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Key value for chemical safety assessment

Effects on fertility

Description of key information

The effects of nonylphenol on fertility and reproductive performance have been investigated in three multi-generation studies. Additional data is available on testicular toxicity.

In a well conducted extended 2-generation study (Chapin 1999) 30 SD rats/sex/dose were exposed to nonylphenol (NP) at dietary concentrations of 0, 200, 650 or 2000 ppm. General toxicity was evident in adults of all generations, seen as a reduction in bodyweight gain at 650 and 2000 ppm and histopathological changes in the kidneys at all dose levels. Consequently a NOAEL could not be derived and a LOAEL for systemic toxicity of 200 ppm (15 mg/kg/d) was suggested for risk assessment in the EU risk assessment report 2002.

Considering the reproduction-related parameters, there were no adverse effects on fertility or mating performance. However, there were slight changes, in the oestrous cycle length at 2000 ppm (F1/F2), timing of vaginal opening at 650 and 2000 ppm (F1,F2,F3) and ovarian weight at 650 (F2) and 2000 ppm (F1, F2, F3). The earlier onset of puberty in females is thought to be due to the estrogenic activity of NP but it must not necessarily be interpreted as an adverse effect per se. Changes in sperm endpoints were seen only in the F2 males; epididymal sperm density was decreased at 650 and 2000 ppm and spermatid count at 2000 ppm. This effect is questionable because the sperm density in all F2 groups, including controls, was considerably greater (by about 25-40%) than reported for F0 and F1 males. Observation of impaired male reproductive tract development in a gavage study (de Jager 1999) suggests additional evidence in favour of the sperm/spermatid count changes related to NP treatment. In this study a detailed evaluation of the male reproductive organs was conducted. Clinical signs of toxicity were not reported. But mortality was observed in 3, 17 and 18 animals from the 100, 250 and 400 mg/kg/day groups. Tubule and lumen diameter and seminiferous epithelial diameter were lower in all dose groups. In addition testicular and epididymal weights were lower at 250 and 400 mg/kg/day and the sperm count was reduced at 400 mg/kg/day. A LOAEL for testicular toxicity of 100 mg/kg/day can be designated (exposure levels which also cause mortality). However, because of the very small group sizes due to mortality, little toxicological significance can be accorded to these findings. The observation of mortality at 100, 250 and 400 mg/kg/day in this gavage study contrasts with the findings of studies involving dietary administration summarised in the Repeated Dose Toxicity section (Hazleton UK, 1989; Cunny 1997; Chapin, 1999). This can be explained by the toxicokinetic of NP. Higher peak concentration can saturate the metabolic capacity of the liver and GI tract (first pass effect) resulting in a decreased detoxification and consequently a higher internal dose. Supporting evidence for testicular toxicity of NP was also provided by El-Dakdoky (2007). The purpose of this study was to investigate the effects of NP on sperm characteristics, fertility index, histopathological and biochemical changes related to oxidative stress in testes. The conclusion was that the exposure of 10 adult male mice to high dose of NP (1/4 LD50) for 35 days had effects on some reproductive organs weight and sperm characteristics (count and motility), testicular MDA, GSH, and SOD but did not influence the mating behavior, male fertility and the developed fetuses. An additional study on testicular toxicity conducted by Lee (1998) was disregarded due to serious shortcomings concerning the study design and an inappropriate route of exposure.

Two multi-generation studies were conducted after completion of the EU RAR 2002: Nagao (2001) and Tyl (2006). In a 2 -generation study according OECD 416 (Nagao, 2001) NP was administered to 25 SD rats/sex/dose at 0, 2, 10, and 50 mg/kg/d by gavage. No adverse changes in clinical signs were observed throughout the study. Significant increases in the liver, kidney and pituitary gland weights in males, and decreases in thymus weight in males and ovary weight in females were observed in the 50 mg/kg group. At necropsy, no treatment-related alterations were observed in any organs including the reproductive tissues in any group. Histopathologic changes were observed in the liver of males and females and kidney of males in F0/F1 animals at 50 mg/kg/d. F1- and F2-offspring of this dose group showed a reduced viability on pnd 0 to 4; the body weight gain of these animals remained unaffected. No effects on preputial separation were observed in males while vaginal opening was accelerated in the female high dose group. No adverse changes in behaviour or learning of the offspring were observed. There were no treatment related changes seen in any reproductive parameter including estrous cycle, mating, fertility, delivery, and lactation except for significant decreases in the numbers of implantation sites in F1 females and in the numbers of F2 pups born alive and a significant decrease in absolute and relative ovary weight in adult F1 females at the high dose group. No treatment related changes were observed in the sperm characteristics. The present data show that exposure to NP for two generations provided indications of estrogen activity in females of the first (F1) generation (acceleration of vaginal opening) and altered kidney and liver structure in rats of the parental (F0) and F1 generations, and an absence of reproductive changes in rats of the F0 and F1 generations. These results confirmed those reported in the Chapin’s study (1999). A NOAEL for reproductive effects of 50 mg/kg/day or greater in parent animals, and 10 mg/kg/day in the next generation can be concluded. The NOAEL for general toxicity is 10 mg/kg/d based on organ weight changes and Histopathologic findings in the liver and kidney.

 

The most recent multi-generation (3.5 generations) study was conducted by Tyl (2006) according OPPTS 837.3800 (GLP). NP (CAS-Number 84852-15-3, 94.25%) was administered to 25 SD rats/sex/dose in two different diets at doses of ~ 0, 1.5, 15, 45, 145 mg/kg/d (Purina 5002, F0, F1, F2, F3) and 0; 45 mg/kg/d (NIH-07; F0,F1,F2). In addition, 17ß estradiol was administered at a concentration of 2.5 ppm (0.1-0.2 mg/kg/day) as positive control for any estrogenic effects.

This study evaluated the potential for dietary administration of NP to affect parental fertility and growth and development of three offspring generations in SD rats, including sperm counts across generations. The latter was included to determine the validity of equivocal reductions observed in the F2 generation by Chapin (1999). In addition the study investigated ambiguous findings of two older studies with regards to specific target organ toxicity (Chapin, 1999 and Cunny, 1997). Details are summarized in IUCLID section 7.5.

With regards to reproductive parameters, there were no treatment-related effects which is in accordance with the conclusions of Chapin (1999) and Nagao (2001). Nagao (2001) reported that NP did not affect sperm parameters or estrous cyclicity at any dose. At 50 mg/kg/day ovarian weights were decreased and a significant decrease in the number of implantation sites and live pups per litter at birth was observed, with reduced survival at this dose on pnd 0-4. NP did not affect acquisition of preputial separation in males, but did accelerate acquisition of vaginal patency in females at 50 mg/kg/day. Chapin (1999) found similar effects on vaginal patency but found no effect on implantation sites. Relative paired epididymal weights (but not absolute weight) were significantly increased in high does F2 and F3 males. This can be attributed to a reduction in male body weights. Importantly, andrological assessments were unaffected across all groups and generations, thus showing that the equivocal effects on sperm count at 650 and 2000 ppm in the F2 generation observed by Chapin (1999) were not substantiated.

Ovarian weights were reduced at 2000 ppm (F0), at 650 and 2000 ppm (F1) in Purina, and at 650 ppm (F0) in NIH-07. This is consistent with Nagao (2001) and Chapin (1999). There were no effects on reproduction in Tyl (2006), Chapin (1999), and Nagao (2001) as a result of these ovarian weight changes, suggesting that the mechanism is unrelated to key reproductive processes performed by the ovary. The positive control, 17b-estradiol, reduced ovarian weights consistent with effects reported in Beigel (1998). The relationship of these 17b-estradiol-induced ovarian weight changes to the reproductive effects of 17b-estradiol is uncertain. However, the effects observed in the 17b-estradiol control, including reduced fertility, gestational, and pregnancy indices, reduced number of implantation sites per litter, reduced numbers of litters, reduced numbers of total and live pups per litter at birth, reduced adult male testes and epididymal weights, and reduced epididymal sperm counts, were consistent with those reported in Beigel (1998). Since no similar findings occurred even at the highest concentration of NP, it is concluded that the weak estrogen-like activity of NP at dietary doses up to 2000 ppm does not result in reproductive effects.

The only treatment-related effect on the offspring observed in Tyl (2006) was a decrease in pup body weight at weaning in the high dose group. The decrease was not present earlier in the lactational period, suggesting that the reduced body weight resulted from direct toxic effect from overexposure to NP that occurs when the pups begin to self-feed on pnd 14.

Overall, these studies provided evidence that nonylphenol exposure over several generations can cause minor perturbations in the reproductive system of offspring, which are compatible with the effects of exogenous oestrogenic activity. These perturbations do not cause functional changes in reproduction of rats at any dose levels tested. No effects on epididymal sperm counts or on any other reproductive endpoints were observed, confirming the conclusions of Chapin (1999) and Nagao (2001) that NP is not a selective reproductive toxicant.

The NOAEL for reproductive toxicity is at or above 2000 ppm (>~ 150 mg/kg/day) in the diet. The NOAEL for systemic toxicity is 200 ppm (~ 15 mg/kg/day), mainly based on adverse effects in male rat kidneys. 

Short description of key information:

- Tyl (2006) conducted a 3.5 generation study in 25 SD rats/sex/dose according OPPTS 837.3800 (GLP) administering CAS-Number 84852-15-3 (94.25%) in two different diets at doses of ~ 0, 1.5, 15, 45, 150 mg/kg/d (Purina 5002, F0, F1, F2, F3) and 0; 45 mg/kg/d (NIH-07; F0,F1,F2)

NOAEL (reproductive toxicity): >= 150 mg/kg/d

NOAEL (systemic): 15 mg/kg/d

LOAEL (systemic): 45 mg/kg/d, based on kidney toxicity in males

- Nagao (2001) conducted a 2-generation OECD 416 study in 25 SD rats/sex/dose administering (CAS 25154-52-3) at doses of 2, 10, 50, 250 mg/kg/day by gavage

NOAEL (reprotox) 50 mg/kg/day (F0); 10 mg/kg/day (F1)

LOAEL (reprotox) >= 250 mg/kg/day (F0); 50 mg/kg/day (F1)

NOAEL (systemic): 10 mg/kg/d

LOAEL (systemic): 50 mg/kg/d. based on histopathologic effects in liver (both sexes) and kidney (males

- Chapin (1999)/NTP (1997) conducted a 3.5 generation study in 30 SD rats/sex/dose according OPPTS 870.3800 (GLP) administering Nonylphenol (CAS 84852-15-3) at doses of 0, 200, 650, and 2000 ppm. Calculated intakes were 0, 15, 50, 160 mg/kg/d during non-reproductive phases rising to 0, 30, 100, 300 mg/kg/d during lactation.

NOAEL (reproductive toxicity): 15 mg/kg/d

LOAEL (reproductive toxicity): 50 mg/kg/d, based on decreased ovarian weight, vaginal opening and sperm parameters (mating performance and fertility were not adversely affected)

LOAEL (systemic): 15 mg/kg/d, based histopathological changes in the kidney

- El-Dakdoky (2007) conducted a non guideline study to evaluate the effect of paternal exposure to NP in 10 male Swiss mice at doses of 20 and 40 mg/kg/d. Mating behaviour and effects on reproductive organs in F0/F1 were assessed.

- Beigel (1998) conducted a 90-day feeding and one-generation reproduction study in SD rats with 17ß-estradiol. Some of the results are serve as reference for the 17ß-estradiol positive control used in Tyl (2006).

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: (P) 49 days
- Weight at study initiation: (P) Males: 123.9 +/- 18.03 g; Females: 117.9 +/- 11.28 g
- Housing: individually in stainless-steel, wire-mesh cages
- Diet: ad libitum, PMI Feeds, Inc., Certified Rodent Diet 5002
- Water: e.g. ad libitum, tap water
- Acclimation period: not reported (quarantine: six days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1
- Humidity (%): 50 +/- 10
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: feed
Vehicle:
acetone
Remarks:
0.005 % based on amount of diet prepared
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or until evidence of copulation
- Proof of pregnancy: intravaginal or extruded copulation plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): On day 14 of gestation for mated females, or at the end of the cohabitation period for females without evidence of copulation, rats were transferred into polycarbonate pans
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
Remarks:
Doses / Concentrations:
0, 0.05, 2.5, 10, 50 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Dose descriptor:
NOAEC
Effect level:
0.05 ppm
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEC
Effect level:
2.5 ppm
Sex:
male/female
Basis for effect level:
other: decrease in body weight, weight gain, food consumption, food efficiancy; evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles; No effects on mating and fertility Indices
Dose descriptor:
conc. level: Concentration used in study but not low enough to classify as LOAEC or NOAEC
Effect level:
10 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
0.05 ppm
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
2.5 ppm
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Reproductive effects observed:
not specified

PARENTAL ANIMALS (P1)

Testes and epididymides:

Absolute testes weights were decreased, and testes were grossly small in 2/10 and 9/10 males fed 10 or 50 ppm of 17-beta-estradiol, respectively. Similar incidences of grossly small epididymides were also present in these groups. Microscopically, slight to moderate diffuse atrophy of interstitial cells was present in both the 10 and the 50 ppm groups. In the 50 ppm group, changes in seminiferous tubules were characterized by marked degeneration and loss of germ cells, most notably elongate spermatids. As a result, the adluminal layer of germ cells was typically composed of round spermatids or spermatocytes. At 10 ppm, changes in the seminiferous tubules were generally very slight and were most commonly characterized by degeneration of individual spermatocytes and spermatids and retention of spermatids in the basement membrane of the tubules. Correlative changes in the epididymides were characterized by oligospermia or aspermia, the presence of germ cell debris in the lumen of tubules, and atrophy of epididymal tubules.

Accessory sex glands:

The weight of the accessory sex gland unit was decreased in males fed 2.5 ppm and greater. The accessory sex glands were grossly atrophied in males fed 10 or 50 ppm 17-beta-estradiol. These gross changes were generally correlative to microscopic atrophy of the prostate and seminal vesicles, components of the accessory sex glands.

Ovaries:

Ovarian weights were decreased and microscopic evidence of ovarian malfunction (atrophy and decreased corpora lutea) was present in females fed 2.5 ppm and above. Microscopically, the severity of atrophy was dose related and was characterized by decrease or absence of corpora lutea and increased follicular atresia (including cystic atresia). Qualitatively there was also an apparent decrease in the number of most of the follicular developmental stages, yet the primordial follicle pool appeared healthy.

Using morphometric analysis, the number of large antral follicles was similar in ovaries from controls and animals treated with 0.05 and 2.5 ppm 17-beta-estradiol. Ovaries from animals treated with 10 and 50 ppm 17-beta-estradiol often contained large cysts and significantly fewer antral follicles than the control animals.

