Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2010 - January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Remarks:
Translated japanese report, GLP not stated
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-ethylphenol
EC Number:
204-598-6
EC Name:
4-ethylphenol
Cas Number:
123-07-9
Molecular formula:
C8H10O
IUPAC Name:
4-ethylphenol
Test material form:
solid
Details on test material:
The test substance p-ethylphenol (another name: 4-hydroxyphenylethane, CAS No.: 123-07-9, molecular formula: C8H10O, molecular weight: 122.17, appearance: white to brown lump or colorless to brown transparent fluid when thawed, purity: 99.4%, boiling point: ca. 219°C, freezing point: 44°C, flash point: 104°C; hereinafter called “PEP”) was stored refrigerated, shielded from light (actual temperature 3-7°C) until it was used for the test.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Sprague-Dawley (SD) [Crl:CD(SD), SPF] rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS
Eight-week-old Sprague-Dawley (SD) [Crl:CD(SD), SPF] rats (62 males and 73 females) were purchased from the Atsugi Breeding Center of Charles River Laboratories Japan, Inc. and accommodated into the Animal Quarters No. 3. These rats were reared for a period of 14 days (including the day of arrival) for the purposes of quarantine and acclimation to the environment). The general condition of each rat was checked every day during this period and the body weight was measured on the day of arrival and the last day of quarantine. During the quarantine and acclimation period, an acclimation number was recorded with a felt pen on the tail of each animal, and an animal card carrying the test No., gender and acclimation No. was attached to each cage for identification of the individual animal. Females were checked as to the estrous cycle every day beginning on the day following arrival. The body weights of animals upon arrival and at the end of quarantine are shown below.

Arrival: 17 November 2010
Body weight upon arrival: 239.7-271.7 g (males), 175.1-201.3 g (females)
End of quarantine: 30 November 2010
Body weight at end of quarantine: 323.3-393.2 g (males), 206.2-259.4 g (females)

During the quarantine and acclimation period, one male (Acclimation No. 59) was found to have a finger injury but the other animals had no abnormality in general condition or in the time course of body weight during the quarantine period. Excluding one male having shown an abnormality in general condition and 6 females having failed to restore a regular 4-day estrous cycle, the animals were grouped according to the body weight-based stratified random sampling technique.

Serial animal numbers were allocated to the thus grouped animals with the animal No. recorded with a felt pen on the tail of each animal and the animal cards of different colors (carrying Test No., gender and Animal No.) attached to the various cages. The 6 females excluded from grouping for a reason of estrous cycle data plus the 10 males and 15 females not allocated to any group were counted as extra animals and later used for other purposes.

HOUSING
The animals were reared in the animal quarters under the settings of acceptable temperature 21.0-25.0°C, acceptable relative humidity 40.0-75.0%, frequency of ventilation ca. 15 times/hour, bright/dark cycle of 12-hour illumination period (from 7 am to 7 pm) and a 12-hour non-illumination period (from 7 pm to 7 am).

The animals were accommodated individually (one animal/cage) into metallic cages with a metal mesh floor (220w x 270d x 190 h mm) (accommodating two animals per cage during the mating period). Each animal was allowed free access to a solid diet (CE-2, CLEA Japan, Inc.) and tap water (Hatano City Tap Water Bureau), although males and post-partum females were required to fast before autopsy. During the period from Day 18 of pregnancy to Day 4 of lactation, all females were individually accommodated into plastic breeding cages for rats (350w x 400d x 180h mm), with paper pulp chips (Paper Clean, Japan SLC, Inc.) provided as bedding when needed. During the rearing period, there was no abnormality in the environments, with temperature and relative humidity kept at 21.5-23.5oC and 44.0-67.0%, resp., in the animal quarters. Analysis of the diet, tap water and bedding provided allowed us to confirm that they were all within the acceptable range set forth in the standard operating procedure.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Females were treated with the test substance during the 2-week pre-mating period, the mating period and the period from Day 1 of pregnancy to Day 4 of postnatal lactation. Females not confirmed as to copulation received treatment until the day before autopsy (42 doses in total). Females having failed to undergo delivery after confirmation of copulation received treatment until the day before autopsy (until the day equivalent to Day 25 of pregnancy). Males received treatment during the pre-mating 2-week period and from the beginning of the 14-day mating period to the day before autopsy (42 doses in total). Each animal was orally treated with the test substance once daily in the morning (between 9:00 and 11:43) on 7 days of the week. The dosing volume was set at 5 mL/kg and calculated on the basis of the body weight measured once a week for all males, the females before and during mating and for females having failed to undergo copulation or calculated on the basis of the body weight measured the last time for females confirmed to have undergone copulation. For the control group, the vehicle (olive oil JP) free of the test substance was administered in the same way. The route of administration was “forced oral administration via a gastric tube for rats” in accordance with the OECD Guidelines for the Testing of Chemicals [421].
Details on mating procedure:
Each male-female pair from the same group began to be accommodated into the same cage in the evening on Day 15 of treatment. From the next morning on, each female was checked for vaginal plug every morning, and the vaginal smear specimen of each female during the mating period was observed under a microscope. The finding of vaginal plug in the vagina or spermatozoa in the vaginal smear specimen allowed the determination “copulation achieved” by the given pair. This day was counted as Day 0 of pregnancy and mating was discontinued on that day, followed by rearing the male and female separately. Depending on the outcome of mating and the presence/absence of pregnancy, calculation was made concerning the number of days from the start of mating to the day of confirmed copulation, the frequency of the return of estrus during that period, the copulation index [(number of pairs having achieved copulation/number of pairs used in mating) x 100, %] and the gestation index [(number of pregnant animals/number of females used for mating) x 100, %].
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of test substance treatment (6 September 2010) and after the end of the treatment period (21 January 2011), the test substance stability was evaluated at the Hatano Research Institute through measuring the infrared absorption spectrum (range measured: 4000-400 cm-1) by the potassium bromide tablet method with the use of a Fourier transform infrared spectrophotometer (FTIR-8300, Shimadzu Corporation), to confirm absence of any change from the earlier to the latter spectrum. For this evaluation, the spectrum yielded from the preceding test of the same test substance (Test No. R-10-002) served as the spectrum before the start of test substance treatment.

