Registration Dossier

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

46 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

high

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

About 90 studies were reviewed in the evaluation of the reproductive toxicity of DEHP. Collectively, these studies were undertaken predominantly in rodents and build on the original observation that DEHP produced testicular atrophy in a subchronic toxicity study. The literature contains many redundant studies, usually at high doses (e.g., 2 g/kg, usually in rats), all of which show similar effects on the testes. A number of more specific studies in the rat have attempted to investigate the mode of action of DEHP using in vivo and in vitro protocols.

Multigeneration reproductive toxicity studies

Rats

Oral

Wolfe and Layton (2004)studied the multigenerational reproductive toxicity of DEHP in Sprague-Dawley rats. The methodology used in this study to a large extent complied with OECD Guideline 416. The number of animals in each test and control group was 17 males and 17 females only (the Guideline recommends a sufficient number of animals in each test and control group to yield preferably not less than 20 pregnant females at or near parturition). However, it is considered that enough pregnancies were produced in the study to assure a meaningful evaluation. Therefore the failure to achieve the desired number of pregnant animals does not invalidate the study. The F0 animals were administered the test article during a 6-week pre-mating period (according to the Guideline a dosing continued for at least 10 weeks before the mating period is required). This, however, is not considered to be a serious deviation since the study was conducted on three generations instead of two, and males of two generations (F1 and F2 animals) were dosed during complete spermatogenic cycle. Thus the study has provided satisfactory information concerning effects on spermatogenesis.

The 10,000-ppm animals only completed the F1 generation and were terminated due to the inability to produce any F2 generation animals. This, however, is not considered to be a serious deviation since the number and choice of original dose levels (10,000-ppm group not included) is considered to be satisfactory for the purpose of the study.

In excess of Guideline requirements, crossover cohabitation was performed on the control and selected F1/F2 animals. This is considered to be scientifically advantageous as it may provide additional data on effects on fertility. Except for females during the lactation period body weight and food consumption of parent animals was measured at limited time points approximately every second week (the Guideline recommends weekly measurements at a minimum). However, the limited number of measurements made in this study is considered to be satisfactory for the purpose of the study.

At the time of termination brain and spleen were not weighed in adults or in pups. Nor where thymus weighed in pups. However, the deviations from the Guideline do not affect the scientific validity of this study. A complete necropsy was performed on all surviving control animals and 10 treated animals from each dose group for each sex (according to the Guideline full histopathology should be performed for all high dose and control animals selected for mating, and organs demonstrating treatment-related changes should also be examined in the low-and mid-dose groups). However, tissue samples from a number of 10 animals/sex/group are in this study considered enough to assure a meaningful histopathological examination. The deviations and/or omissions from the Guideline do not affect the scientific validity of this study.

The study is performed according to GLP (although statement not yet signed), and considered acceptable.

DEHP (purity 99.8%) was administered in the diet at concentrations of 1.5 (Control 1 and 2), 10, 30, 100, 300, 1,000, 7,500 and 10,000-ppm to groups of 17 male and 17 female Sprague-Dawley Crl: CD®BR rats (source: Charles River Laboratories, Portage, Michigan). The control dose level was set at 1.5-ppm, as this was the amount of DEHP found in the control feed. The 10,000-ppm group and their corresponding control group (Control 2) were added to the study structure after the initiation of the original seven dose groups and followed the same study design. Mating pairs were allowed to produce three litters (a, b, c) each.

Animals in the F0 generation began exposure as adults (5 weeks of age) and were bred to produce the F1 generation (F1a, 1b, 1c), the F1 adults (selected from F1c weanlings) were bred to produce the F2 generation (F2a, 2b, 2c), and the F2 adults (selected from F2c weanlings) were bred to produce the F3 generation (F3a, 3b, 3c). The animals were administered the test article during the pre-mating period (6 or 10 weeks), and also during the mating-, gestation- and lactation periods for breeding of the F1, F2 and F3 litters/pups until the day of necropsy (approximately 2 weeks after the last weaning). The F1, F2 and F3 animals received diets containing DEHP after weaning (day 21 post partum) with the same concentration of DEHP as their parents received until necropsy. Additional non-mating males (up to three per litter) were selected from the F1c, and F2c litters, and were maintained following similar procedures as those for mating males, except they were not cohabited with females. The 10,000-ppm animals only completed the F1 generation and were terminated due to the inability to produce any F2 generation animals. A one-week cross over cohabitation was performed on the control and 10,000-ppm F1 animals (up to 17 animals/sex/group), and on the control and 7,500-ppm F2 animals (up to 17 animals/sex/group) in order to determine the affected sex. F1 and F2 animals were then paired with naive animals and received control feed during the cohabitation. Upon separation, the F1 and F2 animals received dosed feed.