Uterus:

Uterine weights were increased in the 10 and 50 ppm groups, although not in a dose-related manner. The absence of a dose response may be the result of mitigation of the estrogenic effects on the uterus by the more severe body weight effects in the 50 ppm group. Grossly, fluid was present in the uterine lumen in 1/10 and 3/10 females in the 10 and 50 ppm groups, respectively. Microscopically, uterine changes were primarily characterized by slight hypertrophy of the epithelium of the endometrium and endometrial glands at 10 ppm and above. The diagnosis of hypertrophy was used to represent an increase in epithelial cell height greater than that typically seen in the estrual or proestrual uterus. Low incidences of slight focal squamous metaplasia were also present in the endometrium and endometrial glands of 50 ppm females.

There were no statistically significant changes in labeling indices of uterine stromal cells at any of the concentrations tested. The labeling indices of uterine epithelial cells were significantly decreased in the 10 and 50 ppm groups after 28 days and, although not statistically significant, remained decreased in the 50 ppm group after 90 days. The height of the uterine epithelium was significantly increased in 50 ppm females following 28 days on test and in the 10 and 50 ppm groups after 90 days on test.

Vagina:

Incidences of estrus based on vaginal histology (Yuan, 1991) were increased in females fed 10 ppm 17-beta-estradiol and greater. In some treated females, the histology of the vaginal mucosa resembled that of a proestrous mucosa except that the striatum granulosum (and stratum cornium) were absent or poorly developed, and the stratum mucinification was thickened. This change is usually correlated with proestrus based on vaginal cytology (Biegel el al., 1998) and, thus, was diagnosed as proestrus with hypertrophy.

P1 AND F1 PREWEANING EVALUATIONS

Reproductive Indices:

Reproductive indices were affected in rats fed 10 and 50 ppm 17-beta-estradiol. There was no evidence that mating occurred in the 50 ppm group, based on the absence of copulation plugs. In rats fed 10 ppm 17-beta-estradiol, the mating

index (number copulated/number cohoused) was decreased to 33.3%; however, no litters were subsequently produced. There

was no effect on mating or fertility in rats fed 0.05 or 2.5 ppm 17-beta-estradiol.

F1 Preweaning Evaluations:

There were decreased numbers of pups/litter born, number of pups born alive, and number of pups alive on day 4 (pre- and postculling) in rats whose parents were fed 2.5 ppm 17 -beta-estradiol. There were also decreased weights in the F1 generation pups in the 2.5 ppm group during the lactation periods, while the decrease in pup weight in the 0.05 ppm

group was present only at birth. There were no statistically significant differences in the anogenital distance in the treatment groups. There was an increase in the time to preputial separation in male pups in the 2.5 ppm group and a decrease in the time to vaginal patency in female pups in the 0.05 and 2.5 ppm groups. Forty-three percent (24/56) of the female pups in the 2.5 ppm group were vaginally patent on the day of weaning.

F1 POSTWEANING EVALUATIONS

Testes and epididymides:

In 2.5 ppm F, males, the absolute testicular and epididymal weights were decreased compared to controls.

Accessory sex glands:

The absolute accessory sex glands weight was decreased in the 2.5 ppm F1 group. There were no gross or microscopic findings in the accessory sex glands of F1 males although equivocal secretory depletion was present in some F1 males fed 2.5 ppm 17-beta-estradiol.

Ovary:

There was a statistically significant decrease in absolute and an increase in relative (to body weight) ovarian weights an in F1 females fed 2.5 ppm 17-beta-estradiol.

Using morphometric analysis, the number of follicles was similar in ovaries from the F1 generation control and animals

treated with 0.05 ppm 17-beta-estradiol. However, the number of large antral follicles was significantly reduced in animals treated with 2.5 ppm 17-beta-estradiol. There were no significant differences in follicle number between P1 and F1 rats fed 0 or 0.05 ppm 17-beta-estradiol. In contrast, the number of antral follicles in rats fed 2.5 ppm 17-beta-estradiol was significantly lower in the F1 generation compared to the P1 generation.

Uterus:

There were no statistically significant changes in uterine weight in the F1 generation females. However, microscopic changes similar to those observed in the subchronic study were observed in the 2.5 ppm F1 females. There were no statistically significant changes in height of the uterine epithelium in F1 females.

Vagina:

Instances of estrus by vaginal histology were increased in F1 females fed 2.5 ppm. Thickening of the mucosa of the proestrual vagina, noted in the subchronic study, was also seen in 2.5 ppm F1 females.

Conclusions:
This study investigated the effect of ß-Estradiol on reproductive development. It is included as reference to distinguish hormon and non-hormon effects in reproductive toxicity studies (e.g. Tyl 2006).
Executive summary:

In a 1-generation reproduction study 17-beta-estradiol (98 -100%) was administered in the diet to 30 Crl:CD BR rats/sex/dose at dose levels of 0, 0.05, 2.5, 10, and 50 ppm for 90 days. Subsequently animals were mated.

Effects of 17-beta-estradiol on fertility, gestational, and pregnancy indices, numbers of total litter and pups born alive at birth, adult male testes and epididymal weights, and epididymal sperm counts were evaluated.

Vaginal patency was accelerated by 8.8 days at 2.5 ppm. This acceleration was expected since female puberty is under E2 control. Preputial sepatation (PPS) in males was significantly delayed by 8.2 days at 2.5 ppm.

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1996-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, comparable to guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
extended to include teh production of an F3 generation
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Portage, MI facility of Charles River Labs
- Age at study initiation: approx. 11 w
- Weight at study initiation: (Range finding study) Males: 339.1-382.0 g; Females: 217.9-251.4 g; (F0) Males: 228.9-276.4 g; Females: 217.9-251.4 g
- Housing: individually in polycarbonate cages with BetaChip hardwood animal bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Quarantine period: 8 days
- Other: all animal procedures met standards set fot by the National Academy's Guide for the Care and Use of Laboratory Animals and AAALAC, and approved by the appropriate IACUC.

ENVIRONMENTAL CONDITIONS
- Temperature: 68-74 °F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
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 one to two weeks
- Mixing appropriate amounts with (Type of food): Powdered Zeigler NIH 07 Small Animal Feed, which was analyzed for for various ingredients (nutrients, nitrosamines, heavy metals, bacteria, etc.)
- Storage temperature of food: room temperature
- Storage conditions of food: protected from light and dispensed into glass jars with stainless steel followers and lids
Details on mating procedure:
- M/F ratio per cage: two animals per cage by sex - one male, one female - during cohabitation; one to four per cage during the holding and rearing phase; individually at all other times
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as day 0 of pregnacy/gestation
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): one to four per cage in housing conditions described above
- Any other deviations from standard protocol: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the dose range-finding study, dietary formulations of NP and NIH-07 feed at concentrations of 75,250, 800, and 3000 ppm were prepared for two weeks of use. The appropriate quantity of NIH-07 diet was accurately weighed into containers for each dose level. For each dose level, an appropriate quantity of feed flour (prepared by sieving the feed through a USS No. 80 standard sieve) was accurately weighed into a glass beaker. A dietary premix was prepared for each dose level by weighing the appropriate quantity of test article into a 250 ml glass beaker containing approximately 5 g of feed flour. The test article and feed flour were mixed with a stainless steel spatula until no feed/chemical agglomerates remained. Additional feed flour was added in increments and mixed until all of the feed flour was added to the mixture. The feed flour test article was poured into a glass 2000 ml beaker and the 250 ml glass beaker was rinsed three times with small portions of the preweighed feed. Each rinse was poured into the 2000 ml beaker. Approximately 500 g of preweighed feed was added in increments and mixed. The dietary premix and appropriate quantity of preweighed feed were poured evenly into the right and left ports of the Patterson-Kelly blender. The premix beaker was rinsed three times with preweighed feed and the remaining preweighed feed was poured into the Patterson-Kelly blender. The dietary formulation was blended for 10 minutes with the intensifier bar, then for 30 minutes without the intensifier bar, and for an additional 10 minutes with the intensifier bar.
For the multigeneration study, dietary formulations of NP and NIH-07 feed at concentrations of 200, 650, and 2000 ppm were prepared every one to two weeks for one to two weeks of use. The appropriate quantity of NM-07 diet was accurately weighed into containers for each dose level. For each dose level, an appropriate quantity of feed flour (prepared by sieving the feed through a USS No. 80 standard sieve) was accurately weighed Into a glass beaker. A dietary premix was prepared for each dose level by weighing the appropriate quantity of test article into a 250 ml glass beaker containing approximately 5 g of feed flour. The test article and feed flour were mixed with a stainless steel spatula until no feed/chemical agglomerates remained. Additional feed flour was added in increments and mixed until all of the feed flour was added to the mixture. The feed flour test article was poured into a glass 2000 ml beaker and the 250 ml glass beaker was rinsed three times with small portions of the preweighed feed. Each rinse was poured into the 2000 ml beaker. Approximately 500 g of preweighed feed was added in increments and mixed. The dietary premix and appropriate quantity of preweighed feed were poured evenly into the right and left ports of the Patterson-Kelly blender. The premix beaker was rinsed three times with preweighed feed and the remaining preweighed feed was poured into the Patterson-Kelly blender. The dietary formulation was blended for 30 minutes with the intensifier bar, then for 40 minutes without the intensifier bar, and for an additional 30 minutes with the intensifier bar. For the first mix, the blending time was 20 minutes when the intensifier bar was used.
Dietary formulations were stored at room temperature protected from light and dispensed into glass jars with stainless steel followers and lids. Animals were supplied with fresh feed at least once a week. Two 150 g samples were collected from each dose level of every formulation upon completion of mixing and stored in heat-sealed polyethylene bags, protected from light in the freezer at -20 °C to -15 °C. Homogeneity samples (150 g each) were collected from each the right, left, and bottom ports for the low and high-dose groups for the first mix for the dose range-finding study and the multigeneration study and when the size of the mix changed. The concentration of NP in the feed was verified prior to the initiation of the dose range-finding study and for the first, second, seventh, and twelfth mixes for the multigeneration study. Samples selected for dose concenuation analysis were fonvarded with a 5 g sample of bulk test article on dry ice to RTI. Archival samples which were not selected for analysis were retained for at least 90 days following preparation then discarded as hazardous waste.
Samples were analyzed by high performance liquid chromatography by RTI. An aliquot of each level of dose formulation and of the control vehicle was analyzed. Homogeneity analyses were conducted at the 75 and 3000 ppm dose levels for the dose range-finding study and at the 200 and 2000 ppm dose levels for the multigeneration study (Table A4-3). Analysis indicated that the samples were within 91.0-113% of the target concentration and within 10% of each other. All dose formulations analyzed were within 88.5-113% of the nominal concenuation (Table A4-3). Dose formulations of NP in NIH-07 feed (75 ppm) were stable for 21 days when stored at room temperature (22-25 °C) in sealed amber glass bottles and for seven days under the conditions that simulate dosing.
Duration of treatment / exposure:
Adult F0 exposure premating (male/female) lasted 6 weeks and 2 weeks during the cohabitation. The mating trial consisted of three treated groups (30/animals/sex/group). F0 male exposure continued for 15 weeks prior to termination. Selected F1 pups were investigated after 30 days, whereas selected F2 and F3 pups were investigated after 21 days. Mating of F1 and F2 started on prenatal day 86(+-10). F3 animals were killed and necropsied at prenatal day 55-58.
Frequency of treatment:
Daily
Details on study schedule:
- The dams were allowed to rear their young to pnd 21
- On pnd 21 each litter was weaned
- F1 parental animals not mated until 9 weeks after selected from the F1 litters.
- One rat/sex/litter was selected on pnd 16 to produce F2 and F3 generation
- Age at mating of the mated animals in the study: 11 to 13 weeks
Remarks:
Doses / Concentrations:
0, 200, 650, and 2000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 15, 50, 160 mg/kg/d
Basis:
nominal conc.
during non-reproductive phase
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg/d
Basis:
nominal conc.
during lactation
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: range-finding study at concentrations of 75, 250, 800, and 3000 ppm nonylphenol in NIH-07 feed for two weeks with 30 animals per sex
- Rationale for animal assignment (if not random): Sprague-Dawley rats were chosen because of the established quality as a breeder and the availability of historical and control toxicological data and its relative resistance to the reproductive effects of nonylphenol.

Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed twice daily for mortality and signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Physical examinations were performed every two weeks.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were obtained at randomization, at initiation of the treatment, and every two weeks thereafter, execept for F0, F1, F2 females after cohabitation since they were either in gestation or lactation.

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):
F0, (as well as F1, and F2) females were subject to daily vaginal lavage and estrous cyclicity evaluation for 3 weeks prior to mating.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
sperm motility, cauda epididymal sperm density, sperm morphology, testicular spermatid head counts, restistant spermatid head counts, spermatid heads per total testis and heads per milligram testis, epididymal sperm density.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 1, 4, 7, 14, and 21 postpartum: yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1, F2, and F3 offspring:
pnd 1, 4, 7, 14, and 21: total number of pups, numer of live and dead pups, number of male and female pups.
pnd 1 and 21: individual weight and anogenital distance
pnd 4, 7, and 14: individual pup weight and total pup weight for each sex
pnd 1: dam and sire were weighed following delivery
pnd 1, 4, 7, 11, 14, 18, and 21: food consumption


GROSS EXAMINATION OF DEAD PUPS:
Yes, all animals found dead were subject to a gross necropsy to investigate the cause of death.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals were sacrificed by CO2 asphyxiation and discarded on pnd 154 (+-7) for F0, F1, and F2 and on pnd 55-58 for F3 animals.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]


HISTOPATHOLOGY / ORGAN WEIGHTS
At scheduled necropsy beside the body weight the following organs were weighted for F0, F1, F2, and F3:
- Liver, kidneys, spleen, ovaries, vagina/cervix/uterus, right whole and cauda epididymis, right testis, ventral prostate, and seminal vesicles ( with coagulating glands).
The following organs were reatined in appropriate fixative for scheduled or possible histopathologic evaluation:
- Liver, kidneys, spleen, stomach, jejunum, duodenum, ileum, ventral prostate, seminal vesicles, female reproductive tract, and gross lesions.
Exemption: In F0 males only only ventral prostate was weighed, whereas dorsolateral and ventral prostate lobes were dissected and weighed in the necropsies for the F1 and F2 adults.
Postmortem examinations (offspring):
SACRIFICE
- The F1, F2 and F3 offspring not selected as F3 animals were sacrified at pnd 21.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Terminal body weights were obtained and a gross necropsy was performed on 20 F1, F2, and F3 animals per sex per group.


