) was administered in the same way.

Positive control:

none

Observations and examinations performed and frequency:

The starting day of administration was counted as administration day 1 and the day following to administration day 28 as recovery day 1, and all dissection examples of the end of the administration period were observed until autopsy on the day following to administration day 28 and all dissection examples of the end of the recovery period were observed until autopsy on the day following to recovery day 14. Every day during the administration period, it was controlled whether the animals were alive or dead, and their outer appearance, their behavior was monitored once before the administration and at least once after the administration, and if abnormalities were recognized, the symptoms and the observation time of their manifestation and their disappearance was recorded. During the recovery period, monitoring was carried out once every day.

BODY WEIGHT
The body weight of the animals was determined on administration day 1, 2, 7, 14, 21 and 28 before administration, on recovery day 1, 7 and 14 and, further, on the day of autopsy following the day after the end of each period with an electronic balance (Sartorius 1401 B MP7-2 or 1407 MP8-1, Carl Zeiss Co., Ltd.).

FOOD CONSUMPTION
The food consumption was determined on administration day 1, 2, 7, 14, 21 and 28 before administration and, in addition, on recovery day 1, 7 and 14, with an electronic balance (Sartorius 1401 B MP7-2 or 1407 MP8-1, Carl Zeiss Co., Ltd.). For the food consumption on each measurement day, the value, obtained by subtracting the residual amount of the supplied amount in front of the cage on the following day, was taken as consumption amount of 1 day.

URINE
In administration week 4 (day 22) and recovery week 2 (recovery day 8) urine was collected under non-fasting conditions, using a metabolism cage for rats (KN-646, B-1 type, Natsume Seisakusho Co., Ltd.), and with the urine, collected in about 3 hours, these test were carried out: pH, proteine, sugar, ketone body, urobilinogen, bilirubin, occult blood, color, sediment. Further, with the urine collected in about 21 hours, these tests were carried out: urine volume, spec. gravity, combined with the water consumption during urine collection.
The collected urine was discarded after the end of the examination.

HEMATOLOGY
The rats were anesthetized at the time of autopsy with ether after 16 - 21 hours of fasting and approximately 1 mL of blood was collected from the abdominal aorta.

For the evaluation of no. of erythrocytes, hematocrit, hemoglobin, corp. volume, corp. hemoglubin, corp. hemoglubin conc., reticulocyte count, blood platelet count, white blood cell count and % white blood cells, blood treated with EDTA*2K (Benoject II vacuum blood collection tube, Terumo Co., Ltd.) was used.

For the evaluation of prothrombin time and activated partial thromboplastin time, plasma obtained by centrifugation and treated with 3.8 % sodium citrate was used for the testing.
The collected blood and serum were discarded after the end of the examination.

For the evaluation of GOT, lactase dehydrogenase and glucose, blood plasma obtained by 10 minutes of centrifugal separation at 3,000 rpm after treatment with about 20 units of heparin sodium (heparin sodium injection, 1,000 units/ml, Mochida Pharmaceutical Co., Ltd) was used for the examination.

For the evaluation of GPT, alkaline phosphatase, total cholesterol, triglycerides, total bilirubin, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorg. phosphorus, total protein, protein fraction and A/G ratio, blood was collected in a test tube containing a separation agent (Sepaclean, Eiken Kizai Co., Ltd.) and the blood serum obtained by 10 minutes of centrifugal separation at 3000 rpm was used for the examination. The obtained blood plasma and blood serum was cryopreserved at -20 °C after the testing.

Sacrifice and pathology:

PATHOLOGY
The outer appearance of the dissection animals from the end of the administration period was examined on the day following administration day 28 and the outer appearance the of dissection animals from the end of the recovery period was studied on the day following observation day 14.
After blood had been collected under ether anesthesia, the animals were killed by exsanguination.