Parameters evaluated over the course of the study included body weights, feed consumption, clinical observations, reproductive performance, anogenital distance, pup survival, sexual development, oestrous cyclicity, sperm endpoints, gross pathology, organ weights, and limited/selected histopathology. Based on measured feed consumption, mg/kg daily doses were calculated to be 0.12, 0.78, 2.4, 7.9, 23, 77, 592 and 775 mg/kg bw/day in the F0 animals; 0.09, 0.48, 1.4, 4.9, 14, 48, 391 and 543 mg/kg bw/day in the F1 animals; and 0.1, 0.47, 1.4, 4.8, 14, 46 and 359 mg/kg bw/day in the F2 animals.

Parental data(general condition and behaviour, bodyweight, food intake)

The incidence of intercurrent deaths amongst treated F0 animals (17 animals/sex/group) was 1, 1, 0, 0, 1, 1, and 0 in the 10, 30, 100, 300, 1,000, 7,500 and 10,000-ppm groups. The respective numbers for the F1 generation (17 animals/sex/group) were 0, 1, 1, 1, 1, 2, and 0 in the 10, 30, 100, 300, 1,000, 7,500 and 10,000-ppm groups. The incidence of intercurrent deaths amongst treated F2 animals (17 animals/sex/group) was 1, 2, 0, 0, 1, and 1 in the 10, 30, 100, 300, 1,000 and 7,500 groups. The incidence of intercurrent deaths amongst animals in the control groups was 0-2. Clinical signs were generally comparable among all groups in all generations and were not treatment-related in incidence or severity (stated by the author). Comment: No data were available to confirm this statement.

Statistically significant reductions in terminal body weights were noted in adult animals at 10,000 (F0 males: 6%; F1 mating males: 16%; F1 non-mating males: 21%; F1 females: 19%) (There were no F2 animals at 10,000-ppm) and at 7,500-ppm (F1 non-mating males: 10%; F2 mating males: 14%; F2 non-mating males: 14%; F2 females: 8-18% during Week 1-6). Statistically significant reductions in dam body weights were also noted at delivery (9-11%) and during lactation (11-20%) in F0 females at 10,000-ppm.

Parental feed consumption was generally comparable in all groups in all generations on a g/animal/day basis, but was statistically significant increased at 7,500 and 10,000-ppm on a g/kg bw/day basis, except during lactation where dam feed consumption was statistically significant decreased in F0 animals at 7,500 (17%) and 10,000-ppm (11%, PND 4-7).

Reproductive toxicity: F1-, F2-and F3-Mating Trial

Pregnancy indices were decreased at 7,500 and 10,000-ppm. None of the F1 mating pairs produced offspring at 10,000-ppm (this finding was correlated with no sperm or spermatids noted in these animals), and at 7,500-ppm statistically significant decreases in the pregnancy indices were noted for the F2 mating pairs. The total number of males per litter as decreased at 10,000-ppm in the F1a litter (26%) and at 7,500-ppm across all F1 litters combined (F1a+F1b+F1c) (approximately 20%). The total number of F1a pups per litter was decreased at 7,500-ppm (22%) and at 10,000-ppm (21%). The total number of pups per litter across all F1 (F1a+F1b+F1c) litters combined (18%) was also decreased at 7,500-ppm. There was also an increase in the number of cumulative days to deliver the F1a litter for F0 animals at 10,000-ppm.

At 10,000-ppm, male and female pup weights, unadjusted and/or adjusted for litter size, were decreased in the F1a, F1b and F1c litters (7-12%). At 7,500-ppm male and female pup weights, unadjusted and adjusted for litter size, were decreased in the F2c litter (14%) and combined F2a, 2b, 2c litters (10%).

Male anogenital distance (AGD) was decreased at 10,000-ppm in the F1a, F1b, and F1c pups (8-15%) and at 7,500-ppm in the F1a and F1b pups (6.6-8%), in the F2a and F2c pups (13- 17%) and in the F3a pups (13%). No changes were noted in the female AGD throughout all the mating trials. Retained nipples were observed in the F3c male pups (11%) at 7,500-ppm. Testes descent, vaginal opening, and preputial separation were delayed at 10,000-ppm in the F1c pups, and at 7,500-ppm in the F1c, F2c and F3c pups.

The relative length of time spent in estrous stages was statistically significant increased for the F0 females at 10, 300, 1,000 and 7,500-ppm. However, no changes were revealed in the number of females with regular cycles, cycle length, number of cycles and in number of cycling females across the dose groups as compared to the control.