GROSS NECROPSY
- Necropsies included examination of the external surface of the body, orifices, reproductive system, and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGTHSThe following organs were weighed :
- Liver, kidneys, spleen, ovaries, vagina/cervix/uterus, right epididymis, right cauda epididymis, and right testis.
Testes, epididymides, and ovaries from all necropsied animals were fixed in Bouin's. Gross lesions were preserved in 10% neutral-buffered formalin..
Statistics:
Most hypotheses were tested using the nonparametric multiple comparisons procedure of Dunn (1964) or Shirley (1977) as modified by Williams (1986). Jonckheere’s test (1954) was used to identify those comparisons necessitating Shirley’s test. For comparisons with only two groups (sperm data using only control and high-dose groups, for example), Wilcoxon’s test was used.
For proportionate data, the Cochran-Armitage was used (Armitage, 1971). Because litter size can affect individual pup weight, litter size was used as a covariate in the pup weight analyses, using parametric covariance procedures developed by Neter et al. (1985).
Feed consumption was evaluated both as per animal per day, and per kilogram body weight per day. Organ weights were analyzed as both absolute and relative to body weight.
For convenience and efficiency, we will refer to animals that consumed 200 ppm NP as 200NP, those that consumed 650 ppm as 650NP, and the high dose group will be 2000NP.
Feed consumption data were analysed as g/animal/day and g/kg body weight/day. Both absolute organ weights and organ weights relative to body weight were analysed.
Reproductive indices:
Estrous Cycle Length
Mating Index
Pregnancy Index
Fertility index
Gestation Length

Offspring viability indices:
Live pups per litter
Proportion of Pups Born Alive
Pup Sex Ratio
Adjusted Live Pup Weight
Pup Survival
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:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Adult feed consumption was slightly reduced initially, but recovered after the first 1-2 weeks.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Females consumed approx. 25% more than males on a body weight basis and the doses decreased over time as the animals grew.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Vaginal opening was accelarated at 650NP and 2000NP .

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects in any reproductive organ of the males were detected

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Indices of mating behaviour or libido were unchanged. Litter size was reduced by 9% at 2000NP; proportion liveborn, absolute or adjusted pup weight and gestation length were unchanged at all doses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males: weights of paired kidneys corrected for bodyweights was increased at 650NP by 7% and at 2000NP by 10%.
Females: organ weights were unchanged across dose groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males: weights of paired kidneys corrected for bodyweights was increased at 650NP by 7% and at 2000NP by 10%.
Females: body weight was reduced at 2000NP by 9%.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males: slight increase in the incidence of renal tubular mineralization
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
15 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: Minor perturbations in the reproductive system of offspring with no adverse effect on fertility or mating performance
Remarks on result:
other: Generation: All generations tested: F0, F1, F2, F3 (migrated information)
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Renal tubular degeneration and/or dilatation across all four generations
Remarks on result:
other: Generation: All generations tested: F0, F1, F2, F3 (migrated information)
Clinical signs:
not specified
Mortality / viability:
no mortality observed
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
VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
no data

BODY WEIGHT (OFFSPRING)
Offspring feed consumption was slightly reduced initially, but recovered after the first 1-2 weeks. Decrease in body weights at both sexes at 650NP and 2000NP.

SEXUAL MATURATION (OFFSPRING)
Females: vaginal opening was accelarated at 650NP and 2000NP in all three generations (F1, F2, F3); Estrous cycle length was increased in F1 and F2 adults at 2000NP - although small effect;
Males: No consistent detectable effect; although no effects in any reproductive organ were detected in F1 and F3 males a clear monotonic downward trend in whole epididymal weight and sperm density was found in the F2 adults. The study states that the male F2 sperm data are consistent enough to be compelling, but not consistent enough to be conclusive. The F2 sperm effects are either statistical/biological “noise”, or imply heretofore unknown pharmacokinetics or toxicodynamics. These sperm data should be interpreted cautiously until the findings are repeated. Therefore , until these treatment related effects are replicated, they state that it is unwarented to conclude that this study shows an adverse effects of a mild estromimetic on male reproductive development.

ORGAN WEIGHTS (OFFSPRING)
Females: Weight of uterus/vagina was increased in F1.
Both sexes: Increased kidney weights

GROSS PATHOLOGY (OFFSPRING)
Increased kidney weights (F1 male/female, F2 males)

HISTOPATHOLOGY (OFFSPRING)
Both sexes, all generations (F1, F2, F3): increased renal tubule histopathology (dilatation of cysts, mineralization)
Dose descriptor:
LOAEL
Remarks:
reproductive effects
Generation:
F2
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Ovarian weight change and epididymal sperm density, respectively
Reproductive effects observed:
not specified

Summary of results is provided in a table in attachment "summary results".

Conclusions:
A NOAEL of 15 mg/kg/d for reproductive effects can be concluded based on minor perturbations in the reproductive system of offspring with no adverse effect on fertility or mating performance.
General toxicity was evident at all dose levels as renal tubular degeneration and/or dilatation across all four generations. No NOAEL could be derived for systemic toxicity. The LOAEL is 15 mg/kg/d.
Executive summary:

In a 3.5 -generation reproduction study 4 -nonylphenol (> 91%)was administered to 30 Sprague-Dawley rats/sex/dose in diet at dose levels of 0, 200, 650, 2000 ppm (0, 9 -35, 30 -100, 100 -350 mg/kg bw/day). 

Fertility or mating performance was not affected in this study. However, exposure to Nonylphenol did cause minor perturbations in the reproductive system of offspring, which are compatible with the predictable or hypothesized effects of estrogenic activity. Estrous cycle length was increased by about 15% (F1, F2) at 160 mg/kg/day. The timing of vaginal opening was accelerated by 1.5-7 days at 50 mg/kg/day in all offspring generations. Absolute, but not relative, ovarian weights were decreased at 50 mg/kg/day (F2), and 160 mg/kg/day (F1, F2, and F3). In males, changes in sperm endpoints were seen in the F2 generation; epididymal sperm density was decreased by about 10% at 50 and 160 mg/kg/day and spermatid count was decreased by a similar amount at 160 mg/kg/day. According the authors these findings may be caused by methodological problems with the epididymal sperm density measurements.

Evidence of general toxicity was seen in adults of all generations, although there were no treatment-related clinical signs, mortalities or adverse effects on food consumption. Relative kidney weights were increased at 50 and/or 160 mg/kg/day in adult males (F0, F1, and F2) and at 160 mg/kg/day in F1 adult females. Histopathological examination revealed an increase, although often without a clear dose-response relationship, in the incidence of renal tubular degeneration and/or dilatation in adult males from all generations at all dose levels. Similar findings were reported for adult females at 160 mg/kg/day (F1, F2, and F3) and at 15 and 50 mg/kg/day in F3. It can be questioned if the increased incidence of renal tubular degeneration and/or dilatation is related to treatment because these changes were not seen to the same extent in a 90-day study (CMA 1997), which was conducted using the same strain of rats. In addition dose-dependent trend was not apparent in all generations/sexes. The lack of concordance between the studies cannot be explained on the basis of a slightly longer exposure period in the multigeneration study because kidney effects were seen in the F 3 generation which was exposed for only 8 weeks, nor on the basis of in utero and neonatal exposure because the effect also occurred in the F 0 generation. Giving special emphasis to the fact that the increased incidence occurred consistently across all four generations in the multigeneration study, it is considered that this cannot be dismissed as background variation. Consequently, no NOAEL could be derived. The LOAEL for repeated exposure is 15 mg/kg/day, based on histopathological changes in the kidneys

A NOAEL for systemic toxicity could not be established. The LOAEL for systemic toxicity is 200 ppm based on renal tubular degradation and/or dilatation. The NOAEL for reproductive effects is 200 ppm in females based on minor perturbations in the reproductive system of offspring with no adverse effect on fertility or mating performance.

This study is acceptable and satisfies the guideline requirement for a two-generation reproductive study (OECD 416) but was performed as 3.5 generation study.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Documentation insufficient for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on mating procedure:
no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Mated females from gestation day 7 to weaning.
F1 males from weaning until week 10 (covering at least one complete spermatogenic cycle.
Frequency of treatment:
daily
Details on study schedule:
no data
Remarks:
Doses / Concentrations:
0, 100, 250, 400 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 mated females; 20 males from F1
Control animals:
yes, concurrent vehicle
No information was presented on maternal bodyweights, but it was stated that no females showed any physical or behavioural abnormalities. No offspring were born from the mothers receiving 400 mg/kg/day; it is not clear from the report if this was because of maternal deaths or embryonic/foetal resorption.
Dose descriptor:
dose level: Maximum dose used in the study
Effect level:
400 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: See Remark
No offspring were born from the mothers receiving 400 mg/kg/day; the report does not state if this was because of maternal deaths or embryonic/foetal resorption.
There were no malformations or still births among the F1 offspring at 100 and 250 mg/kg/d. No physical or behavioural abnormalities were reported. No mortalities occurred; this is in contrast with the de Jager (1999, testicular toxicity, Part I) where 15 out of 20 animals died at 250 mg/kg/day. F1 bodyweight gain over the course of the study was significantly reduced at 100 and 250 mg/kg/day. F1 absolute but not relative testicular and epididymal weights were decreased at 100 and 250 mg/kg/day. The differences in absolute organ weight are thought likely to be related to the intergroup bodyweight differences. Total cauda epididymal sperm count was reduced at 250 mg/kg/day (by 36%, relative to controls), but at 100 mg/kg/day sperm counts were similar to those of the control group. Seminiferous tubule diameter was slightly lower in both groups. These slight differences were declared to be highly statistically significantly different from the control group. The authors also stated that the tubule lumen diameter and seminiferous epithelium thickness were highly statistically significantly less than the control group in both groups, but no data were presented. Although these quantitative tubular changes were consistent with those of the de Jager (1999, testiculat toxicity) study, in the present study these may be related to the fact that testicular weight was lower in these groups. Histopathology revealed pathological changes in the testes of one F1 male from the 100 mg/kg/day group; in the tubules, cell necrosis, vacuolation and sloughing of the germinal epithelium were described. However, no such histopathological abnormalities were seen at 250 mg/kg/day, so the changes outlined above cannot be attributed to nonylphenol treatment.
This study provides evidence of a reduction in sperm count at 250 mg/kg/day, a dose level, although it is not possible to state whether this is a developmental effect or as a result of direct exposure to the males after weaning. It is not clear if the changes in the tubular measurements represent specific reproductive toxicity or non-specific secondary consequences of the reduction in bodyweight gain.
Dose descriptor:
LOAEL
Remarks:
systemic
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: reduced body weight gain
Dose descriptor:
LOAEL
Remarks:
male fertility
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Changes in seminiferous tubule diameter
Reproductive effects observed:
not specified
Conclusions:
The study presents some doubtful data on seminiferous tubule and lumen diameter and seminiferous epithelium thickness in F1 males. The most reliable result seem to be a reduction in sperm count at 250 mg/kg/day, although it is not possible to state whether this is a developmental effect or as a result of direct exposure to the males after weaning.
Executive summary:

In a one-generation study according OECD 415 para-Nonylphenol (technical grade) was administered to 10 mated female SD rats/dose by gavage at dose levels of 0,100, 250, 400 mg/kg bw/day from gestation day 7 to weaning. 20 F1 males were randomly selected from each group for dosing until week 10 weeks. Testes and epididymal weight were recorded. The total cauda epididymal sperm numbers were determined. The testes were stored for histological examination, which included the identification of the stages of spermatogenesis present and the measurement of the seminiferous tubule diameter, lumen diameter and epithelial thickness.

There were no malformations or still births among the F1 offspring at 100 and 250 mg/kg/d. At 400 mg/kg/d no offspring were born. The report does not state if this was due to maternal deaths or embryonic/fetal resorption. No physical or behavioural abnormalities were seen. No mortalities were reported. Testicular toxicity was observed at all dose levels. A LOAEL of 100 mg/kg bw/day was concluded.

The one-generation study in the rat is classified unacceptable for the assessment of testicular toxicity. The study presents some doubtful data on seminiferous tubule and lumen diameter and seminiferous epithelium thickness in F1 males. The most reliable result seem to be a reduction in sperm count at 250 mg/kg/day, although it is not possible to state whether this is a developmental effect or as a result of direct exposure to the males after weaning.

Endpoint:
one-generation reproductive toxicity
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: Significant methodological deficiencies: Inappropriate dose levles. Little toxicological significance can be accorded to findings on testicular toxicity due to small group/high mortality.
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 weeks
Frequency of treatment:
daily
Details on study schedule:
The test substance was administered for at least one complete spermatogenic cycle (~52 days) starting from week 12 of age. Sampling was carried out at week 22.
Remarks:
Doses / Concentrations:
0, 100, 250, 400 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Dose descriptor:
LOAEL
Remarks:
testicular toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: decrease in seminiferous tubule and lumen diameter and seminiferous epithel diameter
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: mortality (3 of 20)
Reproductive effects observed:
not specified
Conclusions:
A LOAEL for testicular toxicity of 100 mg/kg/day can be designated. This exposure levels also causes mortality. Because of the very small group sizes due to mortality, little toxicological significance can be accorded to these findings.
Executive summary:

In a one-generation study according OECD 415 para-Nonylphenol (technical grade) was administered to 20 male SD rats/dose by gavage at dose levels of 0,100, 250, 400 mg/kg bw/day for 10 weeks. 

Mortality occurred at all dose levels (3, 17, 18). Testicular toxicity was also observed at all dose levels. The LOAEL is 100 mg/kg bw/day.

The one-generation study in the rat is classified unacceptable for the assessment of testicular toxicity. Little toxicological significance can be accorded to these findings due to high mortality at any dose level. 

