Registration Dossier
Registration Dossier
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EC number: 205-426-2 | CAS number: 140-66-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on generations indicated in Effect levels (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- A small but significant reduction in mean testicular size and reduction of daily sperm production was postulated at the highest dose but this effect could not be confirmed in subsequent studies conducted by the same author (R M Sharpe, K J Turner, and J P Sumpter (1998) Endocrine disruptors and testis development. Environ Health Perspect. 106(5): A220–A221. PMCID: PMC1533085)
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Octylphenol was administerd during gestation or during the first 21 days of postnatal life at 10 – 1000 ug/l via the drinking water. In study 1, rats were treated from days 1-22 after births in studies 2 and 3, the mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Route of administration:
- oral: drinking water
- Vehicle:
- ethanol
- Details on exposure:
- Octylphenol was administerd during gestation or during the first 21 days of postnatal life at 10 – 1000 ug/l via the drinking water. In study 1, rats were treated from days 1-22 after births in studies 2 and 3, the mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- study 1: days 1-22 after births
studies 2 and 3: mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth. - Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
0, (10), 100, 1000 µg/L
Basis:
nominal in water - No. of animals per sex per dose:
- 27-49 males
- Control animals:
- yes, concurrent vehicle
- Dose descriptor:
- LOAEC
- Generation:
- F1
- Effect level:
- 1 mg/L drinking water
- Sex:
- male
- Basis for effect level:
- other: Small but significant reduction in mean testicular size and reduction of daily sperm production
- Reproductive effects observed:
- not specified
- Conclusions:
- A small but significant reduction in mean testicular size and reduction of daily sperm production was postulated at the highest dose but this effect could not be confirmed in subsequent studies conducted by the same author (R M Sharpe, K J Turner, and J P Sumpter (1998) Endocrine disruptors and testis development. Environ Health Perspect. 106(5): A220–A221. PMCID: PMC1533085)
- Endpoint:
- fertility, other
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: The decrease in sperm number is not considered to be of biological significance and was not confirmed in the Tyl 1999 2-generation study with a sample size of 30 males per dose.
- Qualifier:
- no guideline followed
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: adult
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data - Route of administration:
- oral: drinking water
- Vehicle:
- unchanged (no vehicle)
- Details on mating procedure:
- Not applicable (study with male rats only), but sperm was examined for fertility parameters.
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 4 months
- Remarks:
- Doses / Concentrations:
10(-5) M
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
10(-7) M
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
10(-9) M
Basis:
nominal conc. - No. of animals per sex per dose:
- no data
- Positive control:
- no data
- Parental animals: Observations and examinations:
- BODY WEIGHT: Yes
- Time schedule for examinations: no data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: no data
Parameters examined are:
water or food consumption; body weight gain; hematocrit; reproductive organ weights; mean serum LH, FSH or testosterone concentrations; germ cell yield or relative numbers of different classes of testicular cells; testicular sperm number and epididymal sperm number - Clinical signs:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- not specified
- Other effects:
- effects observed, treatment-related
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Reproductive performance:
- not specified
- Dose descriptor:
- LOAEC
- Effect level:
- other: 10^(-9) M
- Based on:
- test mat.
- Sex:
- male
- Clinical signs:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Mortality / viability:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Body weight and weight changes:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Sexual maturation:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Histopathological findings:
- not examined
- Description (incidence and severity):
- not applicable (study with male rats only)
- Reproductive effects observed:
- not specified
- Conclusions:
- Disregarded: The decrease in sperm number is not considered to be of biological significance and was not confirmed in the Tyl 1999 2-generation study with a sample size of 30 males per dose.
- Executive summary:
In a reproduction study 4 -tert-octylphenol was administered to an unknown number of male Fisher 344 rats orally in drinking water at dose levels of 10-9M; 10-7M; 10-5M for 4 months. The focus of the study based on substance-related effects on the male reproductive system. No effects were reported on water and food consumption, body weight gain, haematocrit values, reproductive organ weights, mean serum LH, FSH and testosterone concentrations, germ cell yield or relative numbers of different classes of testicular cells; or testicular sperm number.
A statistically significant increase in tail abnormalities which interfered with sperm motility occurred at all doses. According to an ISLI report mentioned in the CEPAD position paper ECBI/108/04 Add.5 the tail abnormalities are a preparation artifact. A slight but significant decrease in epidymal sperm number was found at the highest dose.
The LOAEC is 10-9M, based on test mat.
This study is unacceptable and does not satisfy the guideline requirement for a reproductive study on fertility in male Fisher 344 rats.
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Wright 2000 & Wright 1999
- Principles of method if other than guideline:
- Ewes received twice weekly s.c. injections of octylphenol from Day 70 of gestation to weaning; Day 70 of gestation to birth; birth to weaning; or corn oil from Day 70 of gestation to weaning (control). Blood samples were collected twice weekly to determine progesterone and FSH concentrations from 20 wk of age throughout the first breeding season. Onset of puberty and interestrous intervals were determined from 20 wk of age by twice daily observation for estrus in the presence of a vasectomized ram. The ovaries of each lamb were examined using transrectal ultrasonography from the day of estrus for 15 days. Blood samples were collected every 8 h to examine FSH concentrations and every 2 h to detect the preovulatory gonadotropin surge throughout this estrous cycle.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- other: lamb
- Strain:
- other: Suffolk cross
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: no data
- Age at study initiation: no data about P generation, F1 was born during study (March)
- Weight at study initiation: no data
- Housing: lambs were maintained outdoors with their mothers; housing in accordance with the cruelty to animals act 1876 (Eurepean Community Directive 86/609/EC) licensed by the Department of Health and Children, Ireland. Animals were penned according to treatment.
- Diet (e.g. ad libitum): lambs were weaned at 5 months of age; at 6 months of age: 0.45 kg/day of 19 % protein ration and 0.5 kg/day of hay.
- Water (e.g. ad libitum): lambs were weaned at 5 months of age; free access to water at 6 months of age. - Route of administration:
- intravenous
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: on the day of application
- Details on mating procedure:
- Mating was not included in study.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 1000 µg/kg bw/day s.c. injection
- Frequency of treatment:
- twice weekly
- Details on study schedule:
- Doses were injected from Day 70 of gestation to weaning (n=6); Day 70 of gestation to birth (n=3); or birth to weaning (n=5; gestation = 145 days).
Control animals received vehicle only from Day 70 of gestation to weaning (control; n=5). - Remarks:
- Doses / Concentrations:
1000 µg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- Day 70 - weaning: 6
Day 70 - birth: 3
Birth - weaning: 5
Control: 5 - Control animals:
- yes, concurrent vehicle
- Positive control:
- none
- Litter observations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
- Parameters examined: first behavioral estrus (identified as the first date, the female stood to be mounted), interestrous interval, duration of estrus, and end of breeding season. Parameters were observed in the presence of a vasectomized ram.
- End of breeding season was determined by heat checkings twice daily and transrectal scanning of the ovaries.
- Onset of puberty was defined as the first progesterone rise (date at which the progesterone concentration first reached >= 0.4 ng/mg for two consecutive samples)
BODY WEIGHT: Yes
- Time schedule for examinations: recording at 3-week intervals
BLOOD SAMPLES: Yes (by jugular venepunture)
- Time schedule for examinations: twice weekly from 20 w of age throughout the breeding season
- Parameters examined: FSH, progesterone
- Storage: Blood samples were stored at room temperature for 1 h, then stored in the refrigerator at 4 °C for 24 h and afterwards centrifuged at the same temperature. Serum was decanted and stored at -20 °C until assay.
ENDOCRINE STATUS AMD FOLLICULAR DYNAMICS: Yes
- For determination whether pre-/ or postnatal exposure of OP caused a disruption of folliculogenesis and/or endocrine status: towards the end of the breeding season, hormone concentrations and follicle growth patterns were monitored during a synchronized estrous cycle (15 d). Insertion of a catheter into each ewe's jugular vein to collect blood samples. Samples were taken every 8 h for FSH and every 2 h (commenced 24 h postprostaglandin injection) to detect the preovulatory gonadotropin (FSH and LH) surge. For testing estrus activity, a vasectomized ram was introduced into the pen every 8 h.
OVARIAN FUNCTIONS: Yes (15 d)
- Synchronisation of ovarian cycles by two i.m. injections of Prostaglandin F2alpha (0.5 ml Estrumate) 24 h apart.
- Transrectal ultrasonography was used for examination; 7.5 MHzj linear array transducer (Concept 500; Dynamic Imaging Ltd., Livingston, Scotland)
- Housing during examination periods (5 min): in the dark, animals were restrained in a fostering crate
- Recording of position and diameter of each follicle >= 2 mm and of each corpus luteum.
- Individual follicles that reached diameters of 4 mm and were >= 3 mm for >= 3 days were defined as identified follicles. Emergence was defined as the synchronous growth of of a cohort of identified and unidentified follicles, which were 2 or 3 mm in diameter. The day of ovulation was determined by the collapse of the large antral follicle and formation of a corpus luteum; confirmation by detection of estrus using a vasectomized ram.
RIA ANALYSES: Yes
- Luteiniting hormone: Determination of serum concentrations by radioimmunoassay using a modification of the method described by Sweeney. ON day 1, 200 µl aliquots of of serum or standard, 100 µl of monoclonal antibody, and 100 µl of I125-labeled radioligand were added to tubes. Tubes were vortexed and incubated at room temperature for 24 h. On day 2, 50 µl of secondary antibody were added to tubes, vortexed and incubated at room temperature for 30 min. Addition of 250 µl distilled water and centrifugation at 2500 rpm for 5 min. The supernatant was aspirated and the amount of radioactivity in the pellet was determined by a gamma counter. The sensitivity of the assay was 0.1 ng/ml with 95 % binding. Interassay coefficient of variations at 0.3, 1.7, and 3.6 ng/ml (n=10) were 9.3 %, 9.7%, and 11.7 % resprectively, and intraassay values were 27.6 %, 14.8 %, and 17.7 %, respectively (n=6).
- Follicle-stimulationg hormone: The circulation concentrations of FSH were measured in duplicate aliquots of plasma using an anti-ovine antibody and an iodinated antigen, with a modification to the ovine standards described by Evans et al. The sensitivity of the assay defined by 95 % binding was 0.06 ng/ml. Interassay CVs for the three serum pools with vvalues of 0.3, 1.3, and 3.4 ng/ml (n=15) were 15.2, 15.2 and 18.2 % respectively. Intraassay CVs were 28.85, 15.4, and 11.9 %, respectively (n=6).
- Progesterone: Serum progesterone concentrations were determined using a modification of the time resolved solid phase fluoroimmunoassay DELFIA progesterone kit. Standard supplied with tht ekit was replaced by progesterone (Sigma P0130) standard, which was added to prepared progesterone-free ovine serum. Interassay CVs at 0.2, 1.1, and 2.3 ng/ml (n=10) were 20.3 %, 13.6 %, and 7.5 %, respectively. Intraassay values were 14.8 %, 5.78 %, and 10.8 %, respectively (n=6). - Statistics:
- - ANOVA and Duncan t-test for: Differences in the onset of puberty; first progesterone rise; interestrous interval; body weight throughout the first breeding season and at the onset of puberty; end and duration of the breeding season.