The content (quantity of the test substance contained) was measured in the three sample solutions (0.6, 2 and 6 w/v%) prepared initially on 30 November 2010. The content was judged acceptable if the mean concentration was 90-110% of labeled concentration and the variance in the concentration at each measurement session (percentage of concentration at each session relative to labeled concentration) was within 90.0-110% of the labeled concentration. According to the results, both the mean concentration (101.7-105.3% of the labeled concentration) and the variance in the concentration at each measurement session (98.5-101.0% of the labeled concentration) were within the acceptable range.

The test substance concentration in each sample solution was measured in the following way. Each sample solution was taken in a volume of 0.5 mL and diluted with acetone and then with acetonitrile to yield a test solution (concentration close to 10 μg/mL). Separately, three standard solutions (ca. 5, 10 and 20 μg/mL) were prepared by taking about 50 mg of the test substance thawed at ca. 50°C or less and dissolving it appropriately with a diluent [acetone/acetonitrile mixture (1:10 v/v)]. Each test solution and standard solution were assayed by high performance liquid chromatography (HPLC), to calculate the PEP concentration in each sample solution with the use of a calibration curve depicted from the standard solution.
Duration of treatment / exposure:
Males: 42 d
Females: during the 2-week pre-mating period, the mating period and the period from Day 1 of pregnancy to Day 4 of postnatal lactation.
Females not confirmed as to copulation received treatment until the day before autopsy (42 doses in total).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
13
Details on study design:
This study was designed to evaluate the reproductive and developmental toxicity of p-ethylphenol in male and female rats orally treated with the test substance during the pre-mating 2-week period, during the mating period (2 weeks at maximum), during 2 weeks after the end of the mating period (males only) and during the perinatal period (from Day 1 of pregnancy to Day 4 of lactation; females only).
Positive control:
none

Examinations

Parental animals: Observations and examinations:
The general condition of each animal was checked once a day during the pre-treatment rearing period and twice a day or more frequently (before and after the dose for a given day) during the treatment period.

BODYWEIGHT
Males were weighed on Days 1, 7, 14, 21, 28, 35 and 42 of treatment and on the day of autopsy. Females were weighed on Days 1, 7 and 14 of treatment, Days 0, 7, 14 and 21 of pregnancy, Days 0 and 4 of lactation and the day of autopsy (Day 5 of lactation). For females not confirmed as to copulation, the body weight was measured on Days 21, 28, 35 and 42 of treatment and the day of autopsy. For females not confirmed as to delivery, the body weight was additionally measured on the day equivalent to Day 26 of pregnancy.