In addition, the following organs and tissues were stabilized and preserved in 10 % neutral buffered formalin solution:
Brain (the cerebrum and cerebellum), pituitary gland, thymus, thyroid glands (right and left), parathyroid glands (right and left), adrenal glands (right and left), spleen, heart, thoracic aorta, tongue, esophagus, stomach (forestomach and glandular stomach), liver, pancreas, duodenum, jejunum, ileum (including Peyer's patch), cecum, colon, rectum, larynx, trachea, lung (including the bronchi), kidney (right and left), bladder, testis (right and left), epididymis (right and left), prostate gland, seminal vesicle (including right and left coagulating gland), ovaries (right and left), uterus (right and left corner part and cervical region), vagina, eyeballs (right and left), Harder glands (right and left), mammary glands (generally the right abdomen, females only), skin (abdomen), sternum (including bone marrow), thighbone (including bone marrow, right), spinal cord (neck), skeletal muscles (right femoral region), mesenteric lymph nodes, mandibular lymph nodes (right and left), submandibular glands (right and left), sublingual glands (right and left), parotid glands (right and left), sciatic nerve (right).
Further, the eyeballs and the Harder's glands were stabilized and preserved in Davidson liquid, the testis and epididymis stabilized with Bouin's solution and preserved in 70 % ethanol.

At the time of autopsy the weight of the following organs was determined with an electronic balance (ER-180A, A&D Co., Ltd.):
Brain, lungs, heart, liver, kidneys (right and left), spleen, adrenal glands (right and left), pituitary gland, thymus, thyroid glands (including parathyroid, right and left), testis (right and left), epididymis (right and left), ovaries (right and left). In addition, in case of organs with the indication right and left, the right and left part were weighed separately.

HISTOPATHOLOGY
Each organ and tissue from all examples, which had been stabilized and preserved at the time of autopsy, was cut into thin slices after embedding in paraffin and Hematoxylin-Eosin stained samples were prepared. Further, from organs with lesions visible to the naked eye, samples containing the lesion area were prepared. Among these samples all examples of the control group and the high dosage group were examined microscopically.
In the 1000 mg/kg group, mucosal hyperplasia of the forestomach was observed frequently in males and females at the time of autopsy. As findings in the microscopic examination, changes such as erosion or squamous cell hyperplasia was seen in the forestomach as well as the larynx, the esophagus and glandular stomach, and since these are considered to be due to influence of the 4-EP administration, the organs of the male and female animals of all other dosage groups were also examined microscopically.
Since hyaline droplets were found in the kidney's proximal tubular epithelium in 4 out of 7 males of the 1000 mg/kg group, a microscopic examination was also carried out with the kidneys of males of the other dosage groups.

Statistics:

Bartlett assays were carried out about the results of the body weight, the body weight gain, the body weight gain rate, the food consumption, the quantitative items of the urine analysis, the hematological test, the blood chemical analysis and the relative organ weight, and their homoscedasticity was examined. In case of equal dispersion, analysis was carried out by the method of one-way analysis of variance, and by the Kruskal-Wallis assay method in case of nonequivalent dispersion. If significant differences were seen in the results of the method of one-way analysis of variance, a comparison with the control group was carried out by the Dunnett assay method. If significant differences were seen in the results of the analysis by the Kruskal-Wallis method, a comparison with the control group was carried out by the U-assay method of Mann-Whitney. For the absolute and relative organ weight of target organs with a left and a right organ, the comparison with the control group was carried out with the sum of their values.

The results of the quantitative items of the urine analysis were analyzed with the Kruskal-Wallis assay method, in case of significant differences, the comparison with the control group was carried out using the U-assay method of Mann-Whitney.