Endpoint:
one-generation reproductive toxicity
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: see 'Remark'
Remarks:
Lee (1998) exposed neonatal rats to 0.08, 0.8, and 8 mg/kg bw/d for different intervals ranging from pnd 1 to 30 by i.p. injection. There were indications for NP-mediated effects on the male reproductive system. Due to serious shortcomings concerning the study design (as assessed in EU RAR 2002) and in light of the fact that these results proved to be not reproducible in a similar repetitive study by Odum & Ashby (2000) this study is considered as being invalid for evaluation of NP.
Qualifier:
no guideline followed
Limit test:
no
Reproductive effects observed:
not specified
Conclusions:
Lee (1998) exposed neonatal rats to 0.08, 0.8, and 8 mg/kg bw/d for different intervals ranging from pnd 1 to 30 by i.p. injection. There were indications for NP-mediated effects on the male reproductive system. Due to serious shortcomings concerning the study design (as assessed in EU RAR 2002) and in light of the fact that these results proved to be not reproducible in a similar repetitive study by Odum & Ashby (2000) this study is considered as being invalid for evaluation of NP.
Endpoint:
fertility, other
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:
no guideline followed
Principles of method if other than guideline:
NP is applied by intraperitoneal injection of 0, 21.25 and 42.50 mg/kg bw/day to male mice for 35 consecutive days. These males were subsequently mated with untreated females. Effects of NP on sperm characteristics, fertility index, histopathological and biochemical changes related to oxidative stress in testes were examined.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Central Animal House of the National Research Center, Egypt
- Age at study initiation: (P) 5 w
- Weight at study initiation: (P) Males and Females: 25-27 g;
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): standard chow
- Water (e.g. ad libitum): no further details reported
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 50-65
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: To: no data
Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on mating procedure:
- M/F ratio per cage: 1/1 with untreated female
- Length of cohabitation: 7 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 1 of pregnancy
Duration of treatment / exposure:
35 days (males only)
Frequency of treatment:
daily (males only)
Remarks:
Doses / Concentrations:
0, 21.25, 42.5 mg/kg/d
Basis:

No. of animals per sex per dose:
10 male mice per dose group
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale (-> pilot study):
In the present investigation, the acute intraperitoneal (i.p.) LD 50 of nonylphenol (NP) was calculated for adult male mice as 170 mg/kg b.wt according to the simplified method of evaluating dose effect experiments (Litchfield and Wilcoxon 1949).
In order to determine the minimal doses of NP capable of inducing any toxic effect on testis, daily doses of 5.31, 10.62, 21.25, and 42.50 mg/kg b.wt equivalent to 1/20, 1/10, 1/8 and 1/4 LD 50 of NP were administered intraperitonealy for 2 weeks.
The doses of 5.31 and 10.62 mg/kg were neglected as they did not exert any toxic effect on male reproductive organs weight and sperm characteristics.


Positive control:
Not applicable
Sperm parameters (parental animals):
- Parameters examined in P male parental generations:
testis weight, epididymis weight, weight of seminal vesicles

- Determination of sperm motility:
Concentration and sperm morphological abnormalities, the epididymal content of each mouse was obtained after cutting the tail of epididymis and squeezing it gently in sterile clean watch glass and examined
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After mating trials, male mice of all groups were anesthetized and sacrificed; the reproductive organs (testes, epididymis and seminal vesicles) were removed and weighed.
Testes examination: One of the testes was homogenized, the homogenates were centrifuged and the supernatant was separated and used for oxidative stress analysis. Malondialdehyde (MDA) concentration, Glutathione reduced (GSH) level, and superoxide dismutase (SOD) activities were assayed.
Histological examination: the right testes were fixed in 10% neutral formalin, dehydrated in graded series of alcohol, embedded in a paraffin wax, sectioned at 5–7 µm and stained with hematoxyline and eosin. The diameter and germinative cell layer thickness of the seminiferous tubule (ST) from ten different areas of testes were measured by the aid of ‘‘Leica Q500 MC’’ image analyzer computer system.
- Maternal animals: Females were isolated and kept under observation till 18th day of gestation. Pregnant mice were sacrificed on the day 19 of gestation. The uteri were dissected and the implantation sites, number of viable, resorbed and dead fetuses were recorded. The fetuses were examined morphologically to determine any external abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
The data obtained were subjected to statistical analysis. All values were given as mean standard error of measurement (S.E.). To determine the differences among all groups in the whole parameters one-way analysis of variance (ANOVA) and post hoc LSD analysis were performed using the SPSS/PC computer program (version 10). Statistical significance was determined at the level of significance of p < 0.05.
Dose descriptor:
LOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Effects on reproductive organs weight and sperm characteristics (count and motility), testicular MDA, GSH, and SOD; no effect on mating behavior, male fertility and the developed fetuses
Reproductive effects observed:
not specified

The values of reproductive organs weight and sperm characteristics are shown in Tables 1 and 2, respectively.

Table 1: Testis, epididymis and seminal vesicle weights (mg)

Parameters

Right testis    

Right epididymis  

Seminal vesicle

Control

97.3±4.6  

48.4±1.8

166.0±6.9

Oil

96.6±5.3 

46.7±2.8

165.4±6.6

21.25 mg/kg NP

92.1±3.6

43.0±3.8

164.1±6.1

42.50 mg/kg NP

81.7±2.9*

39.7±2.4*

161.8±4.8

* Indicates significant compared to control group (p < 0.05)

Table 2: Epididymal sperm count, sperm motility and abnormal sperm rate

Parameters

Sperm count

(x 106) sperm/m3

Sperm motility (%)  

Sperm abnormal

rate (%)

Control

27.6±0.54

85.9±1.6

4.23±0.30

Oil

27.1±1.02

85.7±0.6

3.80±0.12

21.25 mg/kg NP

26.3±0.73

78.3±2.8*

4.60±0.37

42.50 mg/kg NP

25.6±0.66*

72.7±2.6**

4.90±0.36

*, ** Indicates significant compared to control group at (p < 0.05), (p < 0.01), respectively.

The higher dose of NP significantly reduced testes and epididymis weight and sperm count (p < 0.05) and motility (p < 0.01), while the lower dose only decreased significantly sperm motility (p < 0.05) in comparing to controls.

No changes in the seminal vesicles weight and in the sperm abnormal rate were noticed in all experimental groups.

Male exposure to 1/8 and 1/4 LD 50 NP for 35 days had no effect on mating behavior or pregnancy rate. Neither causes any significant changes in number of implantation per litter (No. impl./litter), live fetuses, fetal body weight or external visible abnormalities when compared to the control groups (Table 3). These may be attributed to NP had limited toxic effect on spermatogenesis which was not extended to the fertility.

Table 3: Mating index, fertility index and paternal effect on fetuses

Parameters

Mating (%)

Fertility (%)

No. impl./litter

Live fetuses

Fetal body weight (g)

Control

100

100

8.3±0.4

91.0±5.71

1.35±0.03

Oil

85.7

100

7.2±0.9

89.2±6.64

1.33±0.07

21.25 mg/kg NP

85.7

100

7.2±1.1

89.0±5.09

1.25±0.06

42.50 mg/kg NP

85.7

100

7.6±0.8

86.1±5.20

1.23±0.07

 

The diameter and thickness of the germinative cell layer of ST were significantly smaller in the NP exposed groups even at the low dose level (Table 4). These histological measurements further supported the finding of a low testicular mass.

 

Table 4: Mean values of diameter size and germinative cell layer thickness of seminiferous tubules in testes tissue (µm)

Parameters

Diameter size

Germinative cell

layer thickness

Control

237.7±2.81

58.2±2.33

Oil

236.2±5.02

57.5±2.49

21.25 mg/kg NP

221.5±2.20**

46.7±3.52**

42.50 mg/kg NP

213.1±4.27**

34.8±2.22**

** Indicates statistical significant compared to control group at (p < 0.01)

 

Oxidative stress was found in testes tissue following NP exposure indicated by significant increase in testes MDA concentrations (p < 0.05) and decrease in GSH levels and SOD activities (p < 0.001) (Table 5).

Table 5: Epididymal sperm count, sperm motility and abnormal sperm rate

Parameters

MDA

GSH

SOD

Control

76.0±9.9

1.65±0.07

357±6.9

Oil

87.1±9.4

1.64±0.04

351±8.1

21.25 mg/kg NP

108.2±8.0*

1.11±0.03***

224±7.9***

42.50 mg/kg NP

117.3±9.9*

1.16±0.06***

220±8.6***

*, *** Indicates significant compared to control group at (p < 0.05), (p < 0.001), respectively.

 

In the present study, the observed deleterious effects on sperm characteristics and testicular tissue may be attributed to peroxidation of unsaturated fatty acids in the plasma membrane that may lead to alteration of membrane characteristics and function.

Conclusions:
The purpose of this study was to investigate the effects of NP on sperm characteristics, fertility index, histopathological and biochemical changes related to oxidative stress in testes. Exposure of 10 adult male mice to high dose of NP (1/4 LD50) for 35 days had effects on some reproductive organs weight and sperm characteristics (count and motility), testicular MDA, GSH, and SOD but did not influence the mating behavior, male fertility or the developed fetuses.
Executive summary:

In a testicular toxicity study nonylphenol (> 85 %) was administered by i.p. injection to 10 male Swiss mice/dose at dose levels of 0, 21.25, and 42.50 mg/kg bw/day for 35 consecutive days. Males were subsequently mated with untreated females. Control male mice received no treatment.

Exposure to 42.5 mg/kg/d (1/4 LD50) had effects on some reproductive organs weight and sperm characteristics (count and motility), testicular MDA, GSH, and SOD but did not influence the mating behavior, male fertility or the developed fetuses. 

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Atsugi, Japan)
- Age at study initiation: (P) 6 w males, 13 w females; (F1) 3 w
- Weight at study initiation: (P) Males (5 w): mean 117.1s (SD+-2.7); Females (10 ks): mean 207.0 g (SD+-5.3) g; (F1) Males: mean 55.3-56.0 g; Females: 52.7-53.9 g
- Fasting period before study: males 1 w, females 3 w
- Housing: stainless steel, wire-mesh cages with hard-wood animal bedding (ALPHA-dri, Shepherd Specialty Paper, U.S.A.)
- Diet (e.g. ad libitum): ad libitum; CE-2 feed was monitored for contaminants by the vendor. The contents of genistein and daidzein in the diet, tap water, and wood bedding used in the present study were determined (Diet: genistein, ND-2.1 mg/100 g; daidzein, ND-1.9 mg/100 g, tap water and wood bedding: ND).
- Water (e.g. ad libitum): ad libitum; Water quality was monitored prior to the start of the study to ascertain that contaminants were below the levels set forth in the Safe Drinking Water Act.
- Quarantine period: 6-7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25 °C
- Humidity (%): 50-65%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The stability of NP was confirmed by analyses prior to the beginning and the end of the study. Formulations were also stable for up to 10 days in a cold room in the dark.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): CE-2 feed
- Storage temperature of food: cold room

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: individually, unless they were cohabited or delivered or were nursing a litter.
- Length of cohabitation: 7 d
- Females were subjected to vaginal lavage for 2 weeks prior to the start of NP administration. The lavage fluid was applied to a glass slide, air-dried and stained with Wright-Giemsa stain. Cytology was evaluated and the stage of the estrous cycle was determined using the method of Everett [21]. Females showing regular 4-day estrous cycles were used in the present study.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no data
- M/F ratio per cage: individually, unless they were cohabited or delivered or were nursing a litter.
- Length of cohabitation: 7 d
- Proof of pregnancy: vaginal plug and/or presence of sperm referred to as day 0 of gestation
- Females were subjected to vaginal lavage for 2 weeks prior to the start of NP administration. The lavage fluid was applied to a glass slide, air-dried and stained with Wright-Giemsa stain. Cytology was evaluated and the stage of the estrous cycle was determined using the method of Everett [21]. Females showing regular 4-day estrous cycles were used in the present study.
- After successful mating each pregnant female was caged (how): individually in cages
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Male F0 rats were treated for 12 weeks prior to the 2-week cohabitation, until necropsy. Female F0 rats were treated for 2 weeks prior to cohabitation, until necropsy (including during gestation, delivery, and lactation until day 21 after delivery). Male F0 rats were necropsied at 23 weeks of age (after confirmation of fertility by pairing with females). F1 and F2 animals were treated by oral gavage after weaning (postnatal day 22) until necropsy at age of 10 weeks.
Frequency of treatment:
The formulations were administered daily between 13:00 and 15:00 h by oral gavage at each dose to 25 rats of each sex
Details on study schedule:
- F1 parental animals not mated until x weeks (no data) after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [no data] days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Remarks:
Doses / Concentrations:
2, 10, 50 mg/kg/day
Basis:
actual ingested
main study
Remarks:
Doses / Concentrations:
10, 50, 250 mg/kg/day
Basis:
actual ingested
dose-finding study
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
For the dose-finding study, 8-week-old rats of each sex were treated orally with NP at 10, 50, or 250 mg/kg/day for 2 weeks. Subsequently, individual male and female rats within the same dosage group were cohabited to evaluate their reproductive ability. Exposure to NP continued during cohabitation (at most for 7 days), and female exposure continued during gestation and lactation until postpartum day 13. A marked reduction in body weight gain, and significant increases in liver and kidney weights were observed in both sexes, and slight decreases were seen in the fertility index and number of pups born in the 250 mg/kg group, suggesting that NP at 250 mg/kg/day would exert severe toxicity on adult rats over periods longer than the dose range-finding study. No other parameters, including reproduction in male and female rats, were affected by oral administration of NP at 10 or 50 mg/kg/day. Based on the data generated from this dose range-finding study, the doses for the main study were set at 0, 2, 10, or 50 mg/kg/day NP.
- Rationale for animal assignment (if not random): no data
Positive control:
no
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once or twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed twice a week prior to cohabitation. Females were also weighed on days 0, 4, 7, 14, and 20 of gestation, and on postpartum days 0 (the day of delivery), 4, 7, 14, and 21.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in both sexes twice a week prior to cohabitation, and also in females during days 1-2, 7-8, 13-14, and 19-20 of gestation and during postpartum days 3-4, 6-7, and 9-10.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once or twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: male and female rats were observed for clinical signs once or twice daily during the course of the study.

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: All rats were weighed twice a week prior to cohabitation. Females were also weighed on days 0, 4, 7, 14, and 20 of gestation, and on postpartum days 0 (the day of delivery), 4, 7, 14, and 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: Yes
Food consumption was measured in both sexes twice a week prior to cohabitation, and also in females during days 1-2, 7-8, 13-14, and 19-20 of gestation and during postpartum days 3-4, 6-7, and 9-10.

OTHER: BEHAVIOUR and FUNCTION
One offspring of each sex selected randomly from each litter of each group was subjected to behavioral and functional tests (control group: 22 males and 22 females; 2 mg/kg group: 20 each; 10 mg/kg group: 25 each; 50 mg/kg group: 23 each). At 5 to 6 weeks of age, animals were placed in a circular area (150 cm diameter) surrounded by a wall (40 cm height). The light and noise levels averaged 500 lux and 50 dB, respectively, at the
center of the circular area. Incidences of rearing, grooming, defecation, and urination were counted, and latency time and ambulation were recorded automatically using a computer (Unicom, Inc., Japan) during a 3-min trial on three consecutive days. At 6 to 7 weeks of age, offspring were tested in a water-filled straight channel and a Biel maze. On the first day, each animal was subjected to 5 trials in a 150-cm straight channel as a measure of swimming speed. On days 2 through 4, animals were subjected to 3 trials/day for the maze task in the original path as described by Biel [23]. The water temperature of the maze was maintained at 21 to 22 °C. Swimming time (up to 180 s) taken to reach the goal and number of errors (front-body entries into the blind T of the maze, or turns that were not on the direct path to the goal) were recorded. At 7 to 8 weeks of age, offspring were placed into a wheel cage (Nippon Cage, Inc., Japan) 32 cm in diameter and 10 cm in width as a measure of spontaneous activity. Each animal was kept within the wheel for 24 h with free access to food and water in the same animal room. The number of revolutions was automatically recorded with a 20-channel digital counter (Seiko Denki, Inc., Japan). Male and female offspring that were evaluated for behavior and function as described above were subjected to necropsy at 10 weeks of age, and the testes, epididymides, ventral prostate, and seminal vesicle for males, and uterus (at the stages of diestrus, proestrus, or estrus) and ovaries for females were weighed.