FEED CONSUMPTION
The quantity of the diet consumed by each male was measured on Days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 of treatment. The quantity consumed by females was measured on Days 1-2, 7-8 and 14-15 of treatment, Days 0-1, 7-8, 14-15 and 20-21 of pregnancy and Days 3-4 of lactation. For females not confirmed as to copulation, this measurement was performed on Days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 of treatment.
Oestrous cyclicity (parental animals):
All females from each group were checked as to the estrous cycle beginning on the day following arrival. Also after grouping and the start of treatment, vaginal smear specimen preparation was carried out and observation of the estrous cycle was continued until copulation was confirmed after the start of mating by each pair. Furthermore, in each group, the mean number of days until the return of estrus (mean number of days from the beginning of estrus in a given animal to the next estrus) was calculated. Animals experiencing the return of estrus regularly with a cycle of 4-5 days were judged normal.
Sperm parameters (parental animals):
not stated in the report
Litter observations:
On Day 0 of lactation, the number of live males, live females, dead males and dead females among the offspring was counted, and each of the offspring was checked as to gender and external anomaly for calculation of the delivery index [(number of offspring at birth/number of implantation scars) x 100, %], the live offspring index [(number of live offspring at birth/number of implantation scars) x 100, %], the live delivery index [(number of females giving live birth/number of pregnant females) x 100, %] and the live birth index [(number of live births/total number of births) x 100, %]. On Day 0 to 4 of lactation, each offspring was checked as to general condition, to count the number of surviving males, surviving females, dead males and dead females and to calculate the neonatal survival rate [(number of surviving offspring on Day 4 of lactation/total number of surviving offspring on Day 0 of lactation) x 100, %]. Each surviving offspring was weighed on Day 0 and 4 of lactation to calculate the mean body weight for the male group and the female group of each litter and to calculate the sex ratio on Day 0 and 4 of lactation [(number of surviving males/total number of surviving offspring) x 100, %].
Postmortem examinations (parental animals):
On the day following Day 42 of treatment, each male was sacrificed by bloodletting under pentobarbital sodium anesthesia and autopsied. The liver, testis, epididymis, prostate (ventral lobe) and seminal vesicle (including the coagulating gland) were weighed. The testis and epididymis were fixed in Bouin’s fluid (0.1M phosphate-buffered 10% formalin was used for long-term storage), while the liver, stomach, prostate, seminal vesicle and mammary gland were fixed in 0.1M phosphate-buffered 10% formalin.

The testis and epididymis of the males from the control group and the 300 mg/kg group were examined histopathologically. Although the epididymis weight in the 100 mg/kg group was significantly high, we judged it unnecessary to conduct a histopathological examination of the testis and epididymis in the 100 mg/kg or lower dose group. This judgement was based on the general assessment of the finding that there was no inter-group difference in the relative weight of the epididymis as well as the findings from the histopathological and reproductive capability tests. Fixation by the above-mentioned method and subsequent histopathological examination were also carried out on the following organs found to be abnormal during autopsy: the lung from the control group, the testis and epididymis from the 30 mg/kg group, the epididymis from the 100 mg/kg group and the kidney from the 300 mg/kg group.

Autopsy of females after premature death or sacrifice (bloodletting under pentobarbital sodium anesthesia) was carried out as per the following schedule: on the day of death confirmed for females which died prematurely (2 females from the 300 mg/kg group), on the day of the last offspring’s death for females whose offspring died during the lactation period (2 females from the 300 mg/kg group), on Day 5 of lactation for other females having experienced delivery, on the day following Day 42 of treatment for females having failed to achieve copulation (1 female from the 300 mg/kg group), and on Day 26 of treatment for females having achieved copulation but failed to experience delivery (1 female from the 100 mg/kg group). Each ovary was observed under a stereoscopic microscope to count the number of pregnant corpora lutea. On each uterus, the number of implants was counted and the implantation index [(number of implants/number of pregnant corpora lutea) x 100, %] was calculated. In all females other than prematurely dead females, the liver and the ovary were weighed.

The liver, stomach, ovary, uterus, vagina and mammary gland of each female subjected to regular autopsy (Day 5 of lactation) were fixed in 0.1M phosphate-buffer 10% formalin. Each ovary from the control group and the 300 mg/kg group was examined histopathologically. The kidneys from the 30 mg/kg group and the small intestines (duodenum, jejunum and ileum) from the 100 and 300 mg/kg groups which were found to be abnormal during autopsy were also fixed in the same way for subsequent histopathological examination.

In the prematurely dead females and in females whose offspring had died, fixation by the similar method was done not only on the liver, stomach, ovary and lesions (lung, adrenal, spleen, thymus) but also on the brain, lung, heart and kidney for subsequent histopathological examination.

On the basis of the organ weight data and the findings from autopsy, the liver and stomach of the males and female from each group (including the control group) were examined histopathologically.