Reproductive toxicity: F1- and F2 Crossover-Mating Trial

At 7,500 and 10,000-ppm, when treated males were crossed with nulliparous naive females, there were decreased numbers of implantation sites (54% at 7,500-ppm, 98% at 10,000-ppm), and decreased indices of mating, pregnancy (8/17 versus 15/17 at 7,500-ppm; 0/17 versus 11/17 at 10,000-ppm), and fertility (8/14 versus 15/17 at 7,500-ppm; 0/17 versus 11/17 at 10,000-ppm).

At 7,500 and 10,000-ppm, when treated females were crossed with naive males, there was a decrease in AGD in the male pups (11.5% at 7,500-ppm; 17% at 10,000-ppm). Also at 7,500-ppm, male, female, and combined pup weights were decreased, both when unadjusted and adjusted for litter size (8-16%).

Sperm end-points

At terminal necropsies, various sperm end-points were found to be decreased at 7,500-ppm in the F1, F2, and F3 males and at 10,000-ppm in the F0 and F1 males. Epididymal sperm density was decreased at 7,500-ppm in the F2 (64%) and F3 males (94%), and at 10,000-ppm in the F1 males (99.6%). Comment: As a result of technical difficulties the epididymal sperm data for the 10,000-ppm F0 males was not obtained. Total spermatid/cauda was decreased at 7,500-ppm in the F1 (61%), F2 (73%) and F3 males (95%), and at 10,000-ppm in the F1 males (99.8%). Total spermatid/testis was decreased at 7500-ppm in the F1 (69%), F2 (74%) and F3 males (79%), and at 10,000-ppm in the F0 males (31%). At 10,000-ppm no spermatids were present in the testes of F1 males. Spermatid/mg testes was decreased at 7,500-ppm in the F1 (56%), F2 (57%) and F3 males (67%). Decrease in the motile percentage was noted in the F2 males (25%) at 7,500-ppm. Decrease of 12.8% in track speed was revealed along with a 15.6% decrease in the lateral amplitude in the F0 males at 10,000-ppm. Abnormal sperm morphology was seen in the F2 males at 100, 300, 1,000 and 7,500-ppm (stated by the author). Comment: No further data were available.

The results above show statistically significant organ weight changes noted in adult animals in the liver, kidney, and male accessory sex organs at the 7,500 and 10,000-ppm doses, and in the liver at 10 (males only), 300 (females only) and 1,000-ppm. At 10,000-ppm organ weight changes were also observed in adrenal glands (males only), pituitary (males only), uterus and ovaries.

Statistically significant organ weight changes were also noted in F3 animals. A dose-related increase in the absolute and relative liver weights were noted in the 1,000-ppm (21% and 17%, respectively) and 7,500-ppm (51% and 63%, respectively) males. The relative liver weight was also increased (36%) in the 7,500-ppm females. Absolute and relative right testis weights were decreased in the 7,500-ppm males (48% and 45% respectively). Decreases in absolute dorsolateral prostate weight (41%) and relative epididymis weights (35%) were also noted for the 7,500-ppm males.

Comment: no individual animal data on gross observations were available.

All other gross findings seen at necropsy were considered not dose related and incidental. Aplastic testes, epididymis and seminal vesicles, and small testes and epididymis were noted in 1-3 non-mating males at 300-ppm. At 1,000-ppm small prostates were noted in 3 or 4 non-mating males. In comparison of the incidence of these findings to TherImmune´s (the laboratory in question) historical control data, the incidence of the findings in the seminal vesicle and prostate is similar while the incidence for male testis and epididymis is increased (stated by the author).

Comment: No data were available to confirm this statement (historical control data were not included in the draft).

Histopathology

In the testes, minimal to marked atrophy of the seminiferous tubules characterized by loss of germ cells and the presence of Sertoli cell-only tubules, as well as occasional failure of sperm release, were noted at 10,000-ppm in the F0 and F1 males, and at 7,500-ppm in the F1 and F2 males. Minimal atrophy of seminiferous tubules was also observed in F1 males at 100-ppm (1/10) and at 300-ppm (1/10). The changes noted in the testes were correlated with “small testis” observed grossly in the most severe cases of F0 males, and were found in all 7,500 and 10,000-ppm F1 males. In F2 males atrophy of the seminiferous tubules, presents in 10/10 males at 7500-ppm, was correlated to the gross observation of atrophy, and there was failure of sperm release in 1/10 males. Comment: No data were reported for the 1,000-ppm group. Secondary changes were present in the corresponding epididymis including sloughed epithelial cells/residual bodies (3/10 F0 males at 10,000-ppm; 6/10 F1 males at 7500-ppm) and aspermia (1/10 F0 males at 10,000-ppm; 4/10 F1 males at 7,500-ppm; 9/10 F1 males at 10,000-ppm). Secondary changes (including aspermia, oligospermia, residual bodies/sloughed epithelial cells) were also present in the corresponding epididymis of F2 males at 7,500-ppm (number of animals not specified).