- General Linear Models procedure of SAS: Further analysis of the onset of puberty data with body weight as a covariate
- Repeated measures ANOVA using SAS (2/week): Endocrine profiles, FSH, and progesterone
- ANOVA in SPSS, version 8.02: number of waves; interwave interval; number of identified follicles per wave, ovulatory and maximum follicle diamter; the total number of follicles; and the mean 8-h FSH concentrations
- ANOVA in SAS: comparison of characteristics of the preovulatory gonadotropin surges
- Before ANOVA, logarithmic transformations were carried out to yield homogeneity of variance. All values are given as the mean +- SEM - Dose descriptor:
- NOAEL
- Effect level:
- 1 mg/kg bw/day (actual dose received)
- Sex:
- female
- Basis for effect level:
- other: overall effects
- Clinical signs:
- effects observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Reproductive effects observed:
- not specified
- Conclusions:
- Maternal exposure to 1 mg/kg/day OP (s.c) during pre- and/or postnatal life advanced the onset of puberty and first progesterone rise but did not disrupt the inter-estrous interval or endocrine profiles throughout the first breeding season or the pattern of ovarian follicular dynamics.
- Executive summary:
In this study 4 -(tert-octyl)phenol (97 %) was administered to 19 Suffolk cross ewes at a dose of 1 mg/kg bw/day by s.c. injections two times per week. The treated groups received OP from day 70 of gestation to weaning (n=6), from day 70 of gestation to birth (n=3), birth to weaning (n=6) and the control animals were administered corn oil only.
The offspring were examined for effects of maternal exposure on the onset of puberty, endocrine status, and subsequent ovarian follicular dynamics. Blood samples were collected twice per week for determination of progesterone and FSH concentrations from 20 w of age throughout the first breeding season. Onset of puberty and intrestrous intervals were examined from 20 w of age. The ovaries of lambs were examined by transrectal ultrasonography from the day of estrus for 15 days. Blood samples were collected every 8 h to analyse FSH concentrations and every 2 h to observe preovulatory gonadotropin surge.
The onset of puberty and the first progesterone rise was advanced and the FSH preovulatory surge was elevated for longer in the OP-treated lambs compared to the controls. Furthermore, interestrous intervals, FSH profiles and ovarian follicular dynamics did not differ among groups.
In conclusion, maternal exposure to OP during pre- and/or postnatal life advanced the onset of puberty and first progesterone rise but did not disrupt the inter-estrous interval or endocrine profiles throughout the first breeding season or the pattern of ovarian follicular dynamics.
This study is acceptable for the assessment of maternal exposure to low dose octylphenol.
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP; comparable to guideline study; without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
- Version / remarks:
- draft 1996
- Deviations:
- yes
- Remarks:
- : Additional measurements were made on retained F2 offspring.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Sprague-Dawley CD rats (Charles River Laboratories, Raleigh, NC)
- Age at study initiation: (P) 6-7 wks;
- Fasting period before study: quarantine 1 week
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): no data
- Acclimation period: 1 week quarantine
ENVIRONMENTAL CONDITIONS
according to EPA OPPTS 870.3800 (Reproduction and Fertility Effects) - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 5 weeks
- Mixing appropriate amounts with (Type of food): PMI Certified Rodent Diet No. 5002
- preparation: Dosed diet preparations were formulated by dissolving OP in acetone and adding this solution to the appropriate amount of diet. The acetone was allowed to evaporate, and the feed was then mixed in a V-Shell Blender (5 ft 3, Lowe Industries, Inc., Crestwood, IL). Control diets were prepared in the same manner using a similar amount of acetone. Prior to the start of the study, stability of OP in the test diets was confirmed in the storage and feeding containers. - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal smear - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Verification of dosage concentrations was performed by GC with flame ionization (FI) detection.
Analyses showed that the OP in the diet was mixed homogeneously. - Duration of treatment / exposure:
- (P) males and females: 10 weeks pre-breed phase, 3 weeks gestation, 3 weeks lactation
(F1) males and females: 10 weeks pre-breed phase, 3 weeks gestation, 3 weeks lactation - Frequency of treatment:
- daily 7 days a week
- Details on study schedule:
- - F1 parental animals not mated until 10 weeks after selected from the F1 litters. (prebreed phase)
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13 weeks
A schematical picture of the whole study design is given in figure 1 (see attached document) - Remarks:
- Doses / Concentrations:
0, 0.2, 20, 200, 2000 ppm
Basis:
nominal in diet
Verification of dosage concentrations was performed by GC with flame ionization (FI) detection. - Remarks:
- Doses / Concentrations:
0, 0.015, 1.5, 15, 150 mg/kg/day
Basis:
nominal in diet - No. of animals per sex per dose:
- Parental:
(P) 300 animals
groups of animals 30 f/30 m to yield at least 20 pregnant females/group
F1-Generation
On postnatal day (pnd) 4, the size of each F1 litter was adjusted to ten pups by eliminating extra pups by random selection to yield, as nearly as possible, five males and five females per litter.
On pnd 21, each litter was weaned, and at least one F1 male and one F1 female pup per litter, if possible, were randomly selected (30/sex/group) to produce the F2 generation. - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
- For the high dose region, OP intake of parental animals would be just below, at, or above levels previously shown to saturate liver metabolic capacity (200 mg/kg/day), and intake of young animals would exceed this dose.
- 2000 ppm would be expected to result in decreased body weight and possible other effects, thus providing an appropriate high dose.
- 2000 ppm would allow for evaluation of whether the spontaneous resorptions observed in the probe study at this dietary concentration were related to test material toxicity.
- The low dose (0.2 ppm) provided for daily intake of the test material between the two doses that purportedly caused effects on sperm count and testicular weight in an earlier study of Sharpe et al. (1995).
- The 20 ppm dose was considered adequate support for the low dose evaluation if bioavailability of the test material was lower due to dietary exposure compared to drinking water exposure used in the Sharpe et al. study. - Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
Treatment-related effects were limited to consistent and persistent reductions in body weights and weight gains in both sexes
- Time schedule: monitored during treatment
DETAILED CLINICAL OBSERVATIONS: No
There were no treatment- or dose-related clinical observations in either sex in any of the generations.
BODY WEIGHT: Yes
- Time schedule for examinations of F1 Generation (Parents of F2 Generation):
males: day 0, 7.14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84
females: prebreed day: 0, 7.14, 21, 28, 35, 42, 49, 56, 63, 70
females: gestational day: 0, 7, 14, 20
females: postnatal day: 0, 4, 7, 14, 21
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes - Oestrous cyclicity (parental animals):
- For the last 3 weeks of the prebreed exposure period, vaginal smears for estrous cyclicity and normality were taken for all P females.
- Sperm parameters (parental animals):
- Parameters examined in male parental generations:
At the time of sacrifice of F0 and F1 parental males and retained F2 male offspring, testicular homogenization-resistant spermatid head count and calculation of daily sperm production and efficiency of daily sperm production were determined from one frozen testis/male for all males. In addition, number, motility, and morphology of sperm from one cauda epididymis were evaluated in these same animals. - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes/no
- If yes, maximum of 5/sex/litter as nearly as possible; excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
body weights of pups by sex per litter
organ weights
GROSS EXAMINATION OF DEAD PUPS:
There were no treatment- or dose-related gross or microscopic findings for the examined organs, for F0 and F1 parental animals, and for F2 re-
tained adult males. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving paternal animals sacrificed at the end of gestation
- Maternal animals: All surviving animals at the end of lactation phase
GROSS NECROPSY
All F0 and F1 parental animals were subjected to a complete gross necropsy.
The stage of estrus at necropsy was determined for all F0 and F1 females. For parental animals (and retained F2 male offspring) the brain, liver, kidneys, adrenal glands, spleen, ovaries, uterus, testes, epididymides, seminal vesicles with coagulating glands, and the prostate and dorsal prostate were weighed. Specific attention was focused on the examination of the parental reproductive organs, including determining the weight of the prostate and dorsal prostate for all males and ovarian follicle counts for high dose and control F0 and F1 females.
HISTOPATHOLOGY / ORGAN WEIGHTS
Epididymal sperm morphology was examined manually. Histopathologic evaluation of the ovaries with oviducts (2), testis (1), vagina, epididymis (1), uterus with cervix, seminal vesicles (2), and prostate was conducted on the F0 and F1 parental animals and retained F2 male offspring from high dose and control groups. - Postmortem examinations (offspring):
- SACRIFICE
Selected F1 and F2 weanling animals and retained F2 male offspring were subjected to a complete gross necropsy.
GROSS NECROPSY
Selected F1 and F2 weanling animals and retained F2 male offspring were subjected to a complete gross necropsy.
HISTOPATHOLOGY / ORGAN WEIGTHS
For weanling animals, the brain, spleen, thymus, ovaries (2), uterus with cervix and vagina, testes (2), epididymides (2), and seminal vesicles (2) were weighed.
Histopathologic evaluation of the ovaries with oviducts (2), testis (1), vagina, epididymis (1), uterus with cervix, seminal vesicles (2), and prostate was conducted on the F1 parental animals and retained F2 male offspring from high dose and control groups. - Statistics:
- The unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. All statistical analyses employed SAS software (SAS Institute, Inc.). Quantitative continuous data were analyzed using Bartlett's Test for homogeneity of variances (Winer, 1962) followed by appropriate intergroup comparisons and a test for linear trend. Frequency data were analyzed for differences among treatment groups by Chi-Square Tests,
followed by Fisher's Exact Test for intergroup comparisons and a test for linear trend. Comparisons for developmental landmarks (e.g., acquisition of vaginal patency and preputial separation) were made using the Mann-Whitney U Test. In addition, acquisition of reproductive landmarks was analyzed by analysis of covariance, with body weight as the covariate (the actual body weight on the day of acquisition for selected F1 and retained F2 offspring), and the Least Squares Means Test for pairwise comparisons to the control group value. For all statistical tests, the significance limit of 0.05 (one- or two-tailed) was used as the criterion for significance. Analysis of linear trend and for overall and pairwise comparisons of correlated data (i.e., body weights and absolute and relative organ weight data from weanling necropsies) was performed using SUDAAN Software. A test for statistical outliers was performed on male and female body weights and feed consumption in grams per day. If examination of pertinent study data did not provide a plausible biologically sound reason for inclusion of the data flagged as "outlier", the data were excluded from summarization and analysis and were designated as outliers. - Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- reduced body weight at 2000 ppm
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- reduced body weight at 2000 ppm
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- (systemic)
- Effect level:
- 200 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: overall effects
- Dose descriptor:
- LOAEL
- Remarks:
- (systemic)
- Effect level:
- 2 000 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Decreased body weight and weight gain in adults, reduced body weight during latter portion of lactation in offspring, and slightly delayed vaginal opening and preputial separation (considered related to bw decrease)
- Remarks on result:
- other: Generation: P & F1 (migrated information)
- Dose descriptor:
- NOAEL
- Remarks:
- (reproductive parameters)
- Effect level:
- 2 000 ppm (nominal)
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: see 'Remark'
- Remarks on result:
- other: Generation: P & F1 (migrated information)
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Pup body weights per litter were reduced at 2000 ppm for both F1 and F2 offspring for latter lactational time points, pnd 14 and 21 (see tables 6 and 7)
- Sexual maturation:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- (reproductive parameters)
- Generation:
- F1
- Effect level:
- 2 000 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see "Remark"
- Dose descriptor:
- LOAEL
- Remarks:
- systemic
- Generation:
- F1
- Effect level:
- 2 000 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Decreased body weight and weight gain in adults, reduced body weight during latter portion of lactation in offspring, and slightly delayed vaginal opening and preputial separation (consideration related to bw decrease)
- Critical effects observed:
- not specified
- Reproductive effects observed:
- not specified
- Conclusions:
- Dietary exposure to OP for two generations, one litter per generation at 0, 0.2, 20, 200, and 2000 ppm, resulted in effects only at 2000 ppm. No effects on reproductive parameters were observed. No estrogenlike effects on males or females and no low dose effects were evident. The NOAELs for systemic and postnatal toxicity were 200 ppm and at or above 2000 ppm for reproductive toxicity.