In addition, for the approval of the comparison between the groups administered with the test material and the control group, the average and standard deviation of the groups was used and a risk rate of less than 5 % was considered to be statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(1) Administration period
In the 100 and 300 mg/kg groups no abnormalities were observed. In the 1000 mg/kg group, salivation was seen from administration day 6 until the end of administration in 9 males and 6 females. This symptom developed directly after administration and disappeared within 30 minutes after manifestation. In addition, staggering gait was observed in males on administration day 1, 3, 10 and 17, in females on administration day 3, 14 and 16 in 1 animal each, among them, lateral position was also observed on administration day 1 in 1 male and on administration day 3 in 1 female. These symptoms developed several minutes after administration and disappeared within 1 hour after manifestation. Furthermore, soiled perigenital fur was observed on administration day 15 in 1 male and appeared occasionally in females throughout the administration period. Dead animals were not observed in any of the 4-EP administration groups.
(2) Recovery period
Abnormalities were not observed in any of the animals.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(1) Administration period
In the 100 and 300 mg/kg groups, a significant difference compared to the control group was not observed in both males and females. In the 1000 mg/kg group, the body weight of males in the period from administration day 7 to day 28 and of females in the period from administration day 14 to day 28 was significantly low compared to the control group. The body weight gain and the body weight gain rate was significantly low in both males and females.
(2) Recovery period
In the 1000 mg/kg group, the body weight was low throughout the recovery period in both males and females, and was significantly low in males from recovery day 1 until day 14. However, an increase of the body weight exceeding that of the control group was observed in both males and females, and the body weight gain rate was significantly high in both males and females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(1) Administration period
In the 100 and 300 mg/kg groups, a significant difference compared to the control group was not observed in both males and females. In the 1000 mg/kg group, the food consumption of males on administration day 2 and day 7 and of females on administration day 2 was significantly low compared to the control group.
(2) Recovery period
In the 1000 mg/kg group, a significant difference compared to the control group was not observed in both males and females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(1) At the end of administration period
In males, a significant difference compared to the control group was not observed in the 100 mg/kg group. In the 300 mg/kg group, the mean erythrocyte volume was significantly high, but because the value in the 1000 mg/kg group was nearly the same as in the control group, it was considered to be an accidental change.
In females, except that the ratio of stab form neutrophils had accidentally a high value in the 300 mg/kg group, a significant difference compared to the control group was not observed in any of the of the administrated groups.
(2) At the end of recovery period
In the 1000 mg/kg group, a significant difference compared to the control group was not observed in males. In females, the number of erythrocytes was significantly low and the mean quantity of corpuscular hemoglobin was significantly high.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(1) At the end of administration period
In males, the A/G ratio in the 100 mg/kg group was significantly low because the albumin fraction within the protein fractions was low. In the 300 mg/kg group, glucose was significantly high. However, each of these findings were changes without a dose relationship. In the 1000 mg/kg group, GTP was significantly high compared to the control group, and the γ-globulin fraction and sodium were significantly low.
In females, sodium was significantly high in the 100 mg/kg group compared to the control group, but it was a change without a dose relationship. In the 300 mg/kg group, a significant difference compared to the control group was not observed in any of the test items. In the 1000 mg/kg group, significantly high values of γ-GTP, total cholesterol and triglycerides were observed. At the other test items significant differences compared to the control group were not found. Among these, the high value of γ-GTP is thought to be not related to the 4-EP administration in consideration of the narrow variation range of the control group.
(2) At the end of recovery period
In the 1000 mg/kg group, total bilirubin and inorganic phosphorous were significantly high in males. In females, alkaline phosphatase was significantly high.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