Minimal to mild hepatocellular hypertrophy was noted at 10,000-ppm in the F0- (males: 9/10; females: 10/10) and F1 animals (males: 6/10; females 9/10), at 7,500-ppm in the F0- (males: 10/10; females 9/10), F1- (males: 10/10; females: 10/10) and F2 animals (males: 10/10; females: 10/10), and at 1,000-ppm in the F1- (males: 5/10) and F2 animals (number of animals not specified).

Dilatation of the tubules and mineralization occasionally associated with chronic pyelonephritis was observed at 10,000-ppm in the F1 animals (males: 5/10; females: 3/10), at 7,500-ppm in the F1- (males: 3/10; females: 5/10) and F2 animals (males: 4/10; females. 5/10) and at 1,000-ppm in the F1 animals (females 1/10)

Cortex vacuolisation of the adrenals was noted at 10,000-ppm in the F0- (males 6/10 versus 1/10 in the controls) and F1 animals (males 5/10 versus 1/10 in the controls), and at 7,500-ppm in the F1 animals (males 4/10 versus 2/10 in the controls).

Comment: Findings in the adrenal glands (not specified) were also noted in the F1 animals at 1,000-ppm (stated by the author). No further data were available.

Results of the Pathology Working Group’s (PWG’s) reexamination

Sertoli cell vacuolation was observed in the control group as well as in the 1,000-ppm and 7,500-ppm F1 males. It was not observed in the 10,000-ppm animals with diffuse seminiferous tubule atrophy. In the 7,500-ppm males, Sertoli cell vacuolation was observed in seminiferous tubules without atrophy. This vacuolation was similar to that observed in the control group males

Comment: The vacuolation of Sertoli cells observed resulted from distortion during fixation and processing of the tissues according to the PWG. This distortion could have obscured any minimal toxic effects that may be present.

Conclusion

The no-observed adverse effect level (NOAEL) for testicular toxicity in this study was 100-ppm (equivalent to approximately 8 mg DEHP/kg bw/day in the F0 animals and approximately 5 mg DEHP/kg bw/day in the F1 and F2 animals) and was based on decreased absolute and/or relative testis weights noted at 7,500 (F1, F2 and F3 males) and 10,000-ppm (F0 and F1 males), macroscopic pathological findings (small or aplastic testes) noted at 300 (3/45 non-mating F1 males, 1/21 non-mating F2 males), 1,000 (3/25 non-mating F2 males), 7,500 (7/10 mating F1 males, 10/30 non-mating F1 males, 9/10 mating F2 males, 11/20 non-mating F2 males) and 10,000-ppm (2 or 3 of 10 F0 males, 10/10 mating F1 males, 21/21 non-mating F1 males), and microscopic pathological findings (testis seminiferous tubular atrophy) noted at 300 (1/10 F1 males), 7,500 (all F1 and F2 males) and 10,000-ppm (all F1 males, 2 or 3 of 10 F0 males).

Microscopic and/or macroscopic pathological findings and organ weight changes (absolute and/or relative) were also noted in the epididymis, seminal vesicles and prostate. Thus, macroscopically small and/or aplastic epididymides were noted at 300 (2/45 non-mating F1 males, 1/21 non-mating F2 male), 1,000 (3/25 non-mating F2 males), 7,500 (1 or 2 of 10 mating F1 males, 9/10 mating F2 males, 7/20 non-mating F2 males) and 10,000-ppm (21/21 non-mating F1 males). Small seminal vesicles were noted at 300 (1/45 non-mating F1 males) and 7500-ppm (1/10 mating F1 males), and small prostate was noted at 1,000 (3 or 4 of 43 F1 non-mating males), 7,500 (1/10 F0 mating males, 1/10 F1 mating males, 1/30 non-mating F1 males) and 10,000-ppm (1 or 2 of 21 non-mating F1 males). Microscopic pathological changes in the epididymis including sloughed epithelial cells/residual bodies and aspermia/oligospermia were found in F0 and F1 males at 7,500 and 10,000-ppm. Organ weight changes were noted in the epididymis (F1 and F2 males at 7,500-ppm; F0 and F1 males at 10,000-ppm), seminal vesicles (F2 males at 7,500-ppm; F1 males at 10,000-ppm) and prostate (F1 males at 7,500 and 10,000-ppm). At 7,500-ppm changes in epididymis and prostate weights were also noted in F3 males.