OTHER: ADDITIONAL TEST
In the main study, histopathologic changes of the liver and kidneys were found in F 0 adult males or females of the 50 mg/kg group, and the hormonal status in F 0 males was also altered (see the Results section). To clarify these effects of NP, a higher dose (250 mg/kg/day) than that used in the main study was applied to 25 adult males (6 weeks old) for 12 to 13 weeks and females (10 weeks old) for 2 weeks. Ten rats of each sex were administered (13:00 to 15:00 h) corn oil as controls. Male rats exposed to NP and control male rats (16 to 17 weeks of age) were cohabited with untreated female rats of the same strain up to 14 consecutive days to evaluate their reproductive ability. Female rats exposed to NP were not examined for their reproductive ability because four of 25 females died during the administration period (see the Results section). After the evaluation of reproductive function, histopathologic examination of the liver and kidneys, and determination of serum hormone concentrations of males were performed by the same procedure described above.
Oestrous cyclicity (parental animals):
Estrous cycle length
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
The number of sperm in the right cauda epididymis, and the percentage of motile sperm and progressive motile sperm were determined using a Hamilton-Thorne IVOS analyzer (HTM-IVOS; Hamilton Thorne Research, Inc., Beverly, MA) with software version 10.6 as described previously

Parameters examined in [F1,F2] male parental generations:
The testes, epididymides, and seminal vesicle with prostate in males, and ovaries and uterus in females were weighed. The testes, epididymides, and seminal vesicle with prostate from 10 male weanlings from the 50 mg/kg and control groups were examined histologically.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed by ether anesthesia and subjected to necropsy.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
The numbers of live and dead pups were recorded for each litter from postnatal day (PND) 0 to 21, and viability from PND 0 to 4 (preculling) and viability (weaning index) from PND 4 (postculling) to 21 were determined in each litter. Pups were inspected grossly for external abnormalities on PND 0 and were observed daily for clinical signs from birth until weaning. Pups were sexed, their body weights according to sex were recorded on PND 0 in all groups, and the anogenital distance (AGD) was determined using a digital micrometer for each pup in the NP and control groups. Pup body weights were also recorded on PNDs 4, 7, 14, and 21. Following weaning on PND 21, and until 13 weeks of age, offspring were weighed once a week.
Developmental landmarks in the offspring were monitored on a daily basis for individual rats. Randomly selected pups (two pups/sex/litter) in each group were examined for the development of neural re¯exes (righting response, cliffdrop aversion response, negative geotaxis), and for physical development (pinna opening, upper tooth eruption, eyelid opening).
As criteria for sexual maturation, the timing of vaginal opening for female offspring (beginning on PND 28) and preputial separation for male offspring (beginning on PND 35) were assessed (two/sex/litter) and each pup was weighed when these criteria were achieved. On PND 22, randomly selected offspring (two/sex/litter) in each group were kept in the necropsy room at least 2 h prior to necropsy, and anesthetized with pentobarbital. Blood was collected via the inferior vena cava, and pups were subjected to necropsy (13:00 to 15:00 h).

GROSS EXAMINATION OF DEAD PUPS:
[no/yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: surviving animals of F0 at 23 weeks, surviving animals of F1 and F2 that were evaluated for behaviour and function were subjected to necropsy at 10 weeks of age. Male animals of F1 used for mating were killed as soon as mating was finished.
- Maternal animals: surviving animals of F1 and F2 that were evaluated for behaviour and function were subjected to necropsy at 10 weeks of age

GROSS NECROPSY
Male F0 rats at 23 weeks of age were kept in the necropsy room at least 2 h prior to necropsy to minimize the impact of stress on hormone levels, and anesthetized with pentobarbital. Blood was collected from the inferior vena cava, the rats were subjected to necropsy (13:00 to 15:00 h).

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: brain, heart, lung, liver, spleen, kidneys, adrenal glands, thymus, testes, epididymides, ventral prostate, seminal vesicles, thyroid gland, and pituitary gland
Blood collected at necropsy was centrifuged (3000 g, 15 min) and the serum was separated and stored at ~135 °C until determination of the concentrations of testosterone, LH, FSH, TSH, T 3 , and T 4 .
Subsequently, histopathologic examination of the testes, epididymides, ventral prostate, seminal vesicles with coagulating gland, liver, kidneys, spleen, heart, lung, mammary glands, thymus, thyroid gland, parathyroid gland, pituitary gland, and adrenal glands was performed in 10 males each from the 50 mg/kg group and the control group. The liver and kidneys were examined histologically in 10 males each from the 2 and 10 mg/kg groups, because abnormalities were found in these organs in the 50 mg/kg group.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
In the afternoon (13:00 to 15:00 h) on postpartum day 22, dams were anesthetized with pentobarbital and were subjected to necropsy.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The following organs were weighed:
brain, heart, lung, liver, spleen, kidneys, adrenal glands, thymus, ovaries, uterus, thyroid gland, and pituitary gland
Blood was collected via the inferior vena cava, and pups were subjected to necropsy (13:00 to 15:00 h).
Subsequently, histopathologic examination of the ovaries, oviducts, uterus, vagina, heart, liver, kidneys, urinary bladder, spleen, lung, thymus, mammary glands, thyroid gland, parathyroid gland, pituitary gland, and adrenal glands was performed in 10 dams each from the 50 mg/kg group and the control group. The liver was examined histologically in 10 dams each from the 2 and 10 mg/kg groups, because abnormalities in the liver were observed in the 50 mg/kg group.

The testes, epididymides, and seminal vesicle with prostate in males, and ovaries and uterus in females were weighed. The testes, epididymides, and seminal vesicle with prostate from 10 male weanlings from the 50 mg/kg and control groups were examined histologically. The ovaries and uterus from 10 rats from the 50 mg/kg and control groups were also examined histologically. Blood collected at necropsy was centrifuged (3000 g, 15 min) and the serum was separated and stored at 2135 °C until determination of the concentrations of testosterone, LH, FSH, TSH, T 3 , and T 4 in male off-
spring, and of prolactin, LH, FSH, TSH, T 3 , T 4 , and 17b-estradiol (E 2 ) in female offspring.
Statistics:
Data were analyzed, where appropriate, to determine the statistical significance of differences between the control and NP-treated groups; P < 0.05 and P < 0.01 were taken to indicate statistical significance. Copulation and fertility indices were analyzed by Fisher's exact probability test. Differences in histopathologic findings between the control and NP-treated groups, the graded data (negative 2, very slight 6, slight 1; moderate 11;
severe 111) and total numbers of positives were analyzed by Mann-Whitney's U test and one-tailed Fisher's exact probability test, respectively. Individual data or mean values of each litter were treated as a single samples, and homogeneity of variance of these samples among groups was analyzed initially using Bartlett's test. When homogeneity of variance was confirmed, one-way analysis of variance was applied to detect significance
among groups. If a significant difference was detected among groups, Dunnett's test was applied for multiple comparisons. When variance was not homogeneous or there was any group the variance of which was zero, the Kruskal-Wallis analysis of ranks was applied. If significance was detected among groups, Dunnett's test was applied for multiple comparisons. In an additional study, serum hormone concentrations were compared by Stu
dent's t test to determine significant differences.
Reproductive indices:
Estrous Cycle Length
Mating Index
Pregnancy Index
Fertility Index
Gestation Length
Implantation sites
Offspring viability indices:
Live Pups per Litter
Proportion of Pups Born Alive
Pup Sex Ratio
Live Pup Weight
Pup Survival
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Both sexes: salivation in 50 mg/kg/group

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effects

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined, admistration via gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males: Increased kidney and liver weight; increased relative weight in brain, lung, and liver as well as in thyroid gland and pituitary gland.
Decreased thymus weight
Females: Absolute and relative ovary weights were significantly decreased

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males (50 mg/kg/group): 2x dark kidneys; 1x enlarged thyroid gland

HISTOPATHOLOGY (PARENTAL ANIMALS)
Females: changes in the kidney and liver
Both sexes: 8x centrilobular hypertrophy of the hepatocytes;
Males (50 mg/kg/group): In the kidney, eosinophilic bodies were observed in 7 males of the control group and 3 of the 50 mg/kg group, and the extent of this change in the 50 mg/kg group was significantly decreased as compared with that in the controls. In addition, similar changes in the kidney were found in 6 males of the 2 mg/kg group and in 4 males of the 10 mg/kg group, and the extent of this change was slightly less in the 10 mg/kg group. In other organs, including the reproductive organs, no treatment-related changes were observed in any male rat.

OTHER FINDINGS (PARENTAL ANIMALS)
Serum hormone concentrations: increase in TSH level
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: lack of reproductive effects
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: systemic toxicity: overall effects
Remarks on result:
other: Generation: P, F1, F2 (migrated information)
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: systemic toxicity: increased kidney and liver weight, decreased thymus weight, and histopathologic changes in the kidney (males) and liver liver (both sexes)
Remarks on result:
other: Generation: P, F1, F2 (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:
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
VIABILITY (OFFSPRING)
Whereas no effects on viability were observed in F1 pups, significant decreases were observed in F2 pups in the numbers of implantation sites and pups born alive per litter, and pups alive on pnd 4 as compared with the controls. There were no treatment related changes in sex ratio, body weight, or viability during lactation in any group.

CLINICAL SIGNS (OFFSPRING)
Salivation was observed in male and female offspring of the 50 mg/kg group following daily administration of NP after weaning.

BODY WEIGHT (OFFSPRING)
Body weight gain was significantly reduced from pnd 21 to 91 in the 50 mg/kg/group.

SEXUAL MATURATION (OFFSPRING)
The timing of vaginal opening as an end point of female sexual maturation was significantly accelerated in the highest dose group, while that of preputial separation as an end point of male sexual maturation was not different between groups, and anogenital distance was not consistently changed in male or female offspring exposed to NP.

ORGAN WEIGHTS (OFFSPRING)
Significant increases in liver and kidney weights (relative weight) in male offspring of the 50 mg/kg group were noted, which were similar to those seen in parent (F0) animals exposed to NP at 50 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING)
Males: Relative weights of the kidneys and liver in the 50 mg/kg group were significantly increased as compared with those in the controls. Relative thymus weight in the 2 mg/kg group was significantly decreased as compared with that in the controls, but this effect was not dose-dependent over the dose range examined. There were no significant differences between the groups in reproductive organ weights of male offspring, while significant decreases in the absolute and relative ovary weight were found in the 50 mg/kg as compared with controls.
Females: no effects

HISTOPATHOLOGY (OFFSPRING)
On histopathologic examination of the liver and kidney in postpubertal F1 offspring, centrilobular hypertrophy of the hepatocytes was observed in males and females of the highest dose group, as observed in F0 adult rats. However, no histopathologic changes were detected in the kidneys of male or female offspring exposed to NP even at the highest dose level, whereas a decrease in number of eosinophilic bodies was observed in the kidneys in adult rats.

OTHER FINDINGS (OFFSPRING)
- HORMONE CONCENTRATION
The present study demonstrated that the fetuses or progeny may be substantially more susceptible to insults by environmental chemicals, particularly to endocrine active chemicals, than adults. One of the findings in F1 animals that were exposed to NP across the placenta or via milk in the present study was the change in serum hormone concentrations of male and female rats when determined on pnd 22 or after puberty. In 22-day old rats, a significant increase in FSH concentration and decrease in T3 concentration in males, decreases in LH and TSH concentrations in females, and a significant increase in T3 concentrations in females in the 50 mg/kg group were detected as compared with the respective control levels.
In postpubertal rats, a significant decrease in T 3 concentration was observed in male rats in the 2 and 50 mg/kg groups.

- BEHAVIORAL FUNCTION
A significant increase in the incidence of grooming on the first day in males of the 2 mg/kg group, and decreases in the incidence of ambulation on the first and second day, and a decrease in the incidence of rearing on the second day in females of the 2 mg/kg group were observed as compared with controls, while no significant changes were found in any parameter examined in the open-field test in male or female offspring of the 10 or 50 mg/kg groups. With regard to learning ability in the water multiple T-maze, swimming time at the first trial on the second day in male offspring of the 2 mg/kg group, and that at the first trial on the first day in female offspring of the 50 mg/kg group were significantly increased as compared with the controls. There were no significant differences in spontaneous motor activity between the groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: lack of reproductive effects
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: reproductive effects: decreased number of implantations and number of surviving F2 offspring; decreased ovarian weight, premature vaginal opening, changes in hormone status
Reproductive effects observed:
not specified

Two-generational reproductive effects of treatment of SD rats with Nonylphenol (by gavage)

Generation: Treatment group (mg/kg/day)

F0

F1

F2

2

10

50

2

10

50

2

10

50

General Toxicity Parameters

 

 

 

 

 

 

 

 

 

Adult Body Weights

M-,F↓

 

 

 

Mortality/Moribund

0M,0F

0M,0F

0M,0F

0M,1F

0M,0F

0M,0F

 

 

 

Reproductive Parameters

 

 

 

 

 

 

 

 

 

Implantation Sites

 

 

 

 

 

 

 

 

Live Pups per Litter

 

 

 

 

 

 

 

 

Developmental parameters

 

 

 

 

 

 

 

 

 

Anogenital Distance (PND 0)

 

 

 

NE

NE

NE

Day of Vaginal Opening

 

 

 

NE

NE

NE

Day of Preputial Separation

 

 

 

NE

NE

NE

Sperm Parameters

 

 

 

 

 

 

 

 

 

Concentration (x106 /g)

 

 

 

PND22 Weights

 

 

 

 

 

 

 

 

 

Testes (Relative)

 

 

 

NE

NE

NE

Epididymides (Relative)

 

 

 

NE

NE

NE

Prostate and Seminal Vesicle (Relative)

 

 

 

NE

NE

NE

Ovaries (Relative)

 

 

 

NE

NE

NE

Uterus (Relative)

 

 

 

NE

NE

NE

PND22 Hormone Concentrations

 

 

 

 

 

 

 

 

 

Testosterone

 

 

 

NE

NE

NE

Prolactin

 

 

 

NE

NE

NE

LH

 

 

 

F↓

NE

NE

NE

FSH

 

 

 

M↑

NE

NE

NE

TSH

 

 

 

M↓,F↓

F↓

NE

NE

NE

T3

 

 

 

F↓

M↓,F↑

NE

NE

NE

T4

 

 

 

M↓,F↓

NE

NE

NE

Estradiol

 

 

 

NE

NE

NE

Behavioral and Functional Tests

 

 

 

 

 

 

 

 

 

Water Multiple T-Maze

 

 

 

M-,F↑

 

 

 

Adult Organ Weight

 

 

 

 

 

 

 

 

 

Brain (Relative)

M↓,F-

M↑,F-

 

 

 

Heart (Absolute)

M↑,F-

 

 

 

Lung (Relative)

M↑,F-

 

 

 

Liver (Relative)

M↑,F-

M↑,F-

 

 

 

Kidneys (Absolute)

M↑,F

-

 

 

Kidneys (Relative)

M↑,F

M↑,F-

 

 

 

Adrenal Glands (Absolute)

M↑,F-

 

 

 

Thymus (Absolute)

M↓,F-

 

 

 

Thymus (Relative)

M↓,F-

M↓,F-

 

 

 

Epididymides (Absolute)

 

 

 

Ovaries (Absolute)

 

 

 

Ovaries (Relative)

 

 

 

Uterus (Absolute)

 

 

 

Uterus (Relative)

 

 

 

Thyroid Gland (Relative)

M↑,F-

 

 

 

Pituitary Gland (Relative)

M↑,F-

 

 

 

Adult Histopathology

 

 

 

 

 

 

 

Liver:

 

 

 

 

 

 

 

Hypertrophy, Hepatocyte, Centrilobe

M↑,F-

 

 

 

Kidney:

 

 

 

 

 

 

 

Eosinophilic Bodies

M↓,F-

 

 

 

Basophilic Tubule in Cortex

 

 

 

Adult Hormone Concentrations

 

 

 

 

 

 

 

Testosterone

 

 

 

FSH

M↓

 

 

 

TSH

M↑

 

 

 

T3

M↓

 

 

 

T4

 

 

 


M: male; F: female.