Postmortem examinations (offspring):
Each dead offspring was checked for external anomaly, autopsied and fixed for preservation in 0.1M phosphate-buffered 10% formalin. Each surviving offspring was checked for external anomaly and sacrificed by bloodletting under inhalational sevoflurane anesthesia for subsequent autopsy on Day 4 of lactation. During autopsy, internal organs were checked for anomaly. Each offspring found to have any external anomaly were also fixed in the same way for preservation.
Statistics:
The percentage of animals showing changes in the estrous cycle, the copulation index and the fertility index were tested by Fisher’s exact test (significance level: 5%).
Regarding the histopathological findings in each PEP dose group, the significance of differences between the 300 mg/kg group and the control group was tested by Mann-Whitney U-test on the graded findings and by Fisher’s exact test on the total of positive grades (significance level: 5%). In females, the statistical test was performed only on the data from the animals autopsied on Day 5 of lactation.
Regarding the other data, the homoscedasticity in each group was first tested by the Bartlett Method using the data from each animal or the litter average serving as one sample (significance level: 5%). If this test revealed homoscedasticity, one-way analysis of variance was subsequently conducted (significance level: 5%) and, if this analysis revealed any significant inter-group difference, multiple comparison by the Dunnett’s Method was carried out (significance level: 5%). If the variance was 0 in any group or was not same, the Kruskal-Wallis Rank Test was carried out (significance level: 5%) and, if this test revealed any significant inter-group difference, a multiple comparison was made by the Dunnett’s Method (significance level: 5%).
Reproductive indices:
All females confirmed to have achieved copulation were allowed to undergo spontaneous delivery. Delivery was checked every day from the day equivalent to Day 21 of pregnancy to confirmation of delivery. If delivery was completed by 11:00 am of a day, the day was counted as Day 0 of lactation (day of delivery). The status of delivery was observed directly in females for which such observation was possible. Also in the females for which direct observation of the delivery status was not possible, the presence/absence of abnormality was judged on the basis of the general post-delivery condition and the condition of the offspring. After delivery, the status of lactation was checked every day from Day 1 to 4 of lactation. The duration of pregnancy (number of days from Day 0 of pregnancy to the day of delivery) was determined on each female having undergone delivery. Then, in each group, calculation was made of the delivery index [(number of females giving live birth/number of pregnant females) x 100, %] and the implantation index based on the number of implants observed at the time of autopsy on Day 4 of lactation and the number of pregnant corpora lutea [(number of implants/ number of pregnant corpora lutea) x 100, %].
Offspring viability indices:
On Day 0 of lactation, the number of live males, live females, dead males and dead females among the offspring was counted, and each of the offspring was checked as to gender and external anomaly for calculation of the delivery index [(number of offspring at birth/number of implantation scars) x 100, %], the live offspring index [(number of live offspring at birth/number of implantation scars) x 100, %], the live delivery index [(number of females giving live birth/number of pregnant females) x 100, %] and the live birth index [(number of live births/total number of births) x 100, %]. On Day 0 to 4 of lactation, each offspring was checked as to general condition, to count the number of surviving males, surviving females, dead males and dead females and to calculate the neonatal survival rate [(number of surviving offspring on Day 4 of lactation/total number of surviving offspring on Day 0 of lactation) x 100, %]. Each surviving offspring was weighed on Day 0 and 4 of lactation to calculate the mean body weight for the male group and the female group of each litter and to calculate the sex ratio on Day 0 and 4 of lactation [(number of surviving males/total number of surviving offspring) x 100, %].

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Among the males, the 300 mg/kg group sporadically showed reduced spontaneous locomotor activity after the dose on Day 1 (start of treatment) to Day 37 of treatment as well as transient salivation after the dose on Day 8 to Day 37 of treatment (in 1 or 2 animals, each sign).
 
Among the females, one from the 300 mg/kg group (Animal No. F04002) died on Day 22 of pregnancy and another one from the same group (Animal No. F04008) died during labor on Day 23 of pregnancy. In the same group, reduced spontaneous locomotor activity was noted sporadically (in 1-4 animals) throughout the mating period, the pregnancy period and the lactation period beginning on Day 1 of treatment, and transient salivation was observed sporadically (in 1 or 2 animals) on Day 8 to Day 17 of treatment. Furthermore, in the 300 mg/kg group, contamination of the hypogastric region was observed sporadically (in 5 animals) after the start of mating (Day 16-17 of treatment). The hypogastric contamination subsided in 4 females by the end of the mating period (with success in copulation) but was also observed in the other female on Day 0 and 1 of pregnancy.
 
The reduced spontaneous locomotor activity in most males and females subsided about 4 hours after the dose. However, in one female from the 300 mg/kg group (Animal No. F04006), it persisted until about 6 hours after the dose on Day 0 of lactation. In two females on Day 21 of pregnancy (Animal No. F04008, F04011) and one female on Day 22 of pregnancy (Animal No. F04007), the reduced spontaneous locomotor activity after the dose was also observed even some hours after the dose.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females from the 300 mg/kg group died during labor on Day 22 or 23 of pregnancy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Among the males, there was no significant difference in body weight between the control group and any PEP treatment group at any point of time during the test period.
 
Among the females, the 100 mg/kg group showed a significant reduction in body weight on Day 20 of pregnancy (P<0.05). However, there was no significant difference between the control group and any PEP treatment group in terms of body weight during the pre-mating period, pregnancy period or lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Among the males and females, there was no significant difference in the amount of diet consumption between the control group and any PEP treatment group at any time during the test period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(1)   Males sacrificed on completion of the observation period
The control group and each PEP treatment group showed a fatty change of periportal liver cells and small granuloma although there was no difference in the extent or frequency of these changes between the control group and any PEP treatment group.
 
In the stomach, proventricular squamous epithelial cell hyperplasia was noted in 3 animals from the 300 mg/kg group (Animal No. M04004, M04010, M04013). Minimal edema of the proventricular lamina propria mucosae was noted in one animal from the control group which had been found during autopsy to have cysts in the proventricular mucosa (Animal No. M01002).
 