The low observed adverse effect level (LOAEL) for testicular toxicity was set at 300-ppm (equivalent to approximately 23 mg DEHP/kg bw/day in the F0 animals and 14 mg DEHP/kg bw/day in the F1 and F2 animals). At this dose level macroscopic pathological findings in testes (aplastic and/or small) were noted in animals of both generations (F1 and F2), and microscopic pathological findings in testes (seminiferous tubular atrophy) were noted in 1/10 F1 males.

Further on, macroscopic pathological findings in male accessory sex organs other than testes (mentioned above) were also present at this dose level and at higher doses. Atrophy of seminiferous tubules in testis was also observed at 100-ppm. However, this effect on testis at 100-ppm was only noted in one animal in one generation (F1) and in the absence of any accompanying findings. At 300-ppm additional parameters and several generations of animals were affected. Effects on male accessory sex organs other than testis could also be taken into consideration at this dose level. Therefore the LOAEL was set at 300-ppm.

The NOAEL for fertility toxicity in this study was 1,000-ppm (equivalent to approximately 77 mg DEHP/kg bw/day in the F0 animals, and 48 and 46 mg DEHP/kg bw/day in the F1 and F2 animals respectively) and was based on impaired fertility and litter parameters noted at 7,500-ppm and above, and decreased various sperm end-points noted at 7500 (F1-, F2-, F3 males) and 10,000-ppm (F0-, F1 males). None of the F1 mating pairs produced offspring at 10,000-ppm (this finding was correlated with no spermatids present in the testes of F1 males at 10,000-ppm). At 7,500-ppm statistically significant decreases in the pregnancy indices were noted for the F2 mating pairs (8/17 vs. 17/17). The total number of males per litter was decreased at 10,000-ppm in the F1a litter (26%) and at 7,500-ppm across all F1 litters combined (F1a+F1b+F1c) (approximately 20%). The total number of F1a pups per litter was decreased at 7,500-ppm (22%) and at 10,000-ppm (21%). The total number of pups per litter across all F1 (F1a+F1b+F1c) litters combined (18%) was also decreased at 7,500-ppm. There was also an increase in the number of cumulative days to deliver the F1a litter for F0 animals at 10,000-ppm.

The NOAEL for developmental toxicity in this study was 100-ppm (equivalent to approximately 8 mg DEHP/kg bw/day in the F0 animals and approximately 5 mg DEHP/kg bw/day in the F1 and F2 animals) and was based on the fact that the testicular effects were much more severe in the F1 and F2 generations than in F0, indicating the developmental phases as sensitive to the testicular toxicity of DEHP.

The NOAEL for effects not related to reproductive toxicity in adult animals was 300-ppm (equivalent to approximately 23 mg DEHP/kg bw/day in the F0 animals, and 14 mg DEHP/kg bw/day in the F1 and F2 animals) and was based on reductions in bodyweights noted in both sexes at 7,500 (F1, F2 animals) and 10,000-ppm (F0, F1 animals), absolute and/or relative organ weight changes noted at 1000-ppm and above (increased liver: 1,000-ppm and above; increased kidneys: 1,000-ppm and above; increased adrenals: 10,000-ppm; increased pituitary: 10,000-ppm), and microscopic pathological findings noted at 1,000-ppm and above (liver hypertrophy: 1,000-ppm and above; cortex vacuolisation of the adrenals: 7,500-ppm and above; dilation of the tubules and mineralization in the kidneys occasionally associated with chronic pyelonephritis: 1,000-ppm and above). Microscopic pathological findings in the adrenal glands were also indicated in F1 animals at 1,000-ppm (no further data).

In conclusion, a NOAEL of 4.8 mg/kg/day is obtained for testicular toxicity and developmental (testicular) toxicity. The NOAEL for fertility is 46 mg/kg/day.

Results from a 2-generation reproduction toxicity study in Wistar rats indicate effects on reproductive performance, several organs, survival (overall, 8 of 50 adult high dose females died or were killed for humane reasons), as well as on development (Schilling et al., 2001a). The study was performed according to current guidelines and in conformity with GLP. Wistar rats (25 rats/sex and generation) were exposed to dietary levels of 0, 1,000, 3,000 or 9,000-ppm DEHP (corresponding to approximately 0, 113, 340 or 1,088 mg/kg bw and day). The F0 animals were exposed as from the age of 37 days, for at least 73 days before mating, and until weaning. F1 pups were raised and mated to produce a F2 generation. Selected F2 male and female animals (10 of each sex) performed a functional observation battery, motor activity, and a water maze test at 21 days of age.

Histopathology of the testis was performed with light microscopy after Bouins fixation, paraplast embedding, and Haematoxylin and Eosin staining. Evaluation of the testis showed focal tubular atrophy to be the most frequent finding. In the F0 animals, the frequency was 0/25, 1/25, 3/25, and 6/25 in the control, low, mid, and high dose groups, respectively. The number of affected tubules/testis, as well as the presence of diffuse tubular atrophy, was increased in the high dose group.