- Executive summary:
In an extended two-generation reproduction study para-tert-Octylphenol (90,2 %) was administered to 30 SD rats /sex/dose at dose levels of 0, 0.2, 20, 200, 2000 (approximately 0.015, 1.5, 15, 150 mg/kg bw/day).
P-Generation showed reduced body weight at 2000 ppm. Pup body weights per litter were reduced at 2000 ppm for both F1 and F2 offspring for latter lactational time points, pnd 14 and 21. F1-Generation showed effects on sexual maturation. Slightly delayed vaginal opening and preputial separation were considered related to body weight decreases. No effects on reproductive parameters, testes weights or morphology, epididymal sperm counts, motility, or morphology, daily sperm production, efficiency of daily sperm production, or prostate or dorsal prostate weights or histopathology were observed. No estrogen-like effects on males or females and no low dose effects were evident.
From this a NOAEL of 200 ppm for systemic and postnatal toxicity and a NOAEL of above 2000 ppm can be concluded.
This study is acceptable and satisfies the guideline requirement for a two-generation reproductive study (OPPTS 870.3800) in rats.
- Endpoint:
- fertility, other
- Remarks:
- based on test type (migrated information)
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- In accordance with Regulation (EC) 1907/2006 Annex XI (1.5) and the relevant ECHA guidance documents, the substances detailed in the table below are grouped for the purposes of read across to reduce the need for unnecessary repeat testing on the basis that the substances are similar on the basis of a common functional groups.
- Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- not specified
- Body weight and weight changes:
- not specified
- Food consumption and compound intake (if feeding study):
- not specified
- Organ weight findings including organ / body weight ratios:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- not specified
- Remarks on result:
- not measured/tested
- Critical effects observed:
- not specified
- Clinical signs:
- effects observed, treatment-related
- Mortality / viability:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 12.5 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: systematic toxicity
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 100 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: reproductive toxicity
- Reproductive effects observed:
- not specified
- Conclusions:
- The read across for 4-tert-octylphenol (CAS: 140-66-9); is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of in reproductive toxicity. Based on the information available for the read across substances, the substance is not expected to cause damage to fertility.
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment (modified OECD 421)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- : additional parameter to focus on endocrine disrupting properties: histology of pup sex organs and assessment of sex hormones; Number of animals reduced to 4/sex (instead of 8/sex); Limit dose was 100 mg/kg/day due to high mortality at 1000 mg/kg/day
- Principles of method if other than guideline:
- Histopathology: Tissues sampled for histopathology were trimmed and processed routinely for paraffin embedding. Organs included uteri and testes from dams and sires, respectively, as well as from offspring. Sections (5µm) were cut and stained with haematoxylin and Eosin (H&E).
Endocrinology: Hormones were measured by radioimmunoassay (RIA).
Estradiol was determined with the Estradiol Double Antibody kit from Diagnostic products Corporation (KE2D1; DPC; Los Angeles CA, USA);
Progesterone was determined with the Coat-A-Count Progesterone RIA kit (TKPG1; DPC);
Testosterone was determined with the Coat-A-Count Testosterone RIA kit (TKTT1; DPC).
Luteinizing hormone (LH) and follicle stimulating hormone (FSH) were analyzed using reagents (LH: 125I-rLH-15, rLH-RP2 and anti-rLH-S6; FSH: 125I-rFSH-15, rFSH-RP1 and anti-rFSH-S11) kindly provided by the NIDDK, National Hormone & Pituitary Program (Dr. Parlow, CA, USA). - GLP compliance:
- not specified
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: no data
- Age at study initiation: 12-15 weeks at onset of dosing
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually in macrolon type 3 and 4 cages
- Diet: standard RMH-GS feedad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 50 %-75 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Amount of vehicle (if gavage): 1 ml per 200 g body weight - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0 / day 1] of pregnancy
- After 14 days of unsuccessful pairing. Males were dosed further and killed and necropsied after a total dosing period of 28 days. Sex organs were removed, weighed, and analyzed histologically. - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- no data
- Duration of treatment / exposure:
- A schematic representation of the treatment is shown in figure 1 (see attached document)
14 days premating
1-14 days 1:1 mating, then separation and treatment of males further 6 days
22 days gestation then birth (non-pregnant females were treated further 28 days after separation)
4-6 days lactation (after this time non-pregnant females, dams and pups were killed and necropsy was performed) - Frequency of treatment:
- daily (7 days a week)
- Details on study schedule:
- Dosing was different from the general protocol: 4-tert-octylphenol was dosed 100 mg/kd bodyweight/day from 12 days premating onward due to extreme toxicity of a tenfold higher dose
- Remarks:
- Doses / Concentrations:
100 mg/kg bodyweight / day
Basis:
nominal conc. - No. of animals per sex per dose:
- four pairs per dosage group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: An initial dose of 1000 mg/kg bw/day was selected on the basis the estrogenicity of octylphenol. Due to the high mortality this dose was reduced to 100 mg/kg bw/day.
- Positive control:
- Ethynyl-estradiol (Sigma E8476) at 0.1 mg/kg bw.day
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked daily
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: tables 1, 2 and 3
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes - Oestrous cyclicity (parental animals):
- no data
- Sperm parameters (parental animals):
- Parameters examined in male parental generations: testis weight
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, weight, stillbirths, live births
GROSS EXAMINATION OF DEAD PUPS:
yes, external malformations - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: Males were dosed further and killed and necropsied after a total dosing period of 28 days.
- Maternal animals: Dosing of females was continued until postpartum day 6, when females were killed and necropsied.
GROSS NECROPSY
no data
HISTOPATHOLOGY / ORGAN WEIGHTS
- Male animals: Sex organs were removed, weighed, and analyzed histologically.
- Maternal animals: Uteri and ovaria were removed and weighed, corpora lutea and implantation sites counted, and uteri were analyzed histologically. - Postmortem examinations (offspring):
- SACRIFICE
- Offspring was killed six days after birth
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- external malformation
HISTOPATHOLOGY / ORGAN WEIGTHS
Pup anogenital distance was measured and pup sex organs were analyzed histologically. - Statistics:
- Calculation of mean values
- Offspring viability indices:
- no data
- Clinical signs:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 100 mg/kg/day affected food consumption and body weight gain in both sexes
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- 100 mg/kg/day affected food consumption and body weight gain in both sexes
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- Two out of 4 dams became pregnant.
- Dose descriptor:
- LOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: body weight gain, food consumption, small increase of corpora lutea (suggesting premature luteinization), LH and testosterone level
- Clinical signs:
- no effects observed
- Mortality / viability:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- slightly higher compared to control (3,6 %)
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not specified
- Reproductive effects observed:
- not specified
- Conclusions:
- The only reproductive effect observed after exposure of 4 wistar rats/sex to 100 mg/kg bw/day was a questionable increase in corpora lutea.
- Executive summary:
In a reproductive toxicity screening limit test (OECD 421) 4 -tert-octylphenol (>= 90 %) was administered to 4 Wistar rats/sex/dose by gavage at a limit dose of 100 mg/kg bw/day.
One dose of this compound at 1000 mg/kg bw was lethal to the animals. This suggests that other toxicity than endocrine-mediated reproductive effects may be more important for4-tert-octylphenol.
As a consequence, new rats were enrolled into the study which were dosed from 12 days premating onward, at 100 mg/kg bw.day. This dosage affected food consumption and body weight gain in both sexes. Two out of 4 dams became pregnant. At necropsy pregnant dams showed a small increase of corpora lutea over controls, suggesting possibly premature luteinization. Histological analysis showed no abnormalities in both sexes. Luteinizing hormone was somewhat low in dams, and testosterone was decreased in sires.
From this screening study, no NOAEL could be derived.
This study is acceptable and satisfies the guideline requirements for a reproductive toxicity screening study OECD 421 (limit test) in Wistar rats.
- Endpoint:
- fertility, other
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Atsugi, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually in metal cages
- Use of restrainers for preventing ingestion (if dermal): no data
- Diet (e.g. ad libitum): ad libitum, CE-2, Clea Japan
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+-1 °C
- Humidity (%): 50+-5 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 12.5, 25, 50 and 100 mg/kg bw
- Amount of vehicle (if gavage): dosing volume was 5 ml/kg bw - Details on mating procedure:
- - Age of mating animals: female rats were 10 to 11 weeks old
- M/F ratio per cage: all females with a single male
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- No further matings.
- After successful mating each pregnant female was weighed and randomly allocated to six test groups
- Dams delivered naturally and nursed their pubs until postnatal day (PND) 21
- Pubs were weighed on day 0, their sex was determined and litters were reduced randomly to 8 (4 pubs/sex/litter if possible), litters of =<8 were not reduced; 3-5 males and females, respectively, were used, the remaining pubs were discarded - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- from PND 1 to PND 5
- Frequency of treatment:
- daily
- Details on study schedule:
- Body weights were determined from PND 1-6 daily, on day 14 and at 3, 5, 7, and 9 weeks of age.
- Remarks:
- Doses / Concentrations:
12.5, 25, 50, 100 mg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- Control group: 28 males, 30 females from 8 litters
12.5 mg/kg group: 28 males, 28 females from 7 litters
25 mg/kg group: 28 males and 28 females from 7 litters
50 mg/kg group: 30 males and 28 females from 8 litters
100 mg/kg group: 30 males and 30 females from 8 litters - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: based on the test results of a preliminary test with concentrations of 0, 50, 100, and 200 mg/kg/day, the actual test concentrations were chosen
- New method was developed by the authors to avoid pubs dying because of injuries in their stomach, esophagus or trachea after gavage:
* Needle (23G) was cut off to a 23 mm length and 20 mm of an indwelling feeding tube for infants (Nutrient catheter, Type 3Fr, Atom Medical Co., Tokyo) was attached to the needle. It then was mounted to the micro syringe (Hamilton Gastight #1710, Hamilton Co., USA) which was used as a gastric tube.
* Pub is held between the thumb and the 3rd finger and test substance is administered very slowly: dosage rate of approx. 10 µl/sec.
* With this method, no pub dies when corn oil, distilled water or saline at 5 µl/g bw is administered and the pubs' growth is still comparable to the control group
- 12 weeks old animals of all groups were permitted to mate on a 1:1 basis with an untreated male or female Sprague-Dawley rat of the same age to evaluate their reproductive performance for up to 2 weeks
- Sign of successful pregnancy is again the presence of sperm in the vaginal smear
- F1 animals that didn't copulate successfully for 2 weeks were remated with new untreated rats for up to 2 weeks. - Positive control:
- none
- Parental animals: Observations and examinations:
- No data.
- Oestrous cyclicity (parental animals):
- No data.
- Sperm parameters (parental animals):
- No data.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on postnatal day 0: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, postnatal mortality, weight gain, deaths during lactation period, viability on PND 6 and 21 (weaning index), male and female reproductive function after puberty, preputial separation and vaginal opening as endpoints of sexual maturation, estrous cycling, sperm count, serum testosterone concentration, histopathologic changes of the reproductive organs of male and female rats.
GROSS EXAMINATION OF DEAD PUPS:
yes - Postmortem examinations (parental animals):
- no data
- Postmortem examinations (offspring):
- SACRIFICE
- Selected pubs were sacrificed at 21 days of age.