In the testis, localized seminiferous tubule atrophy (either bilateral or unilateral) was noted in 4 animals from the 300 mg/kg group (Animal Nos. M04001, M04002, M04006, M04011) and one animal from the control group (Animal No. M01003). Of these 4 animals from the 300 mg/kg group, one (Animal No. M04002) having shown a small-size testis during autopsy was histopathologically found to have a marked atrophy of the unilateral testis accompanied by diffuse hyperplasia of the Leydig cells. One animal from the 30 mg/kg which had been found during autopsy to have a size reduction of the bilateral testes and epididymides (Animal No. M02006) was histologically free of abnormalities.
 
In the epididymis, one animal which had been found during autopsy to have a size reduction of the epididymis (Animal No. M04002) showed cell residues and an evident decrease of sperm count. One animal each from the 100 mg/kg group (Animal No. M03013) and from the 300 mg/kg group (Animal No. M04001) which had been found during autopsy to have yellow white nodules at the epididymis tail had spermatic granuloma.
Other than these findings, the histopathological examination revealed localized bleeding (accompanied by brown pigmentation) of the lung, accompanied by foamy cell accumulation in the alveoli and eosinophil infiltration around the arteries in one animal from the control group which had been found during autopsy to have macroscopic abnormalities (Animal No. M01008). The animal from the 300 mg/kg group which had been found during autopsy to have large-size kidneys (Animal No. M04002) was histologically free of abnormalities.
 
(2)   Females sacrificed on completion of the observation period (autopsied on Day 5 of lactation)
The autopsy on Day 5 of lactation was carried out on 13 females from the control group, 13 females from the 30 mg/kg group, 12 females from the 100 mg/kg group and 8 females from the 100mg/kg group.
 
In the liver, small granuloma was noted in the control group and each PEP treatment group although there was no difference in the extent or frequency of this change between the control group and any PEP treatment group. Localized subcapsular foci of necrosis were noted in one animal each from the control group and the 300 mg/kg group and a fatty change of periportal liver cells was observed in two animals from the control group. In addition, hepatocyte cytoplasm vacuolation in the lobular intermediate zone was noted in one animal from the 30 mg/kg group (Animal No. F02002).
 
In the stomach, proventricular squamous epithelial cell hyperplasia was noted in 3 females from the 100 mg/kg group and all 8 females from the 300 mg/kg group. Of these animals, two animals from the 100 mg/kg group and five from the 300 mg/kg group additionally showed edema of the gastric lamina propria mucosae and two from the 300 mg/kg group had erosion as well. Two other animals from the 100 mg/kg group had edema of the proventricular lamina propria mucosae. Of the 3 animals from the 100 mg/kg group which had been found by autopsy to have an edematous change of the gastric mucosa, one animal (Animal No. M03012) was histopathologically free of abnormalities. The frequency of edema of the proventricular lamina propria mucosae and hyperplasia of squamous epithelial cells was significantly higher in the 300 mg/kg group than in the control group (P<0.01), and these changes were significantly more severe in the 300 mg/kg group than in the control group (edema of the lamina propria mucosae: P<0.05, hyperplasia of squamous epithelial cells: P<0.01).
 
In the ovary, the control group and the 300 mg/kg group were histologically free of abnormalities.
 
Of the 3 animals from the 100 mg/kg group and 2 animals from the 300 mg/kg group which had been found by autopsy to have small intestinal wall hypertrophy as a macroscopic abnormality, two animals each from the 100 mg/kg group and the 300 mg/kg group were histologically found to have duodenal mucosal hypertrophy. None of these four animals had any histological change of the jejunum or ileum. The remaining one animal from the 100 mg/kg group had no histological change in the duodenum, jejunum or ileum. The animal from the 300 mg/kg group found during autopsy to have a diverticulum between the jejunum and the ileum (Animal No. F04010) was histologically found to have a diverticulum partially lacking the muscle layer. One animal from the 30 mg/kg group found during autopsy to have light-colored rough-surfaced kidneys (Animal No. F02002) was histologically found to have numerous eosinophilic tubules in the renal cortex and medulla accompanied by hyaline casts in the cortex and medulla, degeneration/necrosis of the cortical proximal tubular epithelium and dilatation of the cortical distal tubular lumen.
 
(3)   Dams which died prematurely during the observation period
In the 300 mg/kg group, one dam (Animal No. F04002) died on Day 22 of pregnancy and another dam (Animal No. F04008) died on Day 23 of pregnancy.
 
In both dams, the liver showed vacuolation of the hepatocyte cytoplasm of the lobular intermediate zone, accompanied by diffuse hyperplasia of Kupffer cells. In examination of their stomachs, mucosal erosion was noted in the proventriculus (Animal No. F04008) or the glandular stomach (Animal No. F04002). Kidneys of both dams had numerous hyaline casts in the cortex and medulla accompanied by hyaline droplets and vacuolation of the cortical proximal tubular cytoplasm. Both dams showed reduction in the splenic red pulp and white pulp areas. Brown pigmentation was noted but extramedullary hematopoiesis was minimal. The lungs of both dams showed a sign of edema. The ovary, brain and heart of these dams were free of histological abnormalities. In the animal which had been found during autopsy to have thymus size reduction and light-colored adrenals as macroscopic abnormalities (Animal No. F04002), the histopathological examination revealed evident atrophy of the thymus (accompanied by nucleus condensation) and localized ischemia of the adrenal cortex sinusoids.
 