In the F1 adult males, the frequency of focal tubular atrophy was 3/25, 7/25, 4/25, and 14/25 in the control, low, mid, and high dose groups, respectively. Although fewer animals were affected in the mid than in the low dose group, the effects in the mid dose animals were more pronounced than in the low dose animals. Thus, the number of affected tubules/testis was increased in the two highest dose groups. In addition, diffuse tubular atrophy was observed in the high dose group (3/25). Vacuolisation of Sertoli cells was only observed in atrophic tubuli, which were present in all exposed groups. A reduced or absent sperm-/spermatid counts together with sperm abnormalities was observed in the high dose groups in 2 and 1 animal(s) (of 25) in the F0 and F1 adult males, respectively.

A reduced testis weight (absolute and relative) was observed in the high dose F2 pups.

Other findings: F0

Observations on the F0 parental females were mortality (2/25), a decreased food consumption (25%), reduced body weights, body weight loss during lactation (14%), and a retarded body weight gain (25%) in the high dose group. Effects on organ weights and/or histopathology were observed among both females and males. Besides effects on the testis (see above), there was also an affect on the ovaries in the high dose group (reduced number of growing follicles and of corpora lutea, 15%* and 25%**, respectively). Effects on reproductive performance were evident in the high dose group, as illustrated by a reduced fertility index among both females and males, and an increased postimplantation loss.

Other findings: F1 pups

Observations on the F1 pups in the high dose group included reduced number of live (viability index) and total number of pups, increased number of stillborn pups, increased pup mortality, reduced body weights (31%) and body weight gains (36%) until weaning (day 21 post partum).

Feminisation of male pups was indicated by a reduced anogenital distance (14%), a reduced anogenital index (8%), and an increased frequency of areolas/nipple anlagen in male pups.

The timing of sexual maturation was delayed in both females (vaginal opening) and males (preputial separation). The weight of the thymus and spleen were reduced.

Some of these effects were also significant in the mid dose group (e. g., the viability index, the anogenital index, and the weights of thymus and spleen), and although not statistically significant in most cases, there appears to be a trend also including small effects in the low dose group.

Other findings: F1 parental animals

In the high dose group, there was an increased mortality/sacrifices among dams (6/25) and malformed external genital organs in males (2/25). Food consumption, body weights, and body weight gains were reduced in both males and females. The effects on reproductive performance and organ weights/histopathology were almost identical to those in the F0 generation.

In the lower dose groups, there were besides the effects on the testis (see above) also an increased number of stillborn pups in the mid dose group.

Other findings: F2 pups

In the high dose group, the observations in the F2 pups were almost identical to those in the F1 pups, but included a reduced weight of the testis.

In the mid dose group, the effects in F2 pups seemed more severe than in the F1 pups. There were an increased number of stillborn pups, a decreased live birth index and viability index, lower body weights (6%) and body weight gains (7%), a reduced anogenital distance/index (9% and 8%, respectively), an increased presence of aerola/nipple anlagen affecting 49% of the males, and a decreased thymus weight in males. Although not statistically significant, there appears to be a trend also including small effects on the thymus and the testis (see also above) in the low dose group.

Timing of sexual maturation was not studied in the F2 generation.

In the high dose group, a functional observation battery performed on selected animals at day 21 post partum revealed reduced values for grip strength in males, and reduced values for landing foot-splay in both males and females. The body weights of these animals were reduced (27-38%), but it is not clear whether the reduced body weight could account for the functional effects. No effects were observed on the water maze test or on motor activity. No effects were observed in the lower exposure groups.

Evaluation of immunological data

The present study indicates that DEHP induces atrophy of spleen and thymus. There was a significant decrease in spleen weight at all doses in both male and female F1-pups and a significant decrease in thymus weight in the mid and high dose groups in F1 males. In the F1-females a significant reduction of the thymus was only observed at the highest dose level, however, a non-significant but clear dose dependent trend was observed also for the low and mid dose groups. In the F2-pups, splenic weight was significantly reduced in the high dose group with 30 and 34% in males and females, respectively. The effect on the thymus weight in the F2-pups is similar to that in the F1-pups. A significant reduction in the mid and high dose groups of the F2-males, and for the F2-females a non-significant but dose-dependent reduction in the mid and high dose groups.