- F1 animals that mated, were sacrificed on day 12 of gestation, the number of implants and live or dead embryos was counted.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY: Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathologic observations were performed by experimenters blind to treatment conditions. - Statistics:
- - Statistical analyses of the data for the offspring were performed using the litter as the unit.
- Body and organ weight, organ/body weight ratios, number of implants, timing of vaginal opening and preputial separation, sperm concentration and serum testosterone concentration were compared by analysis of variance (one way), Bartlett's test for homogeneity of variance, and the appropriate t-test (for equal and unequal variance) as described by Steel and Torrie using Dunnett's multiple comparison tables or the heterogeneous Student's t-test with Bonferroni's correction to determine the significance of difference.
- Nonparametric dichotomous data were initially analyzed by the x2 test and if significance was observed between groups then by the Fisher's exact test with the Bonferroni adjustment.
- Nonparametric data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate.
- Comparisons between the octylphenol-treated groups and the control group were determined using P>=0.05 and P=<0.01 as levels of significance. - Reproductive indices:
- EVALUATION OF SEXUAL MATURATION AND ESTROUS CYCLING:
males: preputial seperation (beginning on PND 35)
females: vaginal opening (beginning on PND 28)
Daily vaginal lavage fluid was collected from PND week 7 until confirmation of copulation. The fluid was then analysed for the number of days in each stage of the cycle, cycle length, and the number of normally cycling femaled was determined (normal defined as being from 4-6 days including 1-2 days of estrus).
EVALUATION OF REPRODUCTIVE PERFORMANCE:
Males and females that were 12 weeks of age were mated with untreated rats like described above. On day 12 of gestation, animals were sacrificed and at necropsy, the numbers of implants, and live or dead embryos were counted.
SPERM CONCENTRATION, MEASUREMENT OF REPRODUCTIVE ORGAN WEIGHT, AND DETERMINATION OF SERUM TESTOSTERONE CONCENTRATION:
- After measurement of terminal body weight at least 14 days after copulation, male animals were anesthesized under ether and a blood sample was collected via the inferior vena cava to determine the testosterone concentration in blood.
- The testis, epididymic, ventral prostate, and seminal vesicles with coagulating glands of all groups were weighed and the left cauda was analysed.
HISTOPATHOLOGIC EVALUATION OF REPRODUCTIVE ORGANS:
- On PND, randomly chosen males and females were sacrificed and their testes were weighed. Once piece of tissue of each side of the testes and ovaries and 5 pieces of tissue from the uterus were analysed
- After measurement of organ weights at 18 weeks of age, all males and females that did not copulate or were not pregnant were sacrificed and their testes, epididymides, ventral prostate, and seminal vesicles with coagulating glands and the uteri and ovaries were analysed. One piece of tissue from each side of the testes, ventral prostate, seminal vesicles, ovaries, one side of the head and tail of the epididymides, and five pieces of tissues from the uterus were analysed. - Offspring viability indices:
- No data for the F1 generation, for F2 see REPRODUCTIVE PERFORMANCE in Details on results.
- Clinical signs:
- not specified
- Body weight and weight changes:
- not specified
- Food consumption and compound intake (if feeding study):
- not specified
- Organ weight findings including organ / body weight ratios:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- not specified
- Remarks on result:
- not measured/tested
- Critical effects observed:
- not specified
- Clinical signs:
- effects observed, treatment-related
- Mortality / viability:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 12.5 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: systematic toxicity
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 100 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: reproductive toxicity
- Reproductive effects observed:
- not specified
- Conclusions:
- The NOAEL (systemic toxicity) is 12.5 mg/kg bw/day in males and females and the NOAEL (reproductive toxicity) is >= 100 mg/kg bw/day in males and females.
- Executive summary:
In a 2-generation reproduction study p-Octylphenol (min. 96 %) was administered to 288 Sprague Dawley rats/male and females/dose by gavage at dose levels of 0, 12.5, 25, and 100 mg/kg bw/day neonatally for 5 days. Parameters examined were reproductive function after puberty, preputial separation and vaginal opening as endpoints of sexual maturation, estrous cycling, sperm count, serum testosterone concentration, and histopathologic changes of the reproductive organs of male and female rats. p-Octylphenol affected body weight of males and females in all dose groups, the acquisition of puberty was delayed in the 50 and 100 mg/kg bw/day dose groups. Further observations were a significant decrease in absolute and relative prostate weight in all dose groups and absolute epididymal weight the 100 mg/kg/d group, in the same group an increase in relative testes weight and relative seminal vesicle weights in the 50 and 100 mg/kg/d groups. Histopathology was conducted of reproductive organs in all rats but no alterations were found.
Furthermore, this study shows that neonatal exposition of p-Octylphenol to rats does not cause dysfunction of reproductive performance in both sexes and no disruption of the reproductive tract occurred. Significant decreases in body weights of rats in the 25 mg/kg/d and more groups and delays of sexual maturation in the 50 mg/kg/d and greater groups were observed, as well as a decrease in ventral prostate weights in all treated groups.
The NOAEL (systemic toxicity) is 12.5 mg/kg bw/day in males and females and the NOAEL (reproductive toxicity) is >= 100 mg/kg bw/day in males and females.
This study is acceptable and satisfies the major requirement of a 2 -generation reproductive study in rats.
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28/06/1994-24/08/1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study (OECD 421)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- draft version 12.01.1993
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Stain: Sprague-Dawley Crl:CD BR VAF/Plus strain
- Source: Charles River UK Ltd, Manston Road, Margate.
- Age at study initiation: (P) 11-12 wks males and 7-8 wks females; (F1) 0 days
- Weight at study initiation: before acclimation of 6 days (P) Males: 303-341 g; Females: 147-183 g; after acclimation of 6 days: (P) Males: 361-432 g; Females: 187-232 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing:
pre-mating period: males and females separated 4 to a cage unless reduced by mortality. Cages of males were interspersed amongst those holding females to promote development of regular oestrous cycles. In addition, the cages constituting each treatment group were dispersed between batteries so that possible environmental influences arising from their spatial distribution were equilibrated as far as possible for all treatment. Suspended stainless steel cages (Biotech®) equipped with solid sides, and wire grid front, back and top were used during the pre-mating period.
Mating period: rats were housed on the basis of one male to one female in plastic breeding cages (North Kent Plastics, RB-3 type). At the end of the mating period the males were re-housed with their former cage mates in metal cages (Biotech®) and the females were housed in individual breeding cages (North Kent Plastics, RB-3 type) prior to the birth and rearing of young. Suitable nesting material was provided.
- Diet (e.g. ad libitum): Special Diet Services (SDS) laboratory Animal Diet No. 1 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: first period 6 days, second period of 7 days between allocation of animals to groups and commenced of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
IN-LIFE DATES: From: 28.06.1994 To: 08.08.1994 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Homogeneity and stability of the test article in liquid formulation had previously been confirmed in these laboratories as part of the 28-day toxicity study (SAZ 464/942419). A series of suspensions were made dissolving the test substance in corn oil, the concentrations were chosen to give a constant dosage volume of 5 ml/kg bodyweight. All suspensions were prepared daily and administered on the day of preparation. Samples of dose formulations prepared for the first day of the study were analysed for achievement concentration. Analyses were performed at HRC. Control animals received vehicle alone at the same dosage volume as test group animals.
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is use. The test substance is stable in vehicle during storage and forms a homogeneous suspension.
- Concentration in vehicle: 25, 50, 100 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bodyweight - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- After successful mating each pregnant female was caged in individual breeding cages (North Kent Plastics, RB-3 type) prior to the birth and rearing of young. Suitable nesting material was provided. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Formulation Analysis (SAZ 462/942750): This report contains details of the analytical method used and the results obtained for the determination of concentrations of test substance in dose formulations prepared for day 1 of the study. The formulations were prepared as suspensions of 4(1,1,3,3-tetramethyl-butyl)phenol in corn oil by Pharmacy Personnel at Huntingdon Research Centre Ltd. Mean concentrations of 4(1,1,3,3,-tetramethyl-butyl)phenol in dose formulations analysed for Day 1 of the study and the deviation of mean results are recorded: mean results were within 5% of nominal concentrations.
The results for the validation of the method of analysis and determination of the physical and chemical stability of corn oil formulations containing the test substance at concentrations of 1 mg/ml and 200 mg/ml, are presented in HRC Report number SAZ 464/942419 (Repeated dose toxicity: oral)
- Determination of concentrations of test substance in dose formulation analysed for day 1 of the study.
- Determination of chemical stability in corn oil formulations.
- Validation of the method of analysis for the determination of test substance in corn oil formulations
- Determination of the physical stability of the test substance in corn oil formulations.
Formulation Analysis Report (SAZ 464/942419):
Mean results were within +2%/-1%. At nominal concentrations of 1 mg/ml and 200 mg/ml, the test substance produces a homogeneous suspension in the corn oil formulation, which can be maintained for up to 1 hour while magnetically stirred and successfully resuspended following storage at ambient temperature for 4 hours.
At nominal concentrations of 1 mg/ml and 200mg/ml the test substance is chemically stable in the corn oil formulation during storage (ambient temperature during the day, refrigeration overnight) for 24 hours.
Procedural recovery obtained during method validation and determination of stability indicate that the analytical method is both accurate and precise: a mean procedural recovery value of 98.1% ± 1.91% CV (n = 8) was obtained for 1 mg/ml and 98.2% ±
0.99% CV (n = 8) for 200 mg/ml. Results for the analysis of test samples were corrected for the appropriate mean procedural recovery value at analysis.
A typical calibration standard graph confirmed the linearity of detector response for the test substance over the concentration range 2 -10 µ/ml. The absence of a peak at the characteristic retention volume for the test substance in the control sample chromatogram demonstrates the specificity of the HPLC assay.
The analytical results confirm that the doses were accurately formulated for Day 1 of the toxicity study. The results also confirm that specimen formulations were homogeneous and stable for a period representing the time from preparation to completion of dosing. - Duration of treatment / exposure:
- 2 weeks prior to mating, 2 weeks mating period and until litters reached Day 4 post partum
- Frequency of treatment:
- once daily
- Remarks:
- Doses / Concentrations:
0, 125, 250, 500 mg/kg/day
Basis:
actual ingested - No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed initially at grouping up, on the first day of dosing, subsequently ate weekly intervals and finally on the day of sacrifice. During the mating period all females were weighed daily throughout and daily weighing continued until parturition. The occurrence of a positive indication of mating (i.e. sperm or plug) was considered as day 0 of pregnancy. Weights are reported for days 0, 3, 7, 10, 14, 17 and 20 of pregnancy. Weights of pregnant animals without a positive indication of mating were reported for appropriate days taken retrospectively from birth or apparent day of gestation at sacrifice. Dams that littered were weighed once parturition was complete (day 0/1 - reported as day 0) and on day 4.
FOOD CONSUMPTION Yes (g/rat):
Food consumption was recorded weekly for both sexes from initiation of treatment until the animals were placed together for treatment ( weeks 1 to 2). Measurement for males continued weekly during the post mating period (weeks 5 -6). For females was reintroduced on day 1 post partum and recorded on day 4 post partum.
FOOD EFICIENCY
Food conversion ratios were calculated, where possible, over the period weeks 1 to 6 from the bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight.
WATER INTAKE (g/rat)
Water consumption was recorded daily
- Oestrous cyclicity (parental animals):
- Mating performance: vaginal smears were taken daily for 1 week prior to an during the 14-day mating period to enable the number of animals that mated on specific days to be determined. This information was used in conjunction with other data:
to determine whether or not pregnancy had occurred and continued uninterrupted after mating
to detect marked anomalies of the oestrus cycle
to determine the median pre-coital time and duration of pregnancy for dams that littered.