(4)   Dams having lost all offspring
In the 300 mg/kg group, all offspring from one dam (Animal No. F04011) died on Day 0 of lactation and all offspring from another dam (Animal No. 04007) died on Day 1 of lactation.

In the histopathological examination of the liver, one of these dams (Animal No. F04007) showed vacuolation of the hepatocyte cytoplasm of the lobular intermedium zone but the other (Animal No. F04011) was free of abnormalities. The stomach of one dam (Animal No. F04011) had proventricular perforation and edema of the proventricular lamina propria mucosae. The other dam (Animal No. F04007) had erosion of the proventricular mucosa, accompanied by squamous epithelial hyperplasia. The kidneys of both dams showed proximal tubular cytoplasm vacuolation of the cortex. One of the two dams (Animal No. F04011) had hyaline casts in the renal cortex and medulla. Both dams had reduction in the splenic red pulp and white pulp areas, accompanied by brown pigmentation, although extramedullary hematopoiesis was noted in only one of them (Animal No. F04011). In the examination of lungs, one animal (Animal F04007) showed foamy cell accumulation in the alveoli while the other (Animal No. F04011) was free of histological abnormalities. Although autopsy had revealed a macroscopic abnormality of the small intestine (wall hypertrophy) in one of these two dams (Animal No. F04007), no histological abnormality was observed in the ovary, brain, heart or small intestine of this dam.

(5)   Females without copulation or delivery
When the histopathological examination of the ovary, liver and stomach was carried out on one female from the 300 mg/kg group not confirmed as to copulation during the mating period (Animal No. F04013), the ovary was free of abnormalities but the liver had small granuloma and the proventricular mucosa had squamous epithelial hyperplasia.
 
When the liver, stomach, brain, heart, lung and kidney were examined histopathologically in one female from the 100 mg/kg group autopsied on the day equivalent to Day 26 of pregnancy (Animal No. F03003), no abnormality was noted.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no change in the estrous cycle suggesting an influence of PEP treatment. There was one animal from the 300 mg/kg group (Animal No. F04013) not confirmed as to copulation during the mating period but no influence of PEP treatment was noted in the copulation index or the fertility index. There was no significant difference between the control group and any PEP treatment group in terms of the number of days until copulation or the frequency of estrus during that period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
LIVE DELIVERY INDEX AND PREGNANCY PERIOD
The pregnancy period did not differ significantly between the control group and any PEP treatment group.
Although the live delivery index did not differ significantly between the control group and any PEP treatment group, two females from the 300 mg/kg group died during the late stage of pregnancy and all offspring from another female of the same group died on the day of birth.
 
DELIVERY/LACTATION STATUS
One female from the 100 mg/kg group (Animal No. F03003) did not undergo delivery.
Two females from the 300 mg/kg group died during labor on Day 22 or 23 of pregnancy.
One female from the 300 mg/kg group (Animal No. F04007) failed to show any nursing behavior (gathering or licking the newborns upon completion of delivery). Her offspring had no milk spot (a sign of maternal milk pooling) on the abdomen suggesting that they were not being breast-fed. The status of this female after delivery was thus judged as poor. All offspring from this female died on Day 1 of lactation. All offspring (8 males and 4 females) from another female (Animal No. F04011) died on Day 0 of lactation. Damage arising from eating by the dam was observed on the bodies of many dead offspring, giving rise to the judgement that the status of this dam after delivery was poor. One dam from the 30 mg/kg group (Animal No. F02002) showed no abnormality in general condition or nursing behavior but her body weight decreased during the lactation period, consuming very little in terms of food. Furthermore, death of her offspring began to be observed on Day 2 of lactation and the surviving offspring failed to show weigh gain. The autopsy findings and the histopathological findings (mentioned above) from this dam suggested renal dysfunction.
The other pregnant females underwent delivery on Day 22-23 of pregnancy showing no abnormality in the status of delivery or lactation. There was no significant difference in the duration of pregnancy or the live delivery index between the control group and any PEP treatment group.

CORPORA LUTEA, IMPLANTS, IMPLANTATION INDEX
One female from the 100 mg/kg group (Animal No. F03003) was not confirmed as to delivery until Day 25 of pregnancy. Autopsy of this female revealed implantation scars (right: 1, left: 1) and corpora lutea (right: 6, left: 11) but no dead fetus larger than a residual placenta was detected allowing the judgement that all embryos had been absorbed at early stages of pregnancy. The number of corpora lutea in this female was excluded from evaluation because distinction between the pregnant corpora lutea and the corpora lutea arising from ovulation after embryo absorption was difficult.
 