↑: statistically significant increase in the parameter; ↓: statistically significant decrease in the parameter; -: no effect; NE: not examined.


Conclusions:
The NOAEL for reproductive capacity is >= 50 mg/kg/day in parent animals, and 10 mg/kg/day in the next generation. The NOAEL for systemic effects is 10 mg/kg/day in all generations based on organ weight changes and histopathological findings in the liver of both sexes and male kidney at 50 mg/kg/d.
Executive summary:

In a 2-generation reproduction study nonylphenol, NP (> 99%) was administered to 25 Crj:CD (SD) IGS rats/sex/dose by gavage at dose levels of 0, 2, 10, 50 mg/kg bw/day. Litters were culled randomly to eight. Litters of eight offspring or less were not reduced. In addition to standard multi-generation requirements additional behavioural and functional tests were conducted with the offspring.

In the 50 mg/kg/d group histopathologic changes were found in the liver of male and female rats and kidneys of males. Significant increases in the liver, kidney and pituitary gland weights in males, and decreases in thymus weight in males and in ovary weight in females were observed at this dose level. The viability of offspring from postnatal day 0 to 4 in the 50 mg/kg group was reduced as compared with that in the controls, although growth was not affected.

No adverse changes in clinical signs were observed in any rats throughout the study. At necropsy, no treatment-related alterations were observed in any organs including the reproductive tissues in any group. No adverse changes were found in behavior or learning in the offspring of NP-treated groups.

NP did not affect the timing of preputial separation, while vaginal opening was accelerated in the 50 mg/kg group. However, there were no treatment-related changes in any reproductive parameter, including estrous cycle, mating, fertility, delivery, and lactation, except for significant decreases in the numbers of implantation sites and live pups in the 50 mg/kg group. No treatment-related changes were observed in the sperm characteristics.

 

The LOAEL for systemic adult toxicity is 50 mg/kg bw/day in both sexes and all generations, based on results of organ weights and histopathology.  The NOAEL for reproductive capacity is 50 mg/kg bw/day in parental animals and 10 mg/kg bw/day in the next generation under the present experimental conditions.  

 

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study OECD 416 in rats.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 followed
Principles of method if other than guideline:
Intraperitoneal injection of Nonylphenol to verify effects on the reproductive tract of male rats.
Rationale: Verification of surprising effects not reported before P.C. Lee (1998, Endocrine 9, 105-111)
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: Alpk (Wistar derived)
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
arachis oil
Details on mating procedure:
- Alpk:APfSD (Alpk) strain of rats on day 1 of pregnancy (initial body weights 225-275g) were obtained from the AstraZeneca breeding unit (Ald-
erley Park).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The study was carried out using a dose of 8 mg/kg/day of NP, exposed from postnatal days 1-10, used by Lee (1998).
This period was selected because Lee (1998) had shown a > 50% incidence of cryptorchidism with this protocol and had also described reductions in sex organ weights when NP was administered from postnatal days 1-5 or 1-15. Experiment 1 was a simple attempt to reproduce the primary findings of Lee (1998) using a control group and a group exposed to 8 mg/kg/day NP.

(Lee 1998): Lee, P. C. (1998). Disruption of male reproductive tract development by administration of the xenoestrogen, nonylphenol, to male newborn rats. Endocrine 9, 105–111.
Frequency of treatment:
daily from postnatal day 1 to 10
Details on study schedule:
- The pregnant females were allowed to give birth normally (postpartum = d0) and male pups only were dosed via ip from day 1 to day 10 postpartum.
- Litters (4 males per group) received NP (8 mg/kg/day) in arachis oil (5 ml/kg), and controls received arachis oil only (5 ml/kg).
- Furthermore as vehicle DMSO was used (1 ml/kg)
- Female pups were retained with the litter, but not dosed, until weaning at day 28 when the females were culled.
- Testis descent was monitored by observation of the male pups from day 21 until all the testes were descended.
- Testes, epididymides, seminal vesicles (including dorso-lateral prostate), ventral prostate, liver, and kidneys were removed at postmortem and weighed.
- The male pups were killed on day 34 (all males were examined).
Remarks:
Doses / Concentrations:
0, 8 mg/kg/day NP
Basis:
nominal conc.
No. of animals per sex per dose:
4 males
Control animals:
yes, concurrent vehicle
Remarks on result:
not measured/tested
Dose descriptor:
NOAEL
Remarks:
male reproductive tract
Generation:
F1
Effect level:
8 mg/kg bw/day
Sex:
male
Basis for effect level:
other: lack of adverse effect on male reproductive tract
Reproductive effects observed:
not specified

Table 1: The Effect of Neonatal Nonylphenol on Organ Weights of Male Rats

Number  

Terminal body weight 

Testis  

Epididymis  

Seminal vesicles  

Ventral prostate  

analyzed  

(g)  

(g) 

(mg)  

(mg)   (mg)  
Control (Arachis oil)   17 99.2   ± 16.7   0.88    ± 0.20   86.6   ± 16.6   65.0    ± 10.4   56.9    ± 13.0  
NP (8 mg/kg/day) in Arachis oil 17 105.5    ± 12.0   0.93    ± 0.15   95.6   ± 12.5   66.7    ± 11.9   57.5    ± 13.3  
Control (DMSO)   25 134.8    ± 9.0   1.23    ± 0.09   132.2   ± 13.3   74.0    ± 8.0   69.3    ± 11.9  
NP (8 mg/kg/day)   25 134.1    ± 11.0   1.23    ± 0.16   129.4   ± 17.3   69.8    ± 10.1   64.1    ± 12.2  
NP (8 mg/kg/day) and DMSO 25 127.1    ± 7.6   1.16    ± 0.12   126.4   ± 12.8   70.2    ± 10.4   64.8    ± 15.2  

Compounds were administered ip from day 1–10 postpartum (where birth = day 0).

Data are mean absolute weight ± SD.

Conclusions:
None of the effects described by Lee (1998) could be affirmated with the above described experiments. The conclusion is that neonatal exposure (i.p.) of male rats to 8 mg/kg/d Nonylphenol has no effect on the reproductive tract.

Deficiencies in the design an conduct of the investigations by Lee (1998) may be the reason for their surprising results.
The key defincies are:
- ip injection is not an appropriate route of exposure for studies where the neonatal testes are under study;
- the use of DMSO as a vehicle for toxicity studies is questionable, given its membrane permeability and intrinsic toxicity; and in particular,
- ip injection of NP in DMSO provides data for the neonatal rat testes which is of little or no value for human or wildlife risk assessments of this agent
Executive summary:

In an 1-generation reproduction study p-nonylphenol (NP) was administered to 4 male Alpk rats dosed by intraperitoneal injection at dose levels of 0 and 8 mg/kg bw/day. As vehicle and control arachis oil (5 ml/kg) or DMSO (1 ml/kg) was used.

No effects on the reproductive tract of male rats (Testes, epididymides, seminal vesicles (including dorso lateral prostate), ventral prostate, liver, and kidneys) was found.

The study contradicts the results reported by Lee (1998).

This study meets generally accepted scientific standards, is well documented, acceptable for assessment.

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001-06-05 - 2003-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: EPA OPPTS 837.3800 (US EPA OPPTS 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
EPA TSCA GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc. Raleigh NC- Age at study initiation: seven weeks of age at the beginning of exposure, (six weeks of age (42 days) on the scheduled receipt day)
- Weight at study initiation: males: 175-275 g, females: 150-225 g (males: 120-180 g, females: 100-160, on receipt day)
- Quarantine period before study: Animals were quarantined 1 week prior to the start of treatment
- Housing: solid-bottom polycarbonate cages (8''x19''x10,5" high) with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) with Sani-Chip bedding (P. J. Murphy Forest Products, Inc., Montville, NJ)
- Individually housing of each animal during acclimatising period.
- Study animals were housed two per cage (one male, one female from the same dose) during the mating period
- Females were caged individually once they had been successfully mated (or at the end of mating period)
- Diet (e.g. ad libitum): ad libitum, Basic diet: Purina Certified Rodent Chow (No. 5002, PMI Feeds, Inc. St. Louis, MO). In addition NIH-07 (No. 7022 CM, Harlan Teklad, Madison, WI) was used to check the influence of the diet on kidney toxicity effects.
- Water (e.g. ad libitum): tap water (source: City of Durham, Department of Water Resources, Durham, NC) ad libitum in plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes.
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:DIET PREPARATION
- Rate of preparation of diet (frequency): premixes and diets were prepared at a frequency determined by the stability data.
- Mixing appropriate amounts with (Type of food): Purina 5002 / NIH-07
- Storage temperature of food: - 20 °C in appropriate containers until use.
- Diets were prepared in a manner to ensure homogenous distribution in the test material (verified by HPLC)
- Feed in the feed jars was changed at least every seven days.

Water was available ad libitum
Details on mating procedure:
- M/F ratio per cage: 1m/1f
- Length of cohabitation: for a period of up to 14 days (no change in mating partner)
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy/gestation
- When a male died prior to mating: replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually housing
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of dietary concentrations of test material and positive control were performed on all doses and controls for the first three formulations and then for every fourth formulation date thereafter. Standards for acceptable accuracy were: the mean of the analysed samples had to be within 10 % of nominal, and the % RSD (relative standard deviation) for triplicate analyses could not exceed 10 %. Stability analysis: homogeneity and stability of the animal diets at the concentrations used in the study were determined. Diet analysis by high-performance liquid chromatography (HPLC) showed that NP and E2 were mixed homogenously, were stable frozen for at least 49 days and stable for at least 9 days under cage side conditions
Duration of treatment / exposure:
F0 parental animals began exposure at 7 weeks of age and were exposed to their respective diets (25/sex/dose group) ad libitum for a 10 week pre-breed, 2-week mating, 3-week gestation, and 3-week lactation.
Selected F1 and F2 offspring at weaning (one/sex/litter) were directly exposed to their parents' diet for pre-breed, mating, gestation, and lactation.
F3 weanlings (males only, one/litter) were directly exposed to their parents' diets to adulthood until postnatal day (pnd) 110 +- 10.
Frequency of treatment:
Daily
Details on study schedule:
- The dams were allowed to rear their offspring to pnd 21.
- On pnd 21 each F1 litter was weaned.
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- When each F1 litter reached pnd 18 (for females) or pnd 21 (for males), at least one male and one female pup, if possible, were randomly selected to produce the F2 generation.
- Age at mating: 13 to 15 weeks
Remarks:
Doses / Concentrations:
0; 20; 200; 650; 2000 ppm (0; 1,5; 15; 50; 150 mg/kg bw/day)
Basis:
nominal in diet
Purina 5002 diet; F0-F3
Remarks:
Doses / Concentrations:
0, 650 ppm (0, 50 mg/kg bw/day)
Basis:
nominal in diet
NIH-07 diet; F0, F1, F2
No. of animals per sex per dose:
25 animals per sex per dose for F0, F1 and F2 study animals and
25 male animals per dose for F3 study animals (no females)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: historical studies
- Rationale for animal assignment (if not random): confirmation and extend of former findings (historical studies)
Former studies showed effects of kidney toxicity in adult rats exposed to NP.
Furthermore transgenerational effects should be evaluated because they were indicated by a study of Chapin et al. (1999) in the F2 offspring.
Positive control:
Treatment with 17ß-estradiol, 2.5 ppm(25 animals m/f) for F0, F1, F2; (25 males ) for F3
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general condition was checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during prebreed, mating, and post mating holding periods

BODY WEIGHT: Yes
- Time schedule for examinations (sd=study day):
F0, F1, F2 male: sd0, sd7, sd14, sd28, sd35, sd42, sd56, sd63, sd70, sd77, sd84,
F0, F1, F2 female; prebreed and mating: sd0, sd7, sd14, sd28, sd35, sd42, sd56, sd63, sd70, sd84
F0, F1, F2 female; during gestation (gd: gestation day): gd0, gd7, gd14, gd20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as mg 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):
Daily examination during the period of cohabitation (up to 14 days)
Sperm parameters (parental animals):
Parameters examined in all male parental generations:

For adult males, there were no effects on absolute and relative testes or epididymal weights, no effects on cauda epididymal percent motile sperm, percent progressively motile sperm, percent abnormal sperm, or epididymal sperm concentrations. Testicular homogenization-resistant spermatid head counts were also unaffected, as were daily sperm production (DSP) and efficiency of DSP.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day of birth (pnd 0), on pnd 4 and on pnd 21 (weaning) postpartum: yes
- Maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded at weaning.


PARAMETERS EXAMINED
The following parameters were examined in [F1/F2/F3] offspring:
pnd 0: number and sex of pups, stillbirths, live births, grossly examination, physical abnormalities, weight, survival index
pnd 4: number and sex of pups, grossly examination, physical abnormalities, weight, survival index
pnd 21: number and sex of pups, grossly examination, physical abnormalities, weight, survival index

GROSS EXAMINATION OF DEAD PUPS:
Any pup that appeared moribund or died during lactation was necropsied, when possible, to investigate the cause of death, and gross lesions preserved in buffered neutral 10% formalin. Organs were not weighed for these animals.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals of the parental study F0, F1, F2 males were sacrificed after the completion of the delivery period of their litters.
- Maternal animals: All surviving animals were sacrificed after the litter of each generation was weaned.