Duration of pregnancy: the duration of pregnancy for females which littered was taken as the time between the day of successful mating and the day on which pups were first seen. - Sperm parameters (parental animals):
- Parameters examined in all male parental generations:
testis weight, epididymis weight.
Histophatological examination of testes and epididymides. - Litter observations:
- As soon as possible after parturation the young were counted, individually identified within the litter by toe amputation, sexed, weighed and examined for any physical or behavioural abnormalities. Keeping nest disturbance to a minimum, all litters were examined daily for dead and/or abnormal young. Pups were also weighed on day 4 post partum. Dead young were subjected to an autopsy.
- Postmortem examinations (parental animals):
- SACRIFICE
- Male and female animals: All surviving animals shortly after day 4 post partum. Apparent non-pregnant females and males were killed in the week following the latest possible date of birth.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. The uterus of each female which gave birth was visually inspected for implantation sites and the number of sites was recorded. The number of implantation sites was not recorded in error for two females. Uteri of apparently non-pregnant females were examined for evidence of implantation using a modified Salewski technique (Salewski, 1964)
The following organs from all animals killed were dissected free of fat and weighed:
adrenals, brain, epididymides individually (identified as left or right), heart, kidneys, liver, lungs, ovaries, pituitary, prostate (with seminal vesicles + coagulating gland), testes individually (identified as left or right), thymus, spleen.
Appropriate organs were weighed as a pair.
Presentation of tissues: Samples of all the tissues listed below from all animals were preserved in buffered 10% formalin (except testes and epididymides, identified left or right and preserved in Bouin's fixative):
adrenals, brain, epididymides, heart, kidneys, liver, lungs, mammary gland, macroscopically abnormal tissues, ovaries, pituitary, prostate, seminal vesicles (with coagulating gland), teste, thymus, thyroids, uterus (with cervix), vagina.
HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues required for microscopic examination from all P adults of the control group and at the highest dosage in this study were: epididymides, ovaries and testes. These tissues were embedded in the paraffin wax.
A transverse sample of testis and longitudinal sample of associated epididymis was mounted in the same paraffin wax block and labelled left or right. A single 2 µm section of each block was taken and stained with PAS (Periodic Acid Schiff) - haematoxylin.
The ovaries were sectioned at 4 µm and stained with haenotoxylin and eosin.
Following treatment associated histological changes to the testes and epididymides of males at the highest dosage, examination was extended to the intermediate dosage. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed shortly after day 4 post partum.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:
Sex of the pups was confirmed by gonadal inspection.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. The following organs from all animals killed were dissected free of fat and weighed:
adrenals, brain, epididymides individually (identified as left or right), heart, kidneys, liver, lungs, ovaries, pituitary, prostate (with seminal vesicles + coagulating gland), testes individually (identified as left or right), thymus, spleen.
- Statistics:
- Significance test, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters: food consumption, water consumption, weekly bodyweight, bodyweight change, haematology, biochemistry, litter data, organ weights.
Dependent of the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran 1967) followed by William's test (William 1971/2) or non-parametric tests (Kruskal-Wallis (Hollander and Wolfe 1968) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate. For litter data, the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level.
All significant (ie p≤0.05) intergroup differences from the control are reported and were supported by a significant analysis of variance (P≤0.05) unless otherwise indicated (by parentheses of the superscript) in the tables.
Where 75% or more of the values for a given variable are the same, a Fisher's exact test (Fisher 1950) was used, when considered necessary. - Clinical signs:
- effects observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 125 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: post dose salivation; slightly elevated water consumption.
- Dose descriptor:
- LOAEL
- Remarks:
- systemic
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Dose descriptor:
- dose level: dose level:
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Marked toxic effects, increased mortality (13/24 adult animals)
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects on mating performance or development of the litter
- Remarks on result:
- other: Generation: P & F1 (migrated information)
- Dose descriptor:
- LOAEL
- Remarks:
- reproduction
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: lower implantation rate, lower litter size due to increased pre and post natal mortality, reduced litter weight, suggestion of impaired pup growth to day 4; no gross abnormalities amongst the offspring
- Remarks on result:
- other: Generation: P & F1 (migrated information)
- Clinical signs:
- effects observed, treatment-related
- Mortality / viability:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction
- Generation:
- F1
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects on mating performance or development of the litter
- Dose descriptor:
- LOAEL
- Remarks:
- reproduction
- Generation:
- F1
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: lower implantation rate, lower litter size due to increased pre and post natal mortality, reduced litter weight, suggestion of impaired pup growth to day 4; no gross abnormalities amongst the offspring
- Critical effects observed:
- not specified
- Reproductive effects observed:
- not specified
- Conclusions:
- Treatment of 500 mg/kg/day produced a marked toxic effect resulting in the death of 13/24 adult animals and slight effects on mating performance and development of conceptus. Clear treatment-related toxic effects were reported at 250 mg/kg/day. The reproductive performance and development of the conceptus was unaffected at this dose.
- Executive summary:
In a reproduction/developmental toxicity screening study according OECD 421 4-(1,1,3,3 -tetramethyl-butyl)phenol (98,7%) was administered to 12 SD rats/sex/dose by gavage at dose levels of 0, 125, 250, 500 mg/kg bw/day.
Treatment at 500 mg/kg/day produced a marked toxic effect resulting in the death of 13/24 adult animals during the treatment period. There were slight effects on mating performance and development of the conceptus. There was a clear, through less marked, treatment-related effect at 250 mg/kg/day upon the treated adults, although reproductive performance and development of the conceptus was unaffected.
The systemic LOAEL is 250 mg/kg bw/day, based on body weight gain, water consumption, higher liver weights and slightly elevated kidney weights. Macroscopic examination revealed distended caecum (of males) reduced thymus size (in males), enlarged adrenal size (in females) and uniform cortical scarring of kidneys. There were no treatment related effects on mating performance or development of the litter. The systemic NOAEL is 125 mg/kg bw/day. The only changes at this concentration were post dose salivation and slightly elevated water consumption.
The NOAEL for toxicity to reproduction is 250 mg/kg/d based on a lack of effects on mating performance and development of the conceptus.
This study is acceptable and satisfies the guideline requirement for a reproduction/developmental toxicity screening study according OECD 421 in rat.
- Endpoint:
- toxicity to reproduction
- Remarks:
- other: Effect of chronic administration on male reproductive tissues
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Rats were treated s.c. with OP three times per week for 1 or 2 mo (20 and 80 mg/kg). Testis, epididymis, and male accessory sex organ weights and histology as well as testicular sperm counts were analyzed in tissues taken from decapitated rats. Results of epididymal sperm morphology were analyzed in epididymides taken from anesthetized rats
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Route of administration:
- subcutaneous
- Vehicle:
- corn oil
- Details on mating procedure:
- no mating procedure
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- One respectively two month
- Frequency of treatment:
- three times a week (Monday, Wednesday, Friday)
- Remarks:
- Doses / Concentrations:
0, 66 and 264 mg/kg bw/d
Basis:
other: injection of 0.2 ml corn oil containing 0, 20, 80 mg OP - No. of animals per sex per dose:
- 6 males (1 month treatment).
5 males (2 month treatment)). - Control animals:
- yes, concurrent vehicle
- Dose descriptor:
- LOAEL
- Effect level:
- 66 mg/kg bw/day (actual dose received)
- Sex:
- male
- Basis for effect level:
- other: Reduction in the absolute weights of testis, epididymis and seminal vesicle and a reduction in testicular sperm heads (25%) as well as a increase in sperm heads with abnormal morphology after 2 months.
- Reproductive effects observed:
- not specified
- Conclusions:
- A LOAEL of 66 mg/kg/day was deduced based on a reduction in the absolute weights of the testis, epididymis and seminal vesicle (after 2 months) and a reduction in testicular sperm heads (25%) as well as a increase in sperm heads with abnormal morphology.
- Executive summary:
In a chronic administration study 4-tert-octylphenol (98.1 %) was administered to 6 respectively 5 male Fischer 344 rats dosed by three subcutaneous injection per week at dose levels of 0, 20, 80 mg in corn oil (estimated doses approximately 66 and 264 mg/kg bw day)
A small but statistical significantly reduction in body weight and in the absolute and relative weights of testis, epididymis, ventral prostate glands, seminal vesicle and coagulating glands were reported for the higher exposure (264 mg/kg/d). The effects were more severe after 2 months exposure. Furthermore, the histological structure of the male reproductive tract was adversely affected after 2 month, whereas after 1 month only a reduction in epididymis tubule size was reported. A modest reduction (40%) in testicular sperm heads was reported after 1 month, whereas after 2 months exposure very few sperm heads were present in the testis. The number of sperm heads with abnormal morphology was statistically significant increased after 1 and 2 moth exposure. The number of epididymal sperm heads was statistically significant decreased after 1 month, and almost complete eliminated after 2 month.
No changes in body weight were observed for the lower dose (66 mg/kg/d), but a statistically significant reduction in the absolute weights of the testis, epididymis and seminal vesicle were reported after 2 month. After 1 month only a statistically significant reduction in the absolute weight of the ventral prostate was reported. No reductions in the relative weights were reported. No histological changes were reported in the male reproductive tract. A statistically significant reduction in testicular sperm heads (25%) as well as a statistically significant increase in sperm heads with abnormal morphology was reported after 2 months exposure.
No NOAEL could be deduced. The LOAEL is 66 mg/kg/d.
This study is acceptable for the assessment of testicular toxicity at high doses of octylphenol.
- Endpoint:
- toxicity to reproduction
- Remarks:
- other: Effect of chronic exposure to OP on reproductive hormone secretion in adult male rats
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment
- Principles of method if other than guideline:
- Starting 1-3 days after arrival, rats were either not treated (untreated), injected s.c. with corn oil (vehicle), or injected s.c. with OP or estradiol valerate (EV) in vehicle. Injections were given thrice weekly (on Mondays, Wednesdays, and Fridays) for either 1 or 2 mo. The repeated injections were administered at different sites in the nape or inguinal region. Individual body weights and food consumption of rats per cage were monitored periodically during treatments. One half of the rats in each of the 1-mo and 2-mo treatment groups were decapitated. The other halves were anesthetized, and the epididymides and testes were surgically removed. Rats received no further treatments after their last treatment 1-3 days prior to orchidectomy. A blood sample was obtained by cardiac puncture under diethyl ether anesthesia 24 h after orchidectomy, and these rats were decapitated at 3 wk after orchidectomy.
Body weights were recorded just prior to the first injection and on Days 3, 10, 14, 17, and 24 after injection for the 1-mo groups (rats in the untreated group were inadvertently not weighed on Day 24), and on Days 10, 17, 21, 24, 31, 38, 42, 45, and 52 after injection for -the 2-mo groups. They also were recorded on the days of decapitation or orchidectomy and subsequent decapitation. Food (in pellet form) was weighed to the nearest gram and placed in each cage. The amount of food eaten (with spillage under the cages taken into account) during 3-day periods (66-71 h) was recorded for Days 0-3 and 14-17 for the 1-mo groups and for Days 21-24 and 42-45 for the 2-mo groups.