(1) Administration week 4
In the 100 and 300 mg/kg groups, a significant difference compared to the control group was not observed in any of the test items in both males and females. In the 1000 mg/kg group, the urine volume of females was significantly high.
(2) Recovery week 2
In the 1000 mg/kg group, a significant difference compared to the control group was not observed in any of the test items in both males and females.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(1) At the end of administration period
In males, a significant difference compared to the control group was not observed the 100 mg/kg group. In the 300 mg/kg group, the absolute and relative organ weight of the liver was significantly high. In the same group, the relative organ weight of the left side of the thyroid gland was significantly low, but regarding both sides no significant difference was found. In the 1000 mg/kg group, a tendency to high values was observed in the absolute organ weight of the liver, and because the organ weight on the day of autopsy had a low value tendency, the relative body weight was significantly high. In addition, a significantly high value of the relative organ weight of the kidneys was found.
In females, in the 100 and 300 mg/kg groups a significant difference compared to the control group was not observed. In the 1000 mg/kg group, a high value tendency in the absolute organ weight of the liver was found, and because the body weight on the day of autopsy hat a low value tendency, the relative organ weight was significantly high.
(2) At the end of recovery period
In males of the 1000 mg/kg group, the body weight was significantly low on the day of autopsy, and the absolute organ weight of the lungs, pituitary gland, thymus and testis was significantly low, but in regard to the relative organ weight there was no significant difference. The relative organ weight of the brain and epididymis was significantly high. The relative organ weight of the right thyroid gland was significantly high, but regarding both sides no significant difference was found.
In females, even a low value tendency of the body weight was seen on the day of autopsy, no significant difference in the weight of any of the organs was found.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(1) At the end of administration period
In the liver of one female example of the control group yellowish white or dark red patches were seen. In the 100 and 300 mg/kg group, abnormalities were not observed in both males and females. In the 1000 mg/kg group, mucosal hyperplasia of the forestomach was found in males in 5 animals, in females in 6 animals. In addition, in the mucosa of the glandular stomach dark red patches were found in 1 female.
(2) At the end of recovery period
In the control group only a unilateral swelling of the testis was found in 1 male.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(1) At the end of administration period
In males, in the control group 1 example each of bony metaplasia of the lungs, cysts of the kidneys, regeneration of the tubular epithelium and lymphocytic infiltration of the prostate gland and 3 examples of microgranuloma of the liver were observed. In the 100 and 300 mg/kg groups, the forestomach, the glandular stomach, the larynx, the esophagus and the kidneys were examined. In the 100 mg/kg group, acidophilic corpuscles of the proximal tubular epithelium of the kidneys were observed in 2 examples, and hyaline droplets in 3 examples. In the 300 mg/kg group,1 example each of squamous cell hyperplasia in the limiting ridge of the forestomach andsquamous cell hyperplasiain other parts of the forestomach (slight), and 1 example each of regeneration of acidophilic corpuscles, hyaline droplets and tubularepitheliumof the proximal tubular epithelium of the kidneys were observed. In the 1000mg/kg group, 1 example of erosion in the limiting ridge of the forestomach and 4 examples of squamous cell hyperplasia, 2 examples each of degeneration of the squamous epithelium of other parts of the forestomach and edema of the submucosa, in total 7 examples of squamous cell hyperplasia (slight: 4 examples, moderate: 3 examples) were observed. In addition, each 1 example of foam cell accumulation of the lung, microgranuloma of the liver, regeneration of the tubular epithelium of the kidneys, lymphocytic infiltration of the prostate gland and cysts of the pituitary glands, and 4 examples of hyaline droplets of the proximal tubular epithelium of the kidneys were seen.
In females, in the control group 1 example each of localized fibrosis of the lung, vascular dilatation and mineralization of the liver, 2 examples of foam cell accumulation in the lung, and 3 examples of fatty change of periportal hepatocytes in the liver were observed. In the 100 and 300 mg/kg group, the forestomach, the glandular stomach, the larynx and the esophagus were examined, but abnormalities were not found. In the 1000 mg/kg group,1 example each of neutrophilic infiltration of the lamina propria of the larynx, erosion of the larynx and squamous cell hyperplasia of the esophagus, 2 examples each of degeneration of the squamous epithelium and edema of the submucosa of the forestomach, 1 example of granulation in the submucosa, 6 examples of squamous cell hyperplasia (slight: 1 example, moderate: 5 examples) and 1 example of ulcer were observed. In addition, 1 example each of foam cell accumulation in the lung, microgranuloma of the liver and mineralization of the cortico-medullary junction of kidneys, as well as 2 examples of cysts of the kidneys were found.
(2) At the end of recovery period
In males, in the control group 1 example each of metaplasia of the lungs, edema of the submucosa of the rectum, atrophy of the acinar cells of the pancreas, fatty change of periportal hepatocytes and microgranuloma of the liver, hyaline cast, cysts and hyaline droplets of the proximal tubular epithelium of the kidneys, dilatation of the seminiferous tubule of the testis and lymphocytic infiltration in the skeletal muscle, and 2 examples of regeneration of the tubular epithelium of the kidneys were observed. In the 1000 mg/kg group,1 example each of athropy of the acinar cells of the pancreas, hyaline cast, regeneration of the tubular epithelium,intraluminal cellular debris of the eosinophilic body of the kidneys and lymphocytic infiltration of the prostate gland, and 2 examples of microgranuloma of the liver were found.
In females, 2 examples of microgranuloma of the liver were found in the control group. In the 1000 mg/kg group,1 example of foam cell accumulation of the lung and 2 examples of microgranuloma of the liver were observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

4-Ethylphenol was administered orally in doses of 0 mg/kg (control group), 100, 300 and 1000 mg/kg to groups of 7 or 14 animals of each male and female Crj:CD (SD) IGS rats per group repeatedly for 28 days to examine its toxicity. In addition, the observation of the 1000 mg/kg group was continued for 14 days from the day after the end of administration to examine the reversibility of the toxic effects seen at the end of the administration period.