GROSS NECROPSY
- Gross necropsy included examination of the external surfaces: all orifices, carcass, the thoracid, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs.


HISTOPATHOLOGY / ORGAN WEIGHTS
At scheduled necropsy, the following organs were weighed:
- F0, F1, F2 adult females: paired ovaries
- F0, F1, F2 adult males: paired kidneys, paired testes (and one testis for andrology), paired epididymides (and one auda epididymis for andrology)
The following organs were retained in appropriate fixative for scheduled or possible histopathologic evaluation
- F0, F1, F2 adult females: none
- F0, F1, F2 adult males: paired kidneys, one testis, one complete epididymis, and one epididymis minus the cauda
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at pnd 21.
- These animals were subjected to postmortem examinations as follows:
pnd 4: All culled male pups were weighted and euthanized. Paired kidneys were retained in 10% neutral buffered formalin. Female pups were euthanized by decaption and discarded.
pnd 21: The paired kidneys from the nonselected males were removed and fixed at 10% neutral buffered formalin. The nonselected female weanlings were euthanized by CO2 asphyxiation and discarded.


GROSS NECROPSY
- Gross necropsy included examination of the external surfaces: all orifices, carcass, the thoracid, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs.


HISTOPATHOLOGY / ORGAN WEIGTHS
At scheduled necropsy, the following organs were weighed:
- F1, F2 adult females: paired ovaries
- F1, F2, F3 adult males: paired kidneys, paired testes (and one testis for andrology), paired epididymides (and one auda epididymis for andrology)
The following organs were retained in appropriate fixative for scheduled or possible histopathologic evaluation
- F1, F2 adult females: none
- F1, F2, F3 adult males: paired kidneys, one testis, one complete epididymis, and one epididymis minus the cauda
- F1, F2, F3 pnd 4 male culled pups: paired kidneys
- F1, F2, F3 pnd 21 unselected male pups: paired kidneys
- F1, F2, F3 pnd 4 and pnd 21 female pups: none
Statistics:
For comparison of treatment groups standard ANOVA techniques (analysis of variance) were used.
Homogeneity of variance assumption was examined via Levene's Test (Levene, 1960)
If Levene's test indicated lack of homogeneity of variance (p<0.05), robust linear regression methods were used to test all treatment effects. They swere used for overall treatment group differences (Wald Chi-Swuare Tests), as well as the presence of linear tests, followed by individual t-tests for exposed vs. control group comparisons when the overall treatment effect was significant.
Standard ANOVA methods, as well as Levene's test, were implemented in the GLM procedure of SAS(R) Release 6.12 (SAS Institute Inc. 1989 - 1997) and the robust linear regression methods were implemented in the REGRESS procedure of SUDAN (R) Release 7.5.3 (Shah et al. 1997)
Reproductive indices:
- Mating index = no. of females that mated / no. of females paired respectively no. of males that mated / no. males paired
- Gestational index = no. of females with live litters / no. of females pregnant
- Fertility index = no. males siring litters / no. males that mated
- Pregnancy index = no. pregnant females / no. males that mated
Offspring viability indices:
- Stillbirth index = no. dead on pnd 0 / total no. on pnd 0
- Live birth index = no live on pnd 0 / total no. on pnd 0
- 4 day survival index = no surviving 4 days / no. live on pnd 0
- Lactational index = no. surviving 21 days / no. live on pnd 4
Clinical signs:
no effects observed
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 specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Treatment related effects for the 2000 ppm (males and females) and 650 ppm (females only) NP groups and for the 2.5-ppm E2 exposed group in life were limited to reduced body weights and in some cases, associated reduced feed consumption, primarily in the pre-breed exposure period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment related effects were observed on the F1, F2, or F3 males for absolute and relative testes weights, percent motile cauda epididymal sperm, percent progressively motile sperm, or percent abnormal sperm.
Relative (but not absolute) paired epididymal weights were significantly increased for F2 and F3 (but not for F0 and F1) males at 2000 ppm NP
Testicular homogenization-resistant spermatid head counts were also unaffected, as were DSP and efficiency of DSP (calculated from spermatid head counts).
No treatment related effects were observed for cauda epididymal sperm parameters including concentration, motility, or morphology.

For E2-exposed males there were reduced epididymal sperm concentrations in adult F1, F2, and F3 males. Reduced fertility, gestational and pregnancy indices, and reduced numbers of total and live-pups/litter on pnd 0 were also observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reduced absolute and relative paired ovarian weights were observed at 2000 ppm NP for F0, F1 and F2 females, at 650 ppm in Purina feed for F1 females, and at 650 ppm NP in NIH-07 for F0 and F2 females.
For the NP-exposed groups, reproductive indexes, precoital interval, offspring lactational survival indexes, and litter sizes (pre- and postcull) were unaffected.
Gestational length in days was significantly reduced in F1 and F2 females (but not in F0 females) at 650 ppm NP in NIH-07, relative to the NIH-07 control but not to the Purina 5002 control group value. This finding is because the values at 0 ppm NP in NIH-07 in F1 and F2 females were, in fact, increased relative to concurrent and historical control values in Purina 5002.


ORGAN WEIGHTS (PARENTAL ANIMALS)
For males fed Purina 5002 feed, absolute weights of kidneys were significantly increased at F1 Generation at 200, 650 and 2000 ppm
and in the F0 and F2 generation only at 650 ppm NP.
Absolute kidney weight was unaffected in F3 males at 2000 ppm.
Absolute kidney weights were also increased at 650 ppm NP in NIH-07 diet for F0 and F2 males.

Relative kidney weights were increased in F0 and F2 generations at 650 and 2000 ppm and in F1 generation at 650 and 2000 ppm.
Relative kidney weight was increased for all three generations in males given NP at 650 ppm in NIH-07 diet.

For E2-exposed males, absolute and relative paired kidney weights were significantly reduced for F0 and F1 males. Absolute (but not relative) paired kidney weights were significantly reduced for F2 and F3 males. Relative (but not absolute) paired testes weights were significantly increased for E2-exposed F0, F1, F2, and F3 males.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Kidney toxicity was observed histopathologically, manifested as medullary cyst at 2000 ppm NP in Purina and at 650 ppm NP in NIH-07 feed, mineralization at the corticomedullary junction at 650 ppm NP in Purina and NIH-07, and at 2000 ppm NP, and tubular nephropathy at 2000 ppm NP in Purina and 650 ppm NP in NIH-07.

For E2-exposed males increased incidences of mineralization at the corticomendullary junction were present in F0, F1, and F2 males while the incidence of nephropathy was decreased in the E2-treated males compared to the controls.
Absolute and relative ovarian weights were reduced in F0, F1 and F2 for E2-exposed adult females.
Dose descriptor:
NOAEL
Remarks:
as selective reproductive toxicant
Effect level:
>= 150 mg/kg bw/day
Sex:
male/female
Remarks on result:
other: Generation: all (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
VIABILITY (OFFSPRING)
There were no treatment related effects on sex ratios or pup death for NP-exposed animals, but for E2-exposed parents reduced numbers of total and live-pups/litter on pnd 0 were observed (see table 6). Reduced numbers of F1, F2, and F3 litters and reduced numbers of live pups/litter at birth were also observed when exposed to E2.

BODY WEIGHT (OFFSPRING)
Pup body weight per litter was reduced for all F1, F2, And F3 pups at 2000 ppm NP at ond 21 but not at earlier time points.

SEXUAL MATURATION (OFFSPRING)
For E2 exposed animals, anogenital distance at birth was unaffected in both male and female offspring; acquisition of puberty was significantly accelerated by 8.7 days (VP) in female offspring and significantly delayed by 7.0 days (PPS) in male offspring.

ORGAN WEIGHTS (OFFSPRING)
Weights of kidneys were significantly increased at F1 Generation at 200, 650 and 2000 ppm and in the F0 and F2 generation only at 650 ppm.
Absolute kidney weight was unaffected in F3 males at 2000 ppm.
Absolute kidney weights were also increased at 650 ppm NP in NIH-07 diet for F0 and F2 males.

Relative kidney weights were increased in F0 and F2 generations at 650 and 2000 ppm and in F1 generation at 650 and 2000 ppm.
Relative kidney weight was increased for all three generations in males given NP at 650 ppm in NIH-07 diet.


HISTOPATHOLOGY (OFFSPRING)
see results parental animals
Dose descriptor:
NOAEL
Remarks:
male rat kidney toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Kidney toxicity was observed histopatologically, manifested as medullary cysts
Reproductive effects observed:
not specified
Conclusions:
The study confirmed that dietary NP is not a selective reproductive toxicant.The NOAEL is at or above 2000 ppm (>= 150 mg/kg/day).
The study provided a NOAEL for male kidney toxicity of 200 ppm ( 15 mg/kg /day)
Executive summary:

In a 3.5-generation reproduction study Nonylphenol CAS 84852 -15 -3 (94.25% a.i.) was administered to 25 Sprague-Dawley rats/sex/dose in two different diets at dose levels of 0, 20, 200, 650, 2000 ppm (0, 1.5 , 15 , 50, 150  mg/kg bw/day) (Purina 5002 diet) and 650 ppm (NIH-07 diet as reference). On pnd 4, the size of each litter was adjusted to 10 pups by eliminating extra pups by random selection to yield, as nearly as possible, five males and five females per litter.

 

Reproductive toxicity

There were no treatment-related effects on reproductive parameters in this study.

There were not effects on the absolute epididymal weights in any NP group for any generation. Importantly andrological assessments were unaffected across all NP groups and generations.

In this study ovarian weights were reduced at 2000 ppm in F0 females, at 650 and 200 ppm NP in F1 females in Purina and at 650 ppm NP in F0 females in NIH-07.There was no effect on reproduction in the present study as a result of these ovarian weight changes.

 

The only treatment-related effect on the offspring in the present study was a decrease in pup body weight at weaning at the highest dietary concentration. The decrease in weight was not present earlier in the lactation period. Therefore, it was concluded that the reduced body weight resulted from the high intake on a mg NP/kg/day basis (As much as the 2.5 times adult intake) that occurs when the pups begin to self-feed in pnd 14 (as well as possible translactational exposure and/or coprophagia) resulting in a direct toxic effect from overexposure to NP.

 

This study demonstrates a lack of transgenerational effects on epididymal sperm counts or on any other reproductive endpoints and confirms that NP is not a selective reproductive toxicant with a reproductive toxicity NOAEL(repro) of > 2000 ppm (appr. 150mg/kg/day) in the diet.

 

Renal toxicity:

In the present study there were no treatment-related increases in medullary cysts at 200 and 650 ppm NP in Purina 5002 or at 650 ppm NP in NIH-07 in F0, F1, or F2 males. A treatment-related increase in the incidence of medullary cysts was observed in the F0 (2/10), F1 (4/10), and F2 (8/10) males at 2000 ppm NP in Purina 5002.

Mineralization at the corticomedullary junction was observed at 650 ppm and 2000 ppm in Purina 5002 and at 650 ppm NP in NIH-07 in all three male generations evaluated (F0, F1, and F2), with no increased incidence at 200 ppm NP in Purina 5002 (0, 2, and 0 in F0, F1, and F2 males, respectively)

 

This study verified renal toxicity in F0 adult males and F1, F2 male offspring at 650 and 2000 ppm.

Although increased absolute and relative kidney weights were observed in F1 males at 200 ppm NP, they were not associated with increased incidence of the two microscopic findings (medullary cysts and mineralization at the cortico-medullary junction) and there were no renal effects (organ weights or histopathology) in F0 or F2 males at 200 ppm NP.

 

ALOAEL of 650 ppm is concluded based on histopathological findings in the kidney of male rats. The NOAEL is 200 ppm (15 mg/kg bw/days). 

 

This study is acceptable and satisfies the guideline requirements for a 3.5-generation reproductive study OPPTS 837.3800 in rats.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
150 mg/kg bw/day

Effects on developmental toxicity

Description of key information

- IBR Forschungs GmbH (1992) conducted an OECD 414 study (GLP). p-Nonylphenol (93,2%) was administered 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

- Ferguson (2000) conducted a non guideline study to determine the effect of NP on gestation time, birth weights, litter sizes, sex ratios of pups and neurobehavioural tests on progenies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline available
Principles of method if other than guideline:
Para-Nonylphenol was administered to pregnant SD rats in diets beginning on gestational day (GD) 7. At postnatal day (PND) 21, offspring continued on the same maternal diets until PND 77 and were evaluated for behavioural alterations (open-field activity, play behavior, running wheel activity, flavored solution intake).
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
no data
Duration of treatment / exposure:
F0: from gestation day 7
F1: from postnatal day (PND) 21 to 77
Frequency of treatment:
daily
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
Dose / conc.:
2 000 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Key result
Dose descriptor:
LOAEC
Effect level:
25 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined
Abnormalities:
not specified
Developmental effects observed:
not specified

During pregnancy and lactation, dams in the 25-, 500-, and 2,000 ppm groups consumed 9 to 25% less food, which was associated with a 17% less weight gain during GDs 1 to 21 in dams of the 2,000-ppm group, although this effect was not statistically significant. Gestation duration, birth weight, sex ratio of live pups, and number of live or dead pups per litter did not differ between treatment groups. Offspring body weight and food consumption were decreased in the 2,000 ppm group beginning at PND 28; however, an effect of feed aversion could not be eliminated. Behavioral assessments of offspring indicated no consistent NP-related effects in open-field activity at PNDs 22–24, 43–45, and 65–67 nor in running wheel activity at PNDs 63–75. Play behavior at PND 35 and intake of a 0.3% saccharin-flavored solution at PNDs 69–71 did not differ with respect to treatment groups. However, intake of a 3% sodium-flavored solution at PNDs 73–75 was significantly increased in offspring of the 2,000-ppm group and intake of regular water during this same time was also significantly increased.

Conclusions:
Developmental and lifelong exposure to NP resulted in significant decrements in body weight and food consumption of dams and/or offspring, but had little effect on the assessed behaviors.
Executive summary:

Female SD rats (n = 9 -11) were given diets containing para-Nonylphenol at concentrations of 0, 25, 500, or 2000 ppm from gestation day 7 to 21. After lactation, offspring was administerd with the same diet until postnatal day 77. Dams in all dose groups consumed 9-25% less food. There was no difference in gestation period, birth weight, sex ratio of live pups, and number of live pups per litter in any dose group. Behavioral alterations were evident only in increased intake of a sodium solution. There were no consistent effects in open-field activity nor in running wheel activity. Play behavior and intake of a 0.3% saccharin-flavored solution did not differ with respect to treatment groups.