Data were examined by one-way or two-way analysis of variance. Duncan's Multiple Range test was performed when the analysis of variance indicated a significant difference. A p < 0.05 was considered statistically significant. - GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Route of administration:
- subcutaneous
- Vehicle:
- corn oil
- Details on mating procedure:
- no mating procdure
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- One respectively two month
- Frequency of treatment:
- three times a week (Monday, Wednesday, Friday)
- Remarks:
- Doses / Concentrations:
0, 66 and 264 mg/kg bw/d
Basis:
other: s.c. injection of 0.2 ml corn oil containing 0, 20, 80 mg OP - No. of animals per sex per dose:
- There were 12 rats in each group receiving treatment for 1 mo and 10 rats in each group treated for 2 mo.
Six of the rats in each of the 1-mo treatment groups were injected during a period of 25-28 days ("1 mo"),
and five of the rats in each of the 2-mo treatment groups were injected during a period of 54-61 days ("2 mo") and killed by
decapitation, 1-3 days after the last injection, between 0900 and 1100 h.
An equal number of rats per group (one or two) were used on each day of experimentation. - Dose descriptor:
- LOAEL
- Effect level:
- 66 mg/kg bw/day
- Sex:
- male
- Basis for effect level:
- other: Decrease in body weight after 2 months and decreae in Serum LH and FSH concentrations after 1 and 2 month.
- Reproductive effects observed:
- not specified
- Conclusions:
- The results indicate that chronic administration of p-t-octylphenol to adult male rats can adversely affect the secretion of reproductive hormones.
- Executive summary:
In a chronic administration study 4-tert-octylphenol (98.1 %) was administered to 6 respectively 5 male Fischer 344 rats dosed by three subcutaneous injection per week at dose levels of 0, 20, 80 mg in corn oil (estimated doses approximately 66 and 264 mg/kg bw day)
A statistically significant decrease in body weight was reported after 1 and 2 months exposure with the higher dose (264 mg/kg/d). Serum LH, FSH, and testosterone concentrations were statistically significant deceased after 1 and 2 month, and prolactine concentrations statistically significant increased after 1 and 2 months exposure.
A statistically significant decrease in body weight was reported after 2 months exposure to the lower dose (66 mg/kg/d). Serum LH and FSH, concentrations were statistically significant deceased after 1 and 2 month.
No NOAEL could be deduced. The LOAEL is 66 mg/kg/d.
This study is acceptable for the assessment of the secretion of reproductive hormones in male rats at high doses of octylphenol.
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on generations indicated in Effect levels (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented and acceptable for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- s.c. injection of 100 mg/kg/d 4-tert-octylphenol on each of postnatal days 2-12.
Some of the males from the treatment groups above that were reared until adulthood were assessed for mating and fertility at 80-90 days of age.
Determination of Sertoli cell and germ cell nuclear volume per testis; germ cell apoptotic index, and seminiferous tubule lumen formation on days 18-25.
Cross-sections of testes in each treatment group were examined (nuclei of Sertoli cells, spermatogonia, or spermatocytes (apoptotic or nonapoptotic) or over seminiferous tubule lumens were scored.)
Measurement of plasma hormone levels for FSH and inhibin B. - GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Route of administration:
- subcutaneous
- Vehicle:
- corn oil
- Details on mating procedure:
- For Assessment of some males from the treatment group
- M/F ratio per cage: 1/1
- Length of cohabitation: max 7 days
- Proof of pregnancy: vaginal plug referred to as [day 0 / day 1] of pregnancy
- After 7 days of unsuccessful pairing removement of male and monitoring of female to confirm absence of pregnancy.
- After successful mating each pregnant female was caged (how): separately until birth - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- injection on each of days 2 - 13
day 1 = day of birth - Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
0 and 2 mg
Basis:
actual ingested
doses approx. 100 mg/kg bw - No. of animals per sex per dose:
- 7 - 14 male animals per group.
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 100 mg/kg bw/day
- Sex:
- male
- Basis for effect level:
- other: no effect on pubertal spermatogenesis
- Reproductive effects observed:
- not specified
- Conclusions:
- Neonatal exposure of male rats to 100 mg octylphenol/kg bw did not affect pubertal spermatogenesis, long-term changes in testis size, mating or ferility.
- Executive summary:
Effect of neonatal exposure of male rats to 2 mg OP/rat/d (s.c.; 2-12 pnd; equals 100 mg/kg bw) on pubertal spermatogenesis (pnd 18 and 25), and long-term changes in testis size, mating or ferility (90 –100 days of age) were assessed. On postnatal day 18 a significant advanced seminiferous tubule lumen formation was reported as well as a slightly decreased apoptotic rate of germ cells. The testis weight was statistical significant increased. By day 25 the stimulatory effects on spermatogenesis seen on day 18 were no longer evident. In adult rats no changes in testis weight was reported and mating and fertility were reasonably normal.
It was concluded that s.c. injection of 100 mg/kg/d has no adverse significant effect under conditions of this study
This study is acceptable for the assessment of pubertal spermatogenesis in rat.
Referenceopen allclose all
The examined epididymal sperm showed an increased number of tail abnormalities that would interfere with sperm motility. At the highest dose a decreased epididymal sperm number was observed.
Gonadotropin secretion was monitored at 2-h intervals around the time of the expected prevulatory surge during the study. A FSH surge was only observed in one animal in day 70 of gestation to birth group, and this group was omitted from the analysis. No significant differences were observed between groups in the amplitude of the LH surge, but when the FSH concentrations were aligned relative to the peak LH preovulatory surge, FSH was elevated for longer in control than OP-treated animals. On the other side, mean FSH concentration from the blood samples taken prior and during the first breeding season and the mean FSH concentration across the study cycle were not affected by OP. Waves of ovarian follicle growth did not show any difference among the groups in the proportion of ewes having two or three follicle waves per cycles or in the interval between waves. Further treatment-unaffected parameters were the mean number of identified follicles per wave, the mean diameter of the ovulatory follicle, and the diameter of the largest follicle. There were also no difference among the groups detected in the number of small, medium, or large follicles, or combined numbers of follicles.
SEXUAL MATURATION (OFFSPRING)
The mean age of acquisition of vaginal patency in F1 females ranged from 30.5 to 31.8 days, with the mean body weight at acquisition ranging from 97.83 to 91.91 g. The mean age of acquisition of preputial separation in F1 males ranged from 43.1 to 44.7 days, with the mean body weight at acquisition ranging from 220.07 to 207.01 g. When the age at acquisition was analyzed statistically by analysis of variance (ANOVA) and Dunnett's test for pairwise comparisons, age at acquisition of vaginal patency was significantly delayed at 20 ppm (31.9 days) and 2000 ppm (31.8 days) relative to the control value (30.5 days), and the age at acquisition of preputial separation was significantly delayed at 2000 ppm (44.7 days) relative to the control group value (43.1 days).
F1 female body weight at acquisition exhibited a significant dose-related downward trend (P , 0.05) with the mean weight at 200 ppm, 206.09 g (but
not at 2000 ppm, 207.01 g) signi®cantly reduced relative to the control value, 220.07 g.
There were no differences among the control and treated groups receiving 50/mg/kg or less from PND 0-21 but in the 100 mg/kg group, viability on PND 6 and 21 was significantly decreased compared to controls. From PND 1-6, 2 females died in the 50 mg/kg group and six males and five females in the 100 mg/kg group. No other pub died during this period and no pub died after weaning.
CLINICAL SIGNS (OFFSPRING)
BODY WEIGHT (OFFSPRING)
- In male animals in the 50 and 100 mg/kg were significantly lower than those in the control groups throughout the study after the administration period.
- Males of the 25 mg/kg group had significantly lower body weight than the controls at 7 and 9 weeks of age.
- After administration, body weights were decreased in a dose-dependent manner at each time point examined.
- Females in the 50 and 100 mg/kg dose groups had significantly lower body weight than the controls throughout the study after administration period.
- Compared to the controls, body weight was also decreased in females in the 25 mg/kg group at 7 and 9 weeks of age and in the 12.5 mg/kg group at 9 weeks of age.
- At the times of preputial separation in males and vaginal opening in females, body weight was significantly decreased compared to the control group
SEXUAL MATURATION (OFFSPRING)
Times of preputial seperation in males and vaginal opening in females of the 50 and 100 mg/kg groups were significantly delayed compared to the control group.
ESTROUS CYCLE EVALUATION (OFFSPRING)
No significant differences among the treated groups and the controls.
REPRODUCTIVE PERFORMANCE (OFFSPRING)
- Copulation and fertility indices in males and females of all groups did not differ significantly from those of the controls.
- Number of males that failed to copulate or impregnate untreated females: two in 12.5, 25 and 50 mg/kg groups, 3 in 100 mg/kg group; animals copulated and impregnated successfully when remated.
- Numbers of females that did not copulate in the first mating: 1 in the 100 mg/kg group. She did not copulate during the second mating, either.
- Time until copulation didn't differ in treated and untreated males and females.
- Implants/litter in untreated females that mated with treated males did not differ from controls. Implants/litter and numbers of dead embryos in treated females that mated with untreated males did not significantly differ from the control group.
SPERM COUNTS, SERUM TESTOSTERONE LEVELS AND REPRODUCTIVE ORGAN WEIGHTS (OFFSPRING)
- Epididymal sperm and serum testosterone concentrations did not show significant differences among treated and control groups.
- In the 100 mg/kg group: severe atrophy of both testes and epididymides was found in one male; no macroscopic alterations in other males or females in any group
- Terminal body weights, in the 25, 50, and 100 mg/kg groups, absolute epididymides weight in the 100 mg/kg group, and ventral prostate weights in all treated groups were significantly lower than those in the controls
- Absolute weights of paired testes and seminal vesicles with coagulating glands were comparable among all groups.
- Relative weights of the testes in the 100 mg/kg group and those of the seminal vesicle in the 50 and 100 mg/kg groups were significantly increased.
- Relative weights of the ventral prostate in all treated groups were significantly decreased in all animals compared to the control groups.
HISTOPATHOLOGY (OFFSPRING)
Pubs necropsied on PND 21:
- No pathologic changes in reproductive organs of pubs necropsied on PND 21
- No alterations of testes or epididymides in any groups.
- In 2/5 females in the 25, 50, and 100 mg/kg groups, polyovular follicles with two oocytes were found.
- Relative testes and epididymides weights in treated groups were similar to those in the control groups.
18-week-old male rats:
- No obvious abnormalities were found in the testes, epididymides, seminal vesicles or ventral prostate in any treated group.
- Among treated females that did not copulate or were not pregnant, hyperplasia of luminal epithelial cells and infolding of uterine epithelium in an infertile female in the 50 mg/kg group was found.
- No changes were determined in ovaries or uteri or infertile females in the 12.5, 25, or 100 mg/kg groups.
Luteinizing hormone (LH) was somewhat low in dams.
Testosterone was decreased in sires.
There were no differences among the control and treated groups receiving 50/mg/kg or less from PND 0-21 but in the 100 mg/kg group, viability on PND 6 and 21 was significantly decreased compared to controls. From PND 1-6, 2 females died in the 50 mg/kg group and six males and five females in the 100 mg/kg group. No other pub died during this period and no pub died after weaning.
CLINICAL SIGNS (OFFSPRING)
BODY WEIGHT (OFFSPRING)
- In male animals in the 50 and 100 mg/kg were significantly lower than those in the control groups throughout the study after the administration period.
- Males of the 25 mg/kg group had significantly lower body weight than the controls at 7 and 9 weeks of age.
- After administration, body weights were decreased in a dose-dependent manner at each time point examined.
- Females in the 50 and 100 mg/kg dose groups had significantly lower body weight than the controls throughout the study after administration period.
- Compared to the controls, body weight was also decreased in females in the 25 mg/kg group at 7 and 9 weeks of age and in the 12.5 mg/kg group at 9 weeks of age.