As a result, clear effects, which are considered to be caused by the administration of 4-EP, were found in both males and females in the 1000 mg/kg group. In this group, within 1 hour after the end of the dosage, staggering gait or salivation were observed as change of the general appearance, which is considered to be manifested by the administration of 4-EP. These symptoms were not observed during the recovery period. The increase of the body weight was suppressed during the administration period in both males and females, and low values were observed. In the recovery period, the body weight of both males and females was low compared to the control group, but the body weight gain rate at the end of the recovery period exceeded that of the control group and a recovery trend was found. Low values of food consumption were only observed during the administration period in both males and females.

The high values of urine volume of females observed in the urine analysis in administration week 4, are changes common to phenols and considered to be caused by the administration of 4-EP, because similar changes were found in males or females of the high dosage group of a 28 days repeated dose oral toxicity test with similar substances. The same changes were not observed in the analysis in recovery week 2.

In the blood chemical analysis at the end of the administration period, high values of GPT in males and of total cholesterol in females were found. As these changes were also observed in a preliminary examination, they are considered to be caused by the administration of 4-EP. The high values of GPT in males are considered to be changes not based on hepatic impairment, because there are no changes of GOT, alkaline phosphatase, γ-GTP, total bilirubin and the like in the liver function test. On the one hand, high values of total cholesterol were observed in a preliminary examination in males, but found in females in the present examination. Otherwise, the low values of the γ-globulin fraction and sodium in males were thought to be changes not related to the 4-EP administration, because they are not obvious changes in the related items. In addition, the high values of triglycerides in females are variations seen by 1 example of an individual organism which showed especially high values, and were considered to be a change not related to the 4-EP administration. All items with changes observed at the end of the administration period showed no abnormalities at all at the end of the recovery period and were found to be able to recover. As changes, which were only observed at the end of the recovery period, low values of the number of erythrocytes and high values of mean corpuscular hemoglobin in the hematological examination in females, high values of total bilirubin and inorganic phosphorous in the blood chemical examination in males, and high values of alkaline phosphatase in females were observed, but they are all slight changes and were not considered to be delayed effects related to the 4-EP administration,because they are not obvious changes in the related items.

At autopsy, mucosal hyperplasia of the forestomach developed frequently in both males and females, and also squamous cell hyperplasia was observed in the histopathological examination in the forestomach in all males and in 6 out of 7 females. Furthermore, erosion, squamous cell degeneration and submucosa edema, and changes like granulation or ulcer in the submucosa were found. In addition, in the 1 example of females, wherein dark red irregularities were seen in the mucous membrane of the glandular stomach, erosion was observed histopathologically. As other histopathological findings of the gastrointestinal tract, 1 example each of neutrophilic infiltration in the lamina propria of the larynx, erosion of the larynx and squamous cell hyperplasia of the esophagus was observed. Each of these changes have a stimulation effect on the skin/mucosa and were considered as changesrelated to the 4-EP administration.

For the organ weight, a tendency to high values or high values of the absolute and relative organ weight of the liver of males and females was found at the end of the administration period. In males, additionally the relative organ weight of the kidneys was high. The change of the weight of the liver was also found in a preliminary examination and was considered to be an effect of the4-EP administration. However, histopathological abnormalities thought to be caused by the 4-EP administration were not observed in the liver of males and females and in the kidneys of males. In addition, low values of the absolute organ weight of the lungs, pituitary glands, thymus and testis, and high values of the relative organ weight of the brain and epididymis were found in males at the end of the recovery period, but since histopathological abnormalities related to the 4-EP administration were not observed in these organs, they were considered as changes associated with the low values of the body weight. 

Among the other histopathological findings in the 1000 mg/kg group, the incidence of hyaline droplets in the proximal tubular epithelium of the kidneys in males of 57,1 % was high compared to the 0 % of the control group, but the degree of change is slight and it is known, that it can be observed by autogenesis, and because it is within the range of the incident of background data (0 - 71.4 %) from 9 examinations in our test facilities over the past 5 years, it was considered that its toxicological significance is low. In the 100 and 300 mg/kg groups, an increased expression of hyaline droplets in the proximal tubular epithelium of the kidneys was not observed. The expression frequency of other histopathological findings was low and they were kinds of findings which are also seen in the control group, and they were not considered to be changes caused by the 4-EP administration, because they had no connection with the test results of blood and urine.