These results indicate that developmental NP treatment results in maternal and offspring toxicity as evidenced by decreased food intake. However, behavioral alterations were evident only in increased intake of a sodium solution.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991/08/28 - 1991/11/27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted May 12, 1981
Deviations:
not specified
Qualifier:
according to
Guideline:
other: 88/302/EEC
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Versuchstierzucht, 8741 Sulzfeld, Germany
- Age at study initiation: females about 8 w
- Weight at study initiation: a) 183 to 369 g (groups I (a), II, III; b) 203 to 269 g (groups I (b), V)
- Fasting period before study: no data
- Housing: Makrolon cages, cleaned twice a week
- Diet (e.g. ad libitum): as libidum, "Ssniff R" pelleted diet (Alleindiät für Ratten)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a) 7 days, b) 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 °C +- 1,5 °C
- Humidity (%): rel. humidity: 50 to 70%,
- Air changes (per hr): 16 times per hour, (filtered adequately)
- Photoperiod (hrs dark / hrs light): artificial light (120 lux) from 7.00 a.m to 7.00 p.m.


IN-LIFE DATES: 20 days (gestation day 0 - 20)
date of receipt: a) August 21, 1991; b) October 16, 1991
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared freshly each day according to study specific SOP


VEHICLE
- Justification for use and choice of vehicle (if other than water): according test substance data sheet
- Concentration in vehicle: 0 g/l, 15 g/l, 30 g/l, 60 g/l
- Amount of vehicle (if gavage): 5 ml/kg b.w.
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC Analysis of nonylphenol.
The test article was analysed by reverse phase HPLC in corn oil samples. The samples were taken from dosing solutions prepared for the teratogenicity study in Wistar rats, treated orally. The analysed concentration values of the samples show good accordance with the nominal values.
Details on mating procedure:
- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:2
- Length of cohabitation overnight:
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear and / or vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
6-15 day of gestation
Frequency of treatment:
Daily per oral administration, adjusted daily according to the weight development of the animals
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
groups number of females animal no. daily dose (mg/kg b.w.) volume of administration concentration (g(l)
I (a) 32 151-182 0 5 0
I (b) 25 101-125 0 5 0
V 25 551-575 75 5 15
II 25 251-275 150 5 30
III 32 351-382 300 5 60

vehicle: corn oil
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: preliminary dose range finding IBR project no.: 20-04-0502/01-91
- Rationale for animal assignment (if not random): random
- Justification for route of administration: oral in order to obtain high resorption rates and is recommended by OECD guideline
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the entire period of the investigation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the entire period of the investigation.
With regard to: sensory and motor behaviour, coat, urine and fecal excretion, conditions of body orifices.
Viability: checked twice a day
Mortality was recorded on a daily basis.
Additionally, dose response examinations were carried out in appropriate intervals after administration of the test article during treatment.
During gestation females were observed closely for signs of abortion or premature delivery.


BODY WEIGHT: Yes
- Time schedule for examinations: daily during the dosing period
- Recorded only at the beginning of the study (day 0) and additionally at days 6, 10, 15, 20 of gestation
- Body weight development was evaluated over the following phase: 0-6, 6-15, 15-20 and 0-20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and evaluated for body weight development: Yes
- Food consumption was determined by re-weighting non-consumed diet at the end of each weighting interval.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: reproductive organs


OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weights of fetuses and placenta: Yes
- Number of corpora lutea: No data
- Number of implantations and locations: Yes
- Number and location of live and dead fetuses: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (visceral effects)
(Ref. Wilson, J.G. (1965) Methods for administering agents and detecting malformations in experimental animals
in: J.G. Wilson and J. Warkany (eds) Teratology, Principles and Techniques, Univ. of Chicago Press, Chicago, London, pp. 262-277)
- Skeletal examinations: Yes: half per litter
(Ref. Dawson, A.B. (1926), A note on the staining of the skeleton of cleaned specimens with alizarin red S, Strain Technology 1, 123-124
- Head examinations: No data
Statistics:
Analyses of Variance with a subsequent multiple range test for growth (body weight changes and food consumption) and reproduction parameters (numbers of fetuses, resorptions, implantations, corpora lutea and weights of fetuses, placentae and uteri) or if indicated
group mean values were compared by the "Kruskal-Wallis test" and "Mann-Whitney U-test".
The maternal weight on day 20 of gestation was corrected for gravid uterine weight and the corrected rate of body weight gain (day (0-20 of gestation) was calculated.
Statistical evaluation of dose groups II and III was done relative to control group I (a), and dose group V relative to control group I (b)
Indices:
Abortion rate, resorption rate, pre-implantation loss index, post-implantation loss index, live birth index, runts index, variation index, anomaly index, malformation index
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical observation:
- Salivation was noted after treatment of all dose groups
- An increased discharge of urine was found in all animals of the high dose group by estimation of the degree of moisture in the bedding. The onset of this sign was predominantly at day 8 of gestation.

2. Abortion rate
- No females showed signs of abortion or premature delivery in the course of the study.

3. Body weight development
- Body weight gain of group III females was significantly reduces during treatment during the whole study period. Both, the total and the corrected weight gains were affected from day 0 - 20 of gestation.
- Weight gains of group II and V did not differ significantly compared either to group I (A) nor group I (b).

4. Food consumption
In correspondence to the weight gain, food consumption of group III females was significantly decreased between days 6-15 and 0-20 compared to group I (a)

5. Necroscopy findings
A slight dose relationship may be found in the findings of kidneys and spleen in group II and III.
Five females of group III and one female of group II had pale kidneys, 2 group III females showed irregularly shaped kidneys. Two females of group III and one of group II showed reddening of renal pelvis. In 2 group III females and one of group II, the spleen appeared reduced size.
In group IV, treatment with 600 mg/kg was stopped due to the high mortality of animals, pale kidneys (16 of 18 animals) and spleens with reduced size (17 of 18 animals) were found. In 4 animals irregularly shaped kidneys were noted and in 8 animals a reddening of medulla or pelvis.
These findings seem to elucidate the above described dose relationship.
The effect on the kidneys seems to correspond to the increased discharge of urine which was observed during clinical observations in group III.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- The mean number of fetuses did not differ significantly between control and treated groups.
- The mean number of fetuses in the left or right uterine horns did not differ significantly between control and treated groups.
- No biological significant differences were determined among groups concerning the number of fetuses with head or tail presentation
- The sex ratio of male and female fetuses did not differ significantly between control and treated groups.
- The mean fetal body weight did not differ significantly between control and treated groups (irrespective of sex).
- The mean placental weights did not differ significantly between control and treated groups.
- No runts were found in the dose groups.
- There were no dead fetuses in any group.
- The number of resorptions did not differ significantly between control and treated groups.
- Resorption index and post implantation index did not differ significantly between control and treated groups.
- The pre-implantation loss index did not differ in a clearly dose-related manner.
- The mean numbers of implantation did not differ significantly between control and treated groups.
- No relevant differences were found in the left/right intrauterine distribution
- The total number of corpora lutea did not differ significantly between control and treated groups.
- Respective numbers with regard to the left and right ovary did not differ significantly between control and treated groups.

- One malfunction was found in group I (b) and one minor abnormality was found in each of the groups I (a) and V. These findings are considered to be incidental
- Skeletal examination: One malformed fetus was found in each of the control groups.
The incidence of skeletal abnormalities did not reveal any evidence of a treatment effect.
Incomplete ossification of the skull was observed in 76% of the litters and in 41% of the fetuses of group III, which seemed slightly increased compared to group I (a). However, historical data show a mean index of 80,1% of litters and 47,6% of fetuses. => no indication of treatment related differences.
- No abnormalities were found during visceral examination of fetuses.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)

 

 

Group

 

 

Ib

V

I

II

III

1.    Caesarean Section

a. Fetuses

Number of fetuses

mean

15.0

14.0

15.1

15.3

13.5

S.D.

2.3

2.4

4.1

2.0

3.8

Sex Ratio (m/f)

 

1.0

1.0

1.3

0.9

1.1

Weights of fetuses (g)

(Males / Females)

mean

3.91

3.94

3.92

3.96

3.98

S.D.

0.20

0.30

0.31

0.44

0.48

Weights of fetuses (g)

(Males)

mean

4.03

4.01

4.02

4.08

4.11

S.D.

0.21

0.28

0.32

0.46

0.50

Weights of fetuses (g)

(Females)

mean

3.82

3.88

3.82

3.87

3.89

S.D.

0.21

0.31

0.30

0.43

0.49

Dead fetuses

n

0

0

0

0

0

Live Births Index (%)

 

100.0

100.0

100.0

100.0

100.0

Runts Index (%)

mean

0.3

0.0

0.3

0.0

0.0

b. Resorptions

Number per dam

mean

0.9

1.3

1.3

1.5

1.9

 

S.D.

0.9

1.4

1.3

1.2

1.8

Early Resorption

mean

0.9

1.1

1.3

1.4

1.5

 

S.D.

0.9

1.1

1.3

1.3

1.6

Late Resorption

mean

0.0

0.2

0.0

0.1

0.4

 

S.D.

0.2

0.7

0.2

0.4

0.8

Resorption Index (%)

mean

5.7

8.3

7.6

8.8

12.4

Pre-implantation Loss Index (%)

mean

13.5

18.5

7.2

5.6

14.6

c. Implantations

mean

16.0

15.3

16.4

16.9

15.3

S.D.

2.2

2.3

4.3

2.3

3.7

Post-implantation
Loss Index (%)

mean

5.7

8.3

7.6

8.8

12.4

d. Placentae

Mean Weights (g)

mean

0.53

0.53

0.54

0.55

0.58

 

S.D.

0.08

0.05

0.06

0.06

0.07

2.    Fetal Examinations

Malformed Fetuses (skel. + visc.)

 

1

0

1

0

0

Malformation Index (%)
(skel. + visc.)

 

0.3

0.0

0.3

0.0

0.0

Malformed Fetuses (external)

 

1

0

0

0

0

Malformation Index (%) (skel. + visc.+external)

 

0.3

0.0

0.3

0.0

0.0

Litters with Malformations

 

1

0

1

0

0

Variations

Skel.

139

101

114

103

91

Variation Index, skel.

(%)

75.1

60.8

66.3

62.4

61.9

Anomalies

Skel.

34

46

35

40

34

Anomalies Index Skel.

(%)

18.4

27.7

20.3

24.2

23.1

Conclusions:
A maternal NOAEL of 75 mg/kg bw/day can be concluded when administering NP by oral gavage to female rats from day 6 to day 15 of gestation. At 150 mg/kg bw/day 3 of 21 females showed affected kidneys or spleens. However, the dose level of 300 mg/kg caused clear maternal toxic effects like increased mortality, reduced body weight gain and food consumption. With regard to the embryo-fetal development the NOAEL is at or above 300 mg/kg bw/day.
Executive summary:

In a teratogenicity study according OECD 414, Nonylphenol was administered to Wistar Rats (112 pregnant rats and 2219 fetuses) by gavage at dose levels of 0, 75, 150, 300 mg/kg bw/day from day 6-15 of gestation.

Treatment of the pregnant females at a dose level of 75 mg/kg bw was without any general toxicological effect. At a dose level of 150 mg/kg only 3 of 21 females showed affected kidneys or spleens. A dose level of 300 mg/kg bw caused clear maternal toxic effects based on increased mortality, reduced body weight gain and food consumption. With regard to the embryo-fetal development a NOAEL of >= 300 mg/kg was found. 

This teratogenicity study in the Wistar rats is acceptable and satisfies the guideline requirement for a teratogenicity study according OECD 414 in rats conducted under GLP.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Sufficient to address requirements.
Additional information

Developmental toxicity was evaluated by means of an OECD 414 guideline study (IBR, 1992) and additional non guideline studies investigating the effect of prenatal exposure on testicular toxicity (de Jager, 1999) or Neurobehaviour (Ferguson 2000).

In a well conducted OECD 414 guideline study (GLP) 25 femal Wistar rats were exposed to 0, 75, 150, 300 mg NP/kg/d by gavage at gestation days 6-15 (IBR Forschungs GmbH 1992). 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, foetal weights and incidence of both major and minor foetal abnormalities (visceral and skeletal) were not observed. The study provides no evidence of developmental toxicity in the rat at exposure levels which are toxic to the mother; thus the maternal NOAEL was 75 mg/kg/day and the foetal NOAEL was 300 mg/kg/day.

De Jaeger (1999) investigated testicular toxicity. This study was assessed in the EU RAR 2002. Ten female SD rats were exposed by gavage at doses of 100, 250 and 400 mg/kg/d from gestation day 7 until weaning of their litter. Offspring was exposed in utero, during lactation and by gavage until sexual maturity. No explicit information was presented on maternal toxicity but it was stated that no females showed any physical or behavioural abnormalities. No offspring were born in the high dose group. It is not clear from the report if this was because of maternal deaths or embryonic/foetal resorption. There were no malformations or still births among the F1 Offspring. F1 body weight gain was significantly reduced at 100 and 250 mg/kg/day. F1 absolute (but not relative) testicular and epididymal weights were reduced at 100 and 250 mg/kg/day. The differences in absolute organ weight are likely to be related to the intergroup bodyweight differences. Total cauda epididymal sperm count was reduced at 250 mg/kg/day. Seminiferous tubule diameter was slightly lower in both nonylphenol treated groups. Tubule lumen diameter and seminiferous epithelium thickness were reported to be significantly lower at 100 and 250 mg/kg/d, but no data were presented. However, this effect may be related to lower absolute testicular weight. Histopathology revealed pathological changes in the testes of one F1 male from the 100 mg/kg/day group. However, these histopathological abnormalities were not dose dependent, so the changes outlined above cannot be attributed to nonylphenol treatment. The evidence of a reduction in sperm count at 250 mg/kg/day reported is in contrast to a reliable multi-generation study conducted by Tyl (2006). It is not clear if the changes in the tubular measurements represent specific reproductive toxicity or non-specific secondary consequences of the reduction in bodyweight gain. Ferguson (2000) administered 4-NP to pregnant rats at 0, 25, 500, and 2000 ppm in the diet, starting from gestation day 7 until after weaning. NP had no effects on gestation time, birth weights, litter sizes and sex ratios of pups. Neurobehavioural tests on progenies showed no differences compared to the controls. At 2000 ppm NP body weight gain and food consumption were reduced indicating systemic toxicity.

 

According to the results of a valid guideline teratogenicity study (OECD 414) as well as data from supporting studies there is no evidence of developmental toxicity of NP.

Justification for classification or non-classification

There is no indication for effects of NP on male fertility or developmental toxicity in both sexes. Effects observed on female fertility at doses of about 50 mg/kg bw/d (accelerated vaginal opening and reduced ovary weights) lead to some suspicion that NP might have an adverse effect on female fertility [and/or development]. Therefore keeping the current classification of NP as R62/63 (H361fd) is proposed.