- At the times of preputial separation in males and vaginal opening in females, body weight was significantly decreased compared to the control group
SEXUAL MATURATION (OFFSPRING)
Times of preputial seperation in males and vaginal opening in females of the 50 and 100 mg/kg groups were significantly delayed compared to the control group.
ESTROUS CYCLE EVALUATION (OFFSPRING)
No significant differences among the treated groups and the controls.
REPRODUCTIVE PERFORMANCE (OFFSPRING)
- Copulation and fertility indices in males and females of all groups did not differ significantly from those of the controls.
- Number of males that failed to copulate or impregnate untreated females: two in 12.5, 25 and 50 mg/kg groups, 3 in 100 mg/kg group; animals copulated and impregnated successfully when remated.
- Numbers of females that did not copulate in the first mating: 1 in the 100 mg/kg group. She did not copulate during the second mating, either.
- Time until copulation didn't differ in treated and untreated males and females.
- Implants/litter in untreated females that mated with treated males did not differ from controls. Implants/litter and numbers of dead embryos in treated females that mated with untreated males did not significantly differ from the control group.
SPERM COUNTS, SERUM TESTOSTERONE LEVELS AND REPRODUCTIVE ORGAN WEIGHTS (OFFSPRING)
- Epididymal sperm and serum testosterone concentrations did not show significant differences among treated and control groups.
- In the 100 mg/kg group: severe atrophy of both testes and epididymides was found in one male; no macroscopic alterations in other males or females in any group
- Terminal body weights, in the 25, 50, and 100 mg/kg groups, absolute epididymides weight in the 100 mg/kg group, and ventral prostate weights in all treated groups were significantly lower than those in the controls
- Absolute weights of paired testes and seminal vesicles with coagulating glands were comparable among all groups.
- Relative weights of the testes in the 100 mg/kg group and those of the seminal vesicle in the 50 and 100 mg/kg groups were significantly increased.
- Relative weights of the ventral prostate in all treated groups were significantly decreased in all animals compared to the control groups.
HISTOPATHOLOGY (OFFSPRING)
Pubs necropsied on PND 21:
- No pathologic changes in reproductive organs of pubs necropsied on PND 21
- No alterations of testes or epididymides in any groups.
- In 2/5 females in the 25, 50, and 100 mg/kg groups, polyovular follicles with two oocytes were found.
- Relative testes and epididymides weights in treated groups were similar to those in the control groups.
18-week-old male rats:
- No obvious abnormalities were found in the testes, epididymides, seminal vesicles or ventral prostate in any treated group.
- Among treated females that did not copulate or were not pregnant, hyperplasia of luminal epithelial cells and infolding of uterine epithelium in an infertile female in the 50 mg/kg group was found.
- No changes were determined in ovaries or uteri or infertile females in the 12.5, 25, or 100 mg/kg groups.
500 mg/kg/day: a total of 9 male and females were sacrificed or found dead during the treatment, following adverse clinical signs. Clinical signs amongst all animals in the group included post dosing salivation, wet coats, untidy/matted fur, brown stained urogenital region and, under all cages, looses faeces, additionally for mortalities, signs including hunched posture, emaciation, lethargy and abnormal gait were noted.
250 mg/kg/day: Two females were sacrificed around birth - it is uncertain if these were treatment-related. Signs of reactions to treatment included post dosing salivation, wet coats, and, under all cages, loose faeces.
125 mg/kg/day: post dose salivation.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
500 mg/kg/day: Mean bodyweight gain in all animals was reduced and lower weight gain of females was apparent at the end of pregnancy and early lactation. Food consumption was reduced.
250 mg/kg/day: Bodyweight gain was reduced and for females, bodyweight gain was also affected at the end of pregnancy/early lactation.
125 mg/kg/day: no effects
WATER CONSUMPTION (PARENTERAL ANIMALS)
500 mg/kg/day: water consumption was markedly increased.
250 mg/kg/day: water consumption was increased.
125 mg/kg/day: slightly elevated water consumption.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
500 mg/kg/day: Mating performance of the animals following two weeks of treatment was impaired with only 4 of 8 paired females conceiving. However, in view of the marked toxicity at this dosage it is uncertain if this is a direct treatment-related effect or is a consequence of the general poor condition. Libido appears unaffected for males but their fertility was lower. Among females that did conceived, mating performance was generally unaffected although the duration of the pregnancy was longer than expected.
The implantation rate was lower than the controls which, together with increased pre and post natal mortality resulted in lower litter sizes.
250 mg/kg/day: no effects
125 mg/kg/day: no effects
ORGAN WEIGHTS (PARENTAL ANIMALS)
500 mg/kg/day: organ weigh analysis in week 7 revealed increased liver and kidney weights and, more predominantly for males, increased adrenal weights. Weights of specific reproductive organs (testes, epididymides, combined prostate/seminal vesicles/coagulating gland, and ovaries) were reduced, as was thymus weight.
250 mg/kg/day: higher liver weights and slightly elevated kidney weights
125 mg/kg/day: no effects
GROSS PATHOLOGY (PARENTAL ANIMALS)
500 mg/kg/day: Macropathological changes included stained fur, alopecia, raised/thickened areas of forestomach (in females), pale foci and in females, uniform cortical scarring of kidneys and enlarged renal lymph nodes.
250 mg/kg/day: macroscopic examination revealed distended caecum (of males) reduced thymus size (in males), enlarged adrenal size (in females) and uniform cortical scarring of kidneys.
125 mg/kg/day: no effects
HISTOPATHOLOGY (PARENTAL ANIMALS)
500 mg/kg/day: microscopical examination was confined to the reproductive tract and revealed minor changes in the testes and epididymides (trace exfoliation of round spermatids into the seminiferous tubular lumen with minimal numbers of abnormal spermatogenic cells in the caput epididymides and/or trace vacuolation of the seminiferous epithelium.
250 mg/kg/day: microscopic examination was performed on testes and epididymides of males (following the effects at 500 mg/kg/day) and revealed no abnormalities.
125 mg/kg/day: no effects
OTHER FINDINGS (PARENTAL ANIMALS):
500 mg/kg/day: in week 5, haematological investigations revealed increased levels of plasma urea nitrogen, creatinine, bilirubin and glyutamic pyruvate transaminase (GPT) and lower levels of certain electrolytes and circulating albumin.
250 mg/kg/day: there were no obvious haematological or biochemical changes.
125 mg/kg/day: no effects
250 mg/kg/day: there were no effects of treatment on development of the litter.
125 mg/kg/day: here were no effects of treatment on development of the litter.
500 mg/kg/day
A total of 9 male and 4 females were sacrificed or found dead during the treatment period, following adverse clinical signs and bodyweight loss. Clinical signs amongst all animals in the group included post dosing salivation, wet coats, untidy matted fur, brown stained urogenital region and, under all cages, loose faeces, additionally for mortalities, signs including hunched posture, emaciation, lethargy and abnormal gait were noted. Mean bodyweight gain of all animals was reduced and lower weight gain of females was apparent at the end of pregnancy and early lactation. Food consumption was reduced and water consumption was markedly increased. In Week 5, haematological investigations revealed higher numbers of white blood cells and platelets and biochemical investigations revealed increased levels of plasma urea nitrogen, creatinine, bilirubin and glutamic pyruvate transaminase (GPT) and lower levels of certain electrolytes and circulating albumin.
Organ weight analysis in Week 7 revealed increased liver and kidney weights and, more predominantly for males, increased adrenal weights. Weights of specific reproductive organs (testes, epididymides, combined prostatelserninal vesicles/coagulating gland, and ovaries) were reduced, as was thymus weight. Macropathological changes included stained fur, alopecia, raised/thickened areas of forestomach (in females), pale foci and in females, uniform cortical scarring of kidneys and enlarged renal lymph nodes. Microscopic examination was confined to the reproductive tract and revealed minor changes in the testes and epididymides (trace exfoliation of round spematids into the seminiferous tubular lumen with minimal numbers of abnormal spermatogenic cells in the caput epididymis andlor trace vacuolation of the seminiferous epithelium.
Mating performance of the animals following two weeks of treatment was impaired with only4of the8paired females conceiving. However, in view of the marked toxicity at this dosage it is uncertain if this is a direct treatment-related effect or is a consequence of the general poor condition. Libido appeared unaffected for males but their fertility was lower. Amongst females that did conceive, mating performance was generally unaffected although the duration of pregnancy was longer than expected. The implantation rate was lower than the controls which, together with increased pre and post natal mortality resulted in lower litter sizes. Litter weight was reduced and, there was a suggestion of impaired pup growth to Day4.No gross abnormalities were noted amongst the offspring.
250 mg/kg/day
Two females were sacrificed around birth-it is uncertain if these were treatment related. Signs of reactions to treatment included post dosing salivation, wet coats, and, under all cages, loose faeces. Bodyweight gain was reduced and for females, bodyweight gain was also affected at the end of pregnancy early lactation. Water consumption was increased. There were no obvious haematological or biochemical changes but organ weight analysis revealed higher liver weights and slightly elevated kidney weights. Macroscopic examination revealed distended caecum (of males) reduced thymus size (in males), enlarged adrenal size (in females) and uniform cortical scarring of kidneys. Microscopic examination was performed on testes and epididymides of males (following the effects at 500 mg/kg bw/day) and revealed no abnormalities.
There were no effects of treatment on mating performance or development of the litter
125 mg/kg/day
The only changes noted were post dose salivation and slightly elevated water consumption.
No effects of treatment were noted on mating performance or development of the litter.
Results 80 mg p-t-octylphenol, 1 and 2 month:
A statistically significant decrease in body weight was reported after 1 and 2 months exposure. Serum LH, FSH, and testosterone concentrations were statistically significant deceased after 1 and 2 month, and prolactine concentrations statistically significant increased after 1 and 2 months exposure.
Results 20 mg p-t octylphenol 1 and 2 month:
A statistically significant decrease in body weight was reported after 2 months exposure. Serum LH and FSH, concentrations were statistically significant deceased after 1 and 2 month.
On postnatal day 18 a significant advanced seminiferous tubule lumen formation was reported as well as a slightly decreased apoptotic rate of germ cells.
The testis weight was statistical significant increased.
As adults no changes in testis weight was reported and mating and fertility were reasonably normal.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 150 mg/kg bw/day
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
In a reproduction/developmental toxicity screening study according OECD 421 Octylphenol (98,7%) was administered to 12 SD rats/sex/dose by gavage at dose levels of 0, 125, 250, 500 mg/kg bw/day (HRC, 1995). Treatment at 500 mg/kg/day produced a marked toxic effect resulting in the death of 13/24 adult animals during the treatment period. Microscopic examination was confined to the reproductive tract and revealed minor changes in the testes and epididymides (trace exfoliation of round spematids into the seminiferous tubular lumen with minimal numbers of abnormal spermatogenic cells in the caput epididymis and/or trace vacuolation of the seminiferous epithelium). Mating performance was impaired with only 4 of 8 paired females conceiving. However, in view of the marked toxicity at this dosage it is uncertain if this is a direct treatment-related effect or is a consequence of the general poor condition. Libido appeared unaffected for males but their fertility was lower. Amongst females that did conceive, mating performance was generally unaffected although the duration of pregnancy was longer than expected. The implantation rate was lower than the controls which, together with increased pre and post natal mortality resulted in lower litter sizes. Litter weight was reduced and there was a suggestion of impaired pup growth. No gross abnormalities were noted amongst the offspring.