In the 300 mg/kg group, the absolute and relative organ weight of the liver at the end of the administration group was high only in males and squamous cell hyperplasia was observed histopathologically in the forestomach. As these are all changes which had been observed in the 1000 mg/kg group, too, they were considered to be caused by the 4-EP administration.

In the 100 mg/kg group, changes thought to be from 4-EP administration were not observed in any test item.

Consequently, in case 4-ethylphenol is administered orally repeatedly for 28-days to rats, 300 mg/kg is thought to be a dose causing high values of the absolute and relative organ weight of the liver and,histopathologically, squamous cell hyperplasia of the forestomach in males. 1000 mg/kg is considered to be a dose causing in male and female animals the expression of toxic symptoms such as staggering gait, salivation and the like, low values of body weight and food consumption, high values of the relative organ weight of the liver or the kidneys, and macroscopically mucosal hyperplasia, histopathologically squamous cell hyperplasia and the like in the forestomach. At the end of the recovery period, in both males and females a recovery tendency of the body weight and a reversibility of the other changes was observed.

Conclusions:

It is concluded that under the present test conditions the no observed effect level (NOEL) is 100 mg/kg for males and 300 mg/kg for females.

Executive summary:

 

4-Ethylphenol was administered orally in doses of 0 mg/kg (control group), 100, 300 and 1,000 mg/kg to groups of 7 or 14 species of each male and female Crj:CD (SD) IGS rats per group repeatedly for28-daysto examine its toxicity. In addition, the observation of the 1,000 mg/kg group was continued for 14 days from the day after the end of administration to examine the reversibility of the toxic effects which were seen at the end of the administration period. The following results were obtained.

 

1.  In the 1,000 mg/kg group after administration staggering gait or salivation were observed as change in the general appearance during the administration period in males and females.During the recovery period abnormalities were not observed inthe 1,000 mg/kg group. Dead animals were not observed in any group administered with the test material.

2. In the 1,000 mg/kg group low values of the body weight were observed from administration day 7 until administration day 28 in males and from administration day 14 until administration day 28 in females. During the recovery period the body weight values of both males and females of the 1,000 mg/kg group were low compared to the control group, but the body weight gain rate exceeded that of the control group and a tendency of recovery was seen.

3. In the 1,000 mg/kg group low values of food consumption were observed on administration day 2 and administration day 7 in males and on administration day 2 in females.

4. In the urine analyses of administration week 4, high values of the urine volume were observed in females. The same change was not observed in recovery week 2.

5. In the findings of the hematological test no changes considered to be caused by the test material were observed in any of the administered group.

6. In the blood chemical analysis, high values of GPT were found in males and high values of total cholesterol in females in the 1,000 mg/kg group at the end of the administration period. This change was not found at the end of the recovery period.

7. At the end of the administration period, the organ weights in the 1,000 mg/kg group showed high values for the relative organ weight of the liver and the kidneys of males and the relative organ weight of the liver of females. In the 300 mg/kg group high values of the absolute and relative organ weight of the liver of males were observed. In the 100 mg/kg group no change was seen in any organ. At the end of the recovery period low values of the absolute organ weight or high values of the relative organ weight were observed together with low values of the body weight in males in the 1,000 mg/kg group. No change of the organ weights of the liver and the kidneys was found in males and females.

8. At autopsy, mucosal hyperplasia of the forestomach was observed frequently in males and females of the 1,000 mg/kg group, and in the histopathological examination of males of the 300 mg/kg group and males and females of the 1,000 mg/kg group, changes likesquamous cell hyperplasiaand the like were recognized in the forestomach at the end of the administration period. These changes were not observed at the end of the recovery period.

 

It follows from the above, that in case 4-ethylphenol is administrated orally to rats repeatedly for 28 days, 300 mg/kg is considered to be a dose which causes elevated values of the absolute and relative organ weight of the liver and, histopathologically,squamous cell hyperplasia of the forestomachin males, 1,000 mg/kg is thought to be a dose causing the manifestation of poisoning symptoms such as staggering gate and salivation, low values of the body weight and food consumption, high values of the relative body weight of the liver or the kidneys, further, macroscopically,mucosal hyperplasia of the forestomach and, histopathologically,squamous cell hyperplasia and the like in male and female animals.At the end of the recovery period, recovery trends were observed in both male and female animals and recoverability of the other changes was found. 

