4-(1,1,3,3-tetramethylbutyl)phenol

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4-(1,1,3,3-tetramethylbutyl)phenol

EC Number:

205-426-2

EC Name:

4-(1,1,3,3-tetramethylbutyl)phenol

Cas Number:

140-66-9

Molecular formula:

C14H22O

IUPAC Name:

4-(2,4,4-trimethylpentan-2-yl)phenol

Constituent 2

Reference substance name:

Octylphenol

EC Number:

266-717-8

EC Name:

Octylphenol

Cas Number:

67554-50-1

Molecular formula:

C14H22O

IUPAC Name:

2-octylphenol

Details on test material:

- Name of test material (as cited in study report): octylphenol (OP)
- Substance type: pure substance
- Analytical purity: approximately 90,2 %
- Impurities (identity and concentrations): other components primarily being other para substituted OPs
- Identity and purity of the test material were established by spectroscopy, GC-MS, and capillary GC prior to the study, .
- Source: Schenectady International Inc. (Schenectady, NY)
- Composition of test material, percentage of components:
- Stability under test conditions: stability was monitored periodically during the study and at study termination. Analyses showed that the OP in the diet was mixed homogeneously, was stable frozen for at least 54 days and for at least 9 days under cageside conditions

Test animals

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)

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

see attached table

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

200 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

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)

3

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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

see attached table

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.

Effect levels (F1)

1

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"

2

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)

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.