In the highest dose group the reduced spleen and thymus weights were observed in parallel with a significant reduction in male and female F1 and F2 pup body weights. Thus, it is possible that in the highest dose group the effect on spleen and thymus weights could be associated with the reduced body weight. However, the effect on the spleen observed in the low-dose group of both male and female F1 pups and on the thymus weight in the mid dose group of male F1 and F2 pups was not accompanied by a reduced body weight. Therefore, without further testing of the immunotoxicity of DEHP a direct immunotoxic effect of DEHP cannot be excluded. Thus, for the effect on the spleen a LOAEL of 1,000-ppm can be concluded from this study.

Evaluation of testicular data

In this study, significant and severe effects on testicular histology, sperm morphology, fertility, and sexual development of the offspring have been observed in the high dose group of both generations. Several of these effects are also clearly apparent in the mid dose group, e. g., a reduced testis weight in F2, focal tubular atrophy and a feminisation of 49% of the male offspring (as indicated by the presence of aerola/nipple anlagen in the males). Some of these effects are also occurring in the low dose group (e. g., focal tubular atrophy), although few tubuli are affected per testis. However, based on the clear dose-response, we conclude that there is an adverse effect on the testis also in the low dose group (113 mg/kg and day), which thus constitutes the LOAEL of the study.

Overall evaluation

It should be observed that although there has been some focus on the testicular effects, the testis have only been studied by standard methods (Bouins fixation, paraplast embedding, and Haematoxylin and Eosin staining) and no measurements of, e. g. hormone concentrations have been conducted. Still, there were effects on numerous parameters relating to reproductive success in the low dose animals (testicular tubular atrophy, relative weight of testis in F2, pup production, pup viability, anogenital index, pup weight gain, and time of sexual maturation of males and females), effects that were not statistically significant in this dose group but at higher ones. Although some of these effects are small in the low dose group, the relevance is supported by known mechanisms of action and clear dose-responses involving the three dose groups.

Wistar rats have been used in the present study. Although it is not the first time Wistar rats have been used in studies on the toxicity of DEHP, the fact that none of the previous studies giving low LOAELs have used Wistar rats, gives some concern relating to the sensitivity of this strain as compared to, e. g. the more commonly used Sprague-Dawley rat.

The low dose in this study (113 mg/kg and day) is considered a LOAEL, and hence, no NOAEL can be deduced from this study.

Results from a 2-generation range finding study in Wistar rats indicate effects on fertility and developmental toxicity (Schilling et al., 1999a). The study was performed according to current guidelines and in conformity with GLP. Wistar rats (F0 generation = 10 rats/sex) were exposed to dietary levels of 0, 1,000, 3,000 or 9,000-ppm DEHP (corresponding to approximately 0, 110, 339 or 1,060 mg/kg bw/day). F1 pups were raised and mated to produce a F2 generation, which was sacrificed two days after birth. The mean relative liver weight was significantly increased in F0 parental males at 3,000 and 9,000-ppm (at the higher dose level also the absolute liver weight). No treatment related histopathological changes were, however, noted. There was a reduced total number of delivered F1 pups and the viability index was reduced on post partum day 0 and 4 at 9,000-ppm. In F1 male pups a treatment related loss of spermatocytes was found at 3,000 and 9,000-ppm (2/10 and 7/9, respectively). At the highest dose level, the presence of areolas/nipple anlagen was significantly increased and the male sexual maturation (based on preputial separation) was significantly retarded. A reduced anogenital distance was observed in F2 male pups at 9,000-ppm (not investigated in F1 pups).

Mortality occurred in F1 parental males (3/9) at 9,000-ppm in the premating phase, initially also reduced food consumption and reduced mean body weights were noted. At this dose level, the fertility was also reduced in the males (fertility/mating index 83%). The absolute and relative testicular weight and the absolute epididymidal weight were significantly decreased at 9,000-ppm. The prostate weight showed a dose-related decrease from 1,000-ppm. The testes and the epididymides were reduced in size in three out of six animals at 9,000-ppm.

Histopathology revealed focal or diffuse atrophy of spermatogenesis of the testes and diffuse Leydig cell hyperplasia in all males, interstitial oedema in the testes in three out of six animals, and debris of an altered spermatogenesis in the epididymides in five out of five animals. Also aspermia (2/5), missing seminal vesicle (1/6), and areolas/nipple anlagen (1/6) were noted. There was a dose-related increase of stillborn pups from 3,000-ppm and a decrease of delivered F2 pups, statistically significant at 9,000-ppm.

The effects found in F1 parental males indicate that DEHP exerts a specific action on male genital organs such as the testicle and the epididymis, when males are exposed during early development. This is strengthened by the fact that female gonads were unaffected. However, concerning testicular effects in developing male pups only one testicle per litter was studied histopathologically in F1 pups and none of the F2 pups. F1 pups were culled at day 21 and neither undescended testes nor hypospadia were investigated. Neither is there any information on effects on Sertoli cells in F1 parental male rats in this range finding study.