 

Accordingly, it is concluded that under the present test conditions the no observed effect level (NOEL) is 100 mg/kg for males and 300 mg/kg for females.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

18-day repeated dose study in newborn and 28-day repeated dose study in young rats (guideline not stated in the publication)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: SPF Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

In the newborn rat study, pregnant SPF Crj:CD(SD)IGS rats (gestation day 14–15) were purchased from Atsugi Breeding Center, Charles River Japan (Yokohama, Japan) and allowed to deliver spontaneously. The day on which parturition was completed was designated as postnatal day (PND) 0. Pups (newborn rats) were separated from dams on PND 3 and were suckled by foster mothers.

In the young rat study, four-week old males and females of the same strain were purchased from the same farm as in the newborn rat study.

The animals were maintained in an environmentally controlled room set at 20–26°C with a relative humidity of 45–65% and a 12:12 h light/dark cycle. All animals in the newborn and young rat studies were allowed free access to a sterilized basal diet (CRF-1, Oriental Yeast, Tokyo, Japan or Laboratory MR Stock, Nosan Corporation, Yokohama, Japan) and water.

The animals were euthanized by exsanguination under anesthesia using ether.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

See "any other information on materials and methods"

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

- newborn rats: 18 days
- young rats: 28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

newborn and young rats

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

newborn rats

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

newborn and young rats

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

newborn and young rats

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

young rats

No. of animals per sex per dose:

- newborn rats: 6/sex/dose
- young rats: 7/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

See "any other information on materials and methods"

Observations and examinations performed and frequency:

See "any other information on materials and methods"

Sacrifice and pathology:

See "any other information on materials and methods"

Other examinations:

See "any other information on materials and methods"

Statistics:

Continuous data were analyzed with Bartlett’s test for homogeneity of variance.
If the data were homogeneous, one-way analysis of variance and Dunnett’s test were conducted for group comparisons between the control and individual chemical-treated groups.
If not homogenous or in case of quantitative urinalysis data, analysis was performed using the Kruskal–Wallis test. In consequence, if a significant difference was detected, the Dunnett type test or Mann–Whitney’s U-test was conducted.
In the newborn rat study, categorical data for general appearance and reflex ontogeny were analyzed by Fisher’s exact probability test or Mann–Whitney’s U-test. A probability less than 5% was considered statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Mortality:

mortality observed, treatment-related

Description (incidence):

see "any other information on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

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:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see "any other information on results"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

young rat (28-day rep. dose study)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Dose descriptor:

NOAEL

Remarks:

newborn rats (18-day rep. dose study)

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

18-day study of 4EP in newborn rats

In the dose-finding study, deaths occurred at 300 mg/kg/day in one female each on days 6 and 8 of dosing, and at 1000 mg/kg/day in all rats by day 3 of dosing. In these dead rats, hypoactivity was observed and additionally, deep respiration, pale skin and/or dehydration were observed. In the surviving rats, hypoactivity during the dosing period was found in 3/5 males and 1/3 females at 300 mg/kg/day.

Clinical signs, such as hypoactivity, hypothermia, tremor, Straub tail, deep respiration and emaciation, were observed in 10/12 males and all 12 females at 300 mg/kg/day. Hypoactivity in males and females and hypothermia, tremor, Straub tail, deep respiration and emaciation in females were significantly more frequent at this dose and these clinical signs disappeared by day 9 of dosing for males and day 13 of dosing for females.

At 300 mg/kg/day, 2/12 females were found dead on days 10 and 12 of dosing. One of them showed dark red lung and congestive edema of the lung and the other showed distention of the gastrointestinal tract and atrophy of the thymic cortex at necropsy.

The delay in the righting reflex was observed in 4/12 males at 300 mg/kg/day, in 1/12 females at 100 mg/kg/day and in 1/10 females at 300 mg/kg/day.

At 300 mg/kg/day, body weights of males and females were significantly lower on PND 7–21.

Significantly high relative weight of the liver was observed in males and females at 300 mg/kg/day at the end of the dosing period.

There were no changes in the parameters of blood biochemistry or histopathological findings related to liver damage.

There were no effects of 4EP treatment at the end of the recovery maintenance period.

