Phenol, 4-nonyl-, branched

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

publication

Title:

Reproductive Toxicity of Male Mice after Exposure to Nonylphenol

Author:

El-Dakdoky MH, Helal MAM

Year:

2007

Bibliographic source:

Bull Environ Contam Toxicol (2007) 79:188–191

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

p-nonylphenol

EC Number:

203-199-4

EC Name:

p-nonylphenol

Cas Number:

104-40-5

IUPAC Name:

4-nonylphenol

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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)

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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 10^6) sperm/m^3	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.

Applicant's summary and conclusion

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.