There was a clear, through less marked, treatment-related effect at 250 mg/kg/day. There were no obvious haematological or biochemical changes but higher liver weights and slightly elevated kidney weights. Macroscopic examination revealed distended caecum (of males) reduced thymus size (in males), enlarged adrenal size (in females) and uniform cortical scarring of kidneys. Microscopic examination was performed on testes and epididymides of males (following the effects at 500 mg/kg/day) and revealed no abnormalities. Reproductive performance and development of the conceptus was unaffected at this concentration.
The systemic NOAEL was 125 mg/kg bw/day. The NOAEL for reproduction was 250 mg/kg/d based on a lack of effects on mating performance or development of the litter.
A second OECD 421 screening test was performed as limit test (RIVM 1998). Due to the high mortality the intended limit dose of 1000 mg/kg bw/day could not be used. Thus a limit dose of 100 mg/kg bw/day was administered by gavage to 4 Wistar rats/sex from day 12 prior to mating until litter reached day 6 post partum. Two out of 4 females became pregnant. The only reproductive effect observed after exposure was a questionable increase in corpora lutea, suggesting possibly premature luteinization. Histological analysis showed no abnormalities in both sexes. From this screening study no NOAEL could be derived but the result does not contradict the second OECD 421 study.
In an extended two-generation reproduction study according to EPA OPPTS 870.3800 Octylphenol (90,2 %) was feed to 30 SD rats/sex/dose at dose levels of 0, 0.2, 20, 200, 2000 ppm (0, 0.015, 1.5, 15, 150 mg/kg/day)(Tyl 1999). Effects related to OP administration in adult animals were limited to minimal changes in body weight and feed consumption in the high dose group. There were no treatment-related effects in reproductive measurements, reproductive organs, or extensive evaluations of sperm measurements in males (F0, F1, F2). Effects in offspring occurred only at the high dose and were limited to reduced pup body weights and several minor, transient organ weight differences in weanling animals. The reduced pup body weights occurred only during the latter portion of the lactation period when the pups were self-feeding and, therefore, were being directly exposed to high daily doses of OP in the diet. Since organ weight differences were not observed in adult animals from the same generation and from the same litters, and there were no correlated functional or histological alterations, these changes were considered to be of minimal or no toxicological relevance.
Slight increases in the time to acquisition of developmental landmarks (vaginal opening and preputial separation) were observed in offspring from the high dose group. The delays were relatively minor, both less than 2 days, and were considered related to the lower body weight.
In addition, differences in anogenital distance were considered to assess the relevance of positive in vitro estrogen-receptor binding studies in vivo. If OP acts through an estrogen receptor-mediated mechanism, reduced anogenital distance in the F2 males and no effect in F2 females might be expected. In Tyl (1999) the anogenital distance differs by 0.03 to 0.09 mm compared to controls. This is not considered to be of biological significance, based on the following: (1) the magnitude of the differences is minimal, (2) there were no correlating effects of any kind, and (3) there is no known biologically plausible mechanism for OP to result in an increase in anogenital distance in females, since anogenital distance is under androgenic control and Octylphenol is negative as androgen or antiandrogen in the HepG2 androgen receptor assay in vitro. A study by Beigel et al. (1998) describe the effects from dietary estrogenic exposure (17ß-estradiol) during in utero and lactational development of the reproductive systems in the rat. These effects included precocious vaginal opening at 0.05 ppm, up to 9 days at 2.5 ppm, delayed preputial separation up to 8 days, alterations in estrous cycles, changes in reproductive organs at 2.5 ppm and, ultimately at higher doses, complete infertility. None of these changes were observed in either F1 or F2 generations of animals exposed to OP at dietary concentrations from 0.2 to 2000 ppm. Octylphenol is approximately 1000-fold less potent than 17b-estradiol. Sch effects might have been expected at concentrations as low as 50 ppm (equivalent to 0.05 ppm of 17b-estradiol). Thus, it is concluded that OP in the diet shows no estrogenic activity in the rat at a dose range over four orders of magnitude, from 0.2 ppm to a maximum tolerated dose (2000 ppm).
The NOAELs for systemic and postnatal toxicity were 200 ppm and at or above 2000 ppm for reproductive toxicity.
Several other publications are available using s.c.injection as route of exposure. This is considered to be inappropriate for human risk assessment. Atanassova (2000) assessed the effect of neonatal exposure of male rats to 100 mg/kg bw/d (s.c.) on pubertal spermatogenesis, long-term changes in testis size, mating and ferility. Under conditions of this study no adverse effect could be concluded.
Wright (2002) administered octylphenol at 1 mg/kg/d (s.c.) to 19 Suffolk cross ewes day 70 of gestation to weaning. The offspring were examined for effects of maternal exposure on the onset of puberty, endocrine status, and subsequent ovarian follicular dynamics. The onset of puberty and the first progesterone rise was advanced and the FSH preovulatory surge was elevated for longer in the OP-treated lambs compared to the controls but did not disrupt the inter-estrous interval or endocrine profiles throughout the first breeding season or the pattern of ovarian follicular dynamics.
Two additional studies using the same route of exposure concluded adverse effects on testicular toxicity and secretion of reproductive hormones. Bookfor and Blake (1997) concluded testicular toxicity at 66 mg/kg/d, based on a reduction in the absolute weights of the testis, epididymis and seminal vesicle (after 2 months) and a reduction in testicular sperm heads (25%) as well as a increase in sperm heads with abnormal morphology. Blake and Bookfor (1997) concluded an adverse effect on reproductive hormones at the same dose. However, s.c. injection is considered an inappropriate route and the relevance is unclear. The adverse findings were not confirmed in multi-generation OECD/GLP studies.
Short description of key information:
Two screening tests according to OECD 421 are available. HRC (1995) applied 4 -tert-octylphenol (98.7%) by gavage to 12 SD rats/sex/dose at 0, 125, 250, 500 mg/kg bw/day (SAZ 462/942750). A NOAEL of 125 mg/kg/d was concluded for systemic toxicity and a NOAEL of 250 mg/kg/d for reproductive performance and development of the conceptus.
Piersma (1998) conducted a Limit test with 100 mg/kg bw/day. The only reproductive effect was a questionable increase in corpora lutea (premature luteinization). No histological abnormalities were found (RIVM Report 650030 002). Tyl (1999) conducted a 2-generation feeding study according to EPA OPPTS 870.3800. 30 SD rats were exposed to 0, 0.2, 20, 200, and 2000 ppm OCT (90.2%) in the diet. A NOAEL of 200 ppm ( 15 mg/kg/day) was concluded for systemic toxicity, based on decreased body weight and weight gain in adults at 2000 ppm. A NOAEL of 2000 ppm ( 150 mg/kg/day) was concluded for reproductive toxicity because no effects were observed on fertility, pregnancy, gestation index, number of implants, litter size, postimplantation loss, necropsy of weanlings, etc. A delay in vaginal opening and preputial separation at 2000 ppm was considered to be related to decrease in body weight. Additional information is available from several publications that is either not reliable or does not contradict the findings of the OCED GLP studies.
Effects on developmental toxicity
Description of key information
Harazono and Ema (2001) conducted a teratogenicity study in 16 female rats/dose (gestation day 0-8) at 0, 15.6, 31.3, 62.5, 125, 250 or 500 mg/kg/d. A full OECD 414 study is not available for 4-tert-octylphenol but due to the structural similarity between octylpheol and nonylphenol the read across from a well conducted OECD study with nonylpheol is applicable. IBR Forschungs GmbH (1992) conducted such an OECD 414 study (GLP) administering p-Nonylphenol (93,2%) to 25 female Wistar rats/dose at day 6-15 of gestation by gavage at doses of 0, 75, 150, 300 mg/kg/d.
NOAEL (foetal): >=300 mg/kg/d
NOAEL (maternal): 75 mg/kg/d
LOAEL (maternal): 150 mg/kg/day, based on macroscopic changes in the kidney and spleen
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 300 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
The OECD guideline 414 is designed to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism. This may include “assessment of maternal effects as well as death, structural abnormalities, or altered growth in the foetus. The guideline is not intended to examine solely the period of organogenesis, (e.g. days 5-15 in rats) but also effects from pre-implantation, when appropriate, through the entire period of gestation to the day before caesarean section. To assess teratogenicity fetuses are evaluated for soft tissue and skeletal changes. Part of the prenatal developmental toxicity is covered by a well conducted two-generation reproduction study (Tyl 1999). No treatment or dose-related effects have been recorded on parameters connected to prenatal development like number or implants, live or dead pubs per litter, post implantation losses, uterine content or necropsy of pups. In addition the authors conclude that PTOP in diet of rats has no estrogen activity, leading to a NOAEL for reproductive toxicity of 2000 ppm (ca 150 mg/kg/d). This study does not cover all part of prenatal development toxicity as the assessment of skeletal and soft tissue malformation in the offspring is missing.
In a non-guideline developmental toxicity study 4-tert-octylphenol was administered to 16 female Wistar rats per dose by gavage at dose levels of 0, 15.6, 31.3, 62.5, 125, 250 or 500 mg/kg bw/day from days 0 through 8 of gestation (Harazono and Ema (2001)). Death occurred in the high dose groups (7/7 at 500 mg/kg/d; 2/6 at 250 mg/kg/d). Pronounced clinical signs were observed at 125 mg/kg/d. Significant decreases in body weight gain on days 0 -9 were observed at 31.3 mg/kg and above and decreased food consumption at 15.6 mg/kg and above, respectively (maternal toxicity). A significant increase in the relative (but not absolute) incidence of post-implantation loss per litter was observed at 31.3 mg/kg/d and above. No increase in the incidence of fetuses with external malformations or malformations within the oral cavity was found in any OP-treated group. The NOAEL for teratological effects is above 125 mg/kg/d under conditions of this study. A NOAEL for maternal toxicity could not be established if the decrease in food consumption t the lowest dose is considered an sufficient severe adverse effect. Due to the restricted examination parameters (only external malformation of fetuses were considered) the study does not satisfy the requirements for a teratogenicity study according to OECD 414.
A full OECD 414 study is not available for 4-tert-octylphenol but due to the structural similarity between octylpheol and nonylphenol read across from a well conducted OECD study with nonylphenol is applicable. IBR Forschungs GmbH (1992) conducted such a study under GLP administering p-Nonylphenol (93,2%) to 25 female Wistar rats/dose at day 6-15 of gestation by gavage at doses of 0, 75, 150, 300 mg/kg/d. A further group dosed with 600 mg/k/d was terminated prematurely because of high mortality during the first few days of treatment. Clear indications for maternal toxicity (increased mortality, reduced body weight gain and food intake, macroscopic changes in kidney and spleen) were observed at 300 mg/kg/d. At 150 mg/kg/d 3 of 21 females showed affected kidneys or spleens. No maternal toxicity was observed at 75 mg/kg/d. Post-implantation loss, litter size, fetal weights and incidence of both major and minor fetal abnormalities (visceral and skeletal) were not observed. The study provides no evidence of developmental toxicity in the rat at exposure levels which show clear maternal toxic; thus the maternal NOAEL was concluded to be 75 mg/kg/day and the fetal NOAEL was 300 mg/kg/day.
According to the results of a multigeneration study, a non-guideline teratogenicity study as well as read-across data from a well conducted OECD 414 study using Nonylphenol, there is no indication of teratogenic effects of 4-tert-octylphenol in rat.
Justification for classification or non-classification
According to the results of a multigeneration study, a non-guideline teratogenicity study as well as read-across data from a well conducted OECD 414 study using Nonylphenol, there is no indication of teratogenic effects of 4-tert-octylphenol in rat.
Additional information
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