28-Day study of 4EP in young rats

In the dose-finding study, 4/5 males and all 5 females at 2000 mg/kg/day died after the first dosing and the remaining 1/5 males was killed because of moribundity on day 3 of dosing. At 1000 mg/kg/day, 1/5 females showed soiled perineal fur on days 5–7 of dosing and then died on day 8 of dosing. The body weight of females was significantly lower on day 2 of dosing at 1000 mg/kg/day.

Significantly high values of ALT and total cholesterol at 1000 mg/kg/day and significantly high value of ALT at 500 mg/kg/day were detected in males. Significantly low value of alkaline phosphatase and significantly high value of potassium at 1000 mg/kg/day were detected in females.

In the necropsy findings for rats died during the dosing period, acute changes, such as red coloration of the lung, forestomach and kidney, thinning of the mucosa in the glandular stomach, discoloration of the liver and spleen, blood pooling in the urinary bladder and abdominal dropsy were observed at 2000 mg/kg/day and reddish spots of the glandular stomach and atrophy of the thymus and spleen were detected at 1000 mg/kg/day. For the surviving rats, thickening of the mucosa in the forestomach was observed in 2/5 males and 3/4 females at 1000 mg/kg/day at the end of the dosing period.

At 1000 mg/kg/day, significantly high values of the relative liver weight in males and females and a significantly low value of relative spleen weight in females were observed. At 500 mg/kg/day, a significantly low value of relative spleen weight in females was observed.

In the main study, clinical signs, such as salivation, staggering gait, a lateral position and soiled perigenital fur, were observed in 11/14 males and 9/14 females at 1000 mg/kg/day. At this dose, salivation for males and females was observed within 30 min after dosing daily from day 6 to the end of the dosing period. Staggering gait and a lateral position were occasionally observed in males and females for 1 h from a few minutes after dosing, and soiled perigenital fur was occasionally observed for males and females. Significantly low body weights from days 7–28 of dosing in males and from days 14–28 in females were also observed.

In urinalysis, a significantly high volume of urine was observed in females at 1000 mg/kg/day at the end of the dosing period.

In the blood biochemistry, significantly high values of ALT in males and total cholesterol in females at 1000 mg/kg/day were observed.

In the necropsy findings, thinning of the mucosa in the glandular stomach in 5/7 males and 6/7 females and reddish spots in the glandular stomach in 1/7 females were observed at 1000 mg/kg/day at the end of the dosing period. Significantly high values of relative liver weight at 300 and 1000 mg/kg/day in males and at 1000 mg/kg/day in females were observed at the end of the dosing period. Significantly high value of relative kidney weight at 1000 mg/kg/day in males was observed at the end of the dosing period. Erosion, hyperplasia of squamous cells, degeneration of squamous cells and/or edema of the submucosa in the forestomach was observed in all 7 males at 1000 mg/kg/day. Hyperplasia of squamous cells in the forestomach was observed in 1/7 males at 300 mg/kg/day. Hyperplasia of squamous cells in the esophagus, degeneration of squamous cells, edema of the submucosa, granulation of the submucosa, hyperplasia of squamous cells and/or ulcer in the forestomach were observed in 6/7 females at 1000 mg/kg/day.

There were no effects of 4EP treatment at the end of the recovery period except for the lowered body weight of males at 1000 mg/kg/day.

Conclusions:

For the administration of 4EP, the NOAEL in the newborn rat 18-day rep. dose study was concluded to be 30 mg/kg/day based on the delay in the development of the righting reflex at 100 mg/kg/day.
The NOAEL in the young rat 28-day rep. dose study was concluded to be 100 mg/kg/day, based on the lesions in the forestomach at 300 mg/kg/day.

Executive summary:

Newborn rat studies were conducted with oral administration of 4-ethylphenol (4EP) on postnatal days (PND) 4–21 to allow comparison of no observed adverse effect level (NOAEL) with those from 28-day studies of young rats starting at 5–6 weeks of age.

In the newborn rat studies, deaths, hypoactivity, Straub tail, deep respiration and delayed righting reflex were clearly observed after 4EP treatment. In the young rat studies, salivation, staggering gait, changes in the liver including high values of liver weight and alanine aminotransferase or total cholesterol and the lesions in the forestomach were clearly observed after 4EP treatments.

NOAELs of 4EP in the newborn rat studies appeared to be almost 3 times lower than those in the young rat studies.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

Based on information available for 4-ethylphenol, this substance is not to be classified according to Regulation (EC) No 1272/2008 in terms of repeated dose toxicity.