There was no significant difference between the control group and any PEP treatment group in terms of the number of corpora lutea, the number of implants or the implantation index.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Sex:
male
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Sex:
female
Basis for effect level:
clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male born to one dam from the 30 mg/kg group (Animal No. F02012) was without a tail and had imperforate anus when checked for external anomaly on Day 0 of lactation. This male survived until it was sacrificed for autopsy on Day 4 of lactation. Autopsy revealed rectal hypoplasia. The other surviving offspring were free of external anomaly on Day 0 through 4 of lactation. Autopsy of these offspring on Day 4 of lactation revealed no abnormality.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was no significant difference between the control group and any PEP treatment group in terms of the delivery index, live offspring index, live birth index, viability index or male-to-female ratio. However, all offspring from one dam of the 300 mg/kg group died on the day of birth and all offspring from another dam of the same group died by Day 4 of lactation.

Premature death of offspring was noted in the control group and each PEP treatment group. The total number of premature offspring deaths was larger in the 300 mg/kg group and the 30 mg/kg group than in the control group, because all offspring from 2 dams of the 300 mg/kg group died prematurely and one dam of the 30 mg/kg group poorly nursed the offspring.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no significant difference between the control group and any PEP treatment group in terms of the body weight of offspring on Day 0 or 4 of lactation.
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Premature death of offspring was noted in the control group and each PEP treatment group. The total number of premature offspring deaths was larger in the 300 mg/kg group and the 30 mg/kg group than in the control group, because all offspring from 2 dams of the 300 mg/kg group died prematurely and one dam of the 30 mg/kg group poorly nursed the offspring. The prematurely dead offspring included the offspring whose survival was unknown under the dam’s eating attack (unknown offspring) and the offspring not allowing observation of internal organs because of autolysis after death. However, the offspring which could be autopsied were free of external anomaly and the offspring allowing observation of internal organs were free of visceral abnormality.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
mortality

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Discussion

The reproductive and developmental toxicity of PEP was evaluated in rats of both sexes orally treated with PEP at dose levels 30, 100 and 300 mg/kg during the pre-mating two-week period (males and females), during the mating period (males and females), during the 42-day period after completion of the mating period (males only) and during the perinatal period from the beginning of pregnancy to Day 4 of lactation (females only).

 

2 dams from the 300 mg/kg group died prematurely at the late stage of pregnancy and all offspring born to 2 females from the same group died during the lactation period. After the start of treatment, these dams showed changes in general condition probably attributable to PEP treatment. The histopathological findings as to the lung, thymus, stomach, liver and kidney of these dams suggest intense stress, cardiovascular disorders, anaemia and hepatic / renal dysfunction. The histopathological examination of their stomachs revealed changes resembling those observed in the other animals autopsied upon completion of the treatment period. These findings suggest that these dams died prematurely or were unable to maintain nursing because of deterioration of their condition arising from changes in general condition (beginning during pregnancy) and the stress related to pregnancy and delivery.

 

One dam from the 30 mg/kg group showed suppressed increase of her own body weight and offspring’s body weight and reduced diet consumption during the lactation period. Autopsy and histopathological examination of this dam suggested renal dysfunction. However, considering the absence of similar changes in the other animals and the findings from the autopsy and histopathological examination conducted upon completion of the treatment period during this test, the renal dysfunction observed in this dam is unlikely to represent a change caused by PEP.

 

Observation of general condition revealed reduced spontaneous locomotor activity and salivation in males and females from the 300 mg/kg group and seemed to reflect an influence from PEP treatment.

 

The mean body weight on Day 20 of pregnancy was significantly smaller for the females from the 100 mg/kg group, probably because the body weight of one female having only two residual placentas in the uterus remained small throughout the pregnancy period. However, considering that the other females in the 100 and 300 mg/kg groups showed no tendency for increase of early embryo absorption and that there was no difference between the control group and any PEP treatment group in terms of the implantation index or the delivery index, we judged PEP treatment to have no effect in suppressing weight gain or inducing early embryo death during the pregnancy period.

 

In analysis of organ weights, both males and females from the 300 mg/kg group showed an increase in relative liver weight. The increase of liverweight seemed to reflect an influence from PEP treatment. However, no change corresponding to the increase of weight was revealed by the histopathological examination of the liver. In addition, the 100 mg/kg group showed an increase in the epididymis weight. This change seemed to be accidental in nature considering that there was no difference in relative weight, that no such change was observed in the 300 mg/kg group and that no corresponding change was revealed by the autopsy or histopathological examination.

 

When autopsied upon completion of the treatment period, females from the 100 mg/kg group showed an edematous change of the proventricular mucosa and hyperplasia of the small intestinal wall. Histopathologically, females from the 100 mg/kg and higher dose groups showed edema of the proventricular lamina propria mucosae, hyperplasia of the proventricular squamous epithelial cells and hypertrophy of the duodenal mucosa, and males from the 300 mg/kg group showed hyperplasia of the proventricular squamous epithelial cells. Considering that PEP is known to stimulate the skin and mucosa and the previous report of a similar change observed in the 300 mg/kg and higher dose groups, the changes observed in the gastric and duodenal mucosa in the 100 and 300 mg/kg groups seem to reflect an influence from PEP treatment.

 

A small number of males from the 100 and 300 mg/kg groups showed histopathological changes in the testis and epididymis. However, all of these males were shown to be fertile and no influence of PEP treatment was observed in the litter size of the pairs involving these males suggesting that PEP has no influence on the male reproductive system.

 

During the mating period, copulation was not confirmed in one pair from the 300 mg/kg group. The estrous cycle of the female constituting this pair had shown a regular return of estrus during the pre-mating treatment period but the return of estrus in this female ceased after mating. Considering that this pair had no histopathological abnormality of the reproductive organs or no abnormality in general condition, it seemed that the start of mating resulted in temporary suspension of the estrous cycle. The other animals from the same group were confirmed to have achieved copulation, resulting in fertilization. We thus judged that PEP had no influence on the estrous cycle or mating results.

 

No influence of PEP treatment was observed in the pregnancy period, number of corpora lutea, number of implants, implantation index, number of offspring at birth or delivery index. There was no influence of PEP treatment on the offspring’s male-to-female ratio, general condition or body weight, either. In the 300 mg/kg group, 2 dams died during the perinatal period and all offspring of 2 dams died during the lactation period, suggesting influence of PEP treatment on delivery, nursing and survival of offspring. However, considering that no influence of the test substance was observed in the surviving offspring, it seems that the death of offspring reflects the test substance’s influence on dams, rather than on the offspring. A check for external anomaly on Day 0 of lactation revealed one offspring without a tail and having an imperforate anus in the 30 mg/kg group. Because no such anomalies were noted in any other offspring, these anomalies were considered to be natural.

Taken together, these results allow to conclude that the no observed adverse effect level (NOAEL) of PEP in dams under the current testing conditions is 100 mg/kg/day for males and 30 mg/kg/day for females from the standpoint of general toxicity and is 100 mg/kg/day from the standpoint of developmental and reproductive toxicity, with the NOAEL for the next generation being 100 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
The NOAEL (no observed adverse effect level) of p-ethylphenol for dams under the present testing conditions from the standpoint of general toxicity is 100 mg/kg/day in males and 30 mg/kg/day in females, the NOAEL of the test substance from the standpoint of reproductive and developmental toxicity is 100 mg/kg/day and the NOAEL for the next generation is 100 mg/kg/day.
Executive summary:

A simplified reproductive and developmental toxicity test of p-ethylphenol was conducted in accordance with the OECD Guidelines for the Testing of Chemicals, to evaluate its influence on the reproductive ability of male and female animals as well as its influence on the growth of newborn. Crl:CD(SD) Rats of both sexes were orally treated with the test substance at dose levels 0 (vehicle: olive oil JP), 30, 100 and 300 mg/kg. Males were autopsied after 42 days of treatment while females were treated with the test substance every day during the pre-mating 2-week period, the mating period and from Day 1 of pregnancy to Day 4 of lactation. Each dam was allowed to undergo spontaneous delivery. Autopsy was carried out on each offspring on Day 4 of lactation and on each dam on Day 5 of lactation.

 

 Findings from dams

There was no premature death among the males but two females from the 300 mg/kg group died during the perinatal period. All offspring born to two other dams from the same group died during the lactation period. When examined histopathologically, these animals were found to have lung edema, thymus atrophy, reduction of splenic red and white pulp areas, vacuolation of the hepatocyte cytoplasm in the hepatic lobule central zone, hyaline casts in the kidney, hyaline droplets and vacuolation of the proximal tubule, erosion/perforation of the gastric mucosa, edema of the proventricular lamina propria mucosae, hyperplasia of the squamous epithelial cells and so on. As changes in the general condition, males and females from the 300 mg/kg group sporadically showed reduced spontaneous locomotor activity and transient salivation after the dose throughout the test period. Organ weight measurement revealed a significant increase of the relative liver weight in the 300 mg/kg group. Histopathologically, females from the 100 mg/kg or higher dose groups showed edema of the proventricular lamina propria mucosae, hyperplasia of the squamous epithelial cells and thickening of the duodenal mucosa, and males from the 300 mg/kg group showed hyperplasia of the proventricular squamous epithelial cells.

Findings from offspring

In the 300 mg/kg group, the live offspring index, the live delivery index and the viability index (neonatal survival rate) tended to be lower. The number of still births was larger in the same group than in the control group. There was no influence of the test substance on the delivery index, body weight or male-to-female ratio.

 Findings as to reproductive and development toxicity

There was no influence of the test substance on the estrous cycle, mating results, duration of pregnancy, number of corpora lutea, number of implants or implantation index. The delivery index tended to be lower in the 300 mg/kg group.

No observed adverse effect level

The NOAEL (no observed adverse effect level) of p-ethylphenol for dams under the present testing conditions from the standpoint of general toxicity is 100 mg/kg/day in males and 30 mg/kg/day in females, the NOAEL of the test substance from the standpoint of reproductive and developmental toxicity is 100 mg/kg/day and the NOAEL for the next generation is 100 mg/kg/day.