Single generation reproductive toxicity studies

Rat

Inhalation

In a 4-week inhalation study conducted according to OECD guideline 412 and the principles of GLP, male Wistar rats (10 rats per group) were exposed 5 days/week, 6 hours/day to 0, 0.01, 0.05 or 1 mg DEHP/litre (0, 10, 50 or 1,000 mg DEHP/m3) (99.7% pure) as liquid aerosol (Klimisch et al., 1992). The males were mated to untreated females. No effects on male fertility were observed 2 and 6 weeks after the end of exposure and no testicular toxicity was detected histologically.

Oral

In a study comparable to a guideline study,Agarwal et al. (1986a)administered DEHP (> 99% pure) to groups of 24 sexually mature male F344 rats (age about 15 weeks) in the diet at 0, 320, 1,250, 5,000 or 20,000-ppm (equivalent to doses of 0, 18, 69, 284 and 1,156 mg/kg bw) per day for 60 days. The males were mated with undosed females at exposure days 61 to 66. A dose-dependent reduction in total body, testis, epididymis, and prostate weights was observed at 5,000 and 20,000-ppm. The only functional reproductive consequence of exposure of male rats to DEHP was a significantly reduced mean litter size at 20,000-ppm (1,156 mg/kg bw/day). This effect was directly correlated with degenerative changes in the testes, along with decreased testicular zinc content, significant reduction in epididymal sperm density and motility, and increased occurrence of morphologically abnormal sperm. There was a trend towards decreased (not statistically significant) testosterone and increased luteinising hormone (LH) and follicle stimulating hormone (FSH) in serum at 5,000 and 20,000-ppm. The incidence of pregnancy, mean litter weight on day 1, frequency of stillbirth and neonatal death, and mean litter growth up to 7 days of age were unaffected. A NOAEL of 69-mg/kg bw/day is derived.

Mouse

Oral

Tanaka (2003)examined the effects of prenatal DEHP exposure on sex ratio in mice. It appears that the study was conducted to address concerns about DEHP effects on sex. Starting at 5 weeks of age, 20 male and female CD-1 mice/sex/group were fed diets containing 0 or 0.03% DEHP (purity > 97.0%). At 9 weeks of age, each female was mated for 5 days with a male from the same or opposite treatment group (i. e., cross-mating). There were 4 treatment groups consisting of 10 mice/sex: control females × control males, control females × treated males, treated females × control males, and treated females × treated males. Females continued to receive the DEHP-containing or control diets during the mating period and throughout gestation. The study authors estimated that intake of DEHP was 49 mg/kg bw/day in males and ~55–58 mg/kg bw/day in females during the preconception period. Intakes by females were estimated at ~45 mg/kg bw/day during the mating period and ~50 mg/kg bw/day during the gestation period. Females were allowed to litter, and endpoints examined on day of birth were litter size, litter weight, individual offspring weight, and sex ratio. Statistical analyses included ANOVA or Kruskal-Wallis test followed by Bonferroni multiple comparison to assess food intake, litter size, and litter and body weights. Chisquared test was used to evaluate sex ratio based on offspring, and the Steel test was used to assess sex ratio based on litter. As a result of pregnancy failures or abortions, there were 8–10 litters delivered in each treatment group. Compared to the group consisting of control males and females, mean body weights of male offspring were increased in all groups containing a treated female and/or male parent. No significant effects were noted for litter size, litter weight, total or average sex ratio, or female offspring weights. The study authors concluded that the concentrations of DEHP used in this study did not produce adverse effects on sex ratios.

Tanaka et al.(2005)gave DEHP (>97% purity) to CD-1 mice in the diet from 5 weeks of age in the F0 generation to 9 weeks of age in the F1 generation. A single dietary dose level of 0.03% was used, with control animals receiving untreated basal feed (n = 20/sex/treatment group). At 9 weeks of age, 10 DEHP treated females were paired with DEHP-treated males, 10 DEHP-treated females were paired with control males, 10 control females were paired with DEHP-treated males, and 10 control females were paired with control males. The females’ diet was available to males during the 5-day cohabitation phase. Females reared their own unadjusted litters, which were weaned at 4 weeks of age. One female and male from each litter were retained and fed their dam’s diet until 9 weeks of age. All F1 offspring underwent neurobehavioral testing during the lactation period, including surface righting and negative geotaxis on PND 4 and 7, cliff avoidance on PND 7, swimming behavior on PND 4 and 14, and olfactory orientation on PND 14. Exploratory behavior was assessed in 1 male and 1 female from each litter at 3 weeks of age. Post-weaning tests included multiple-T water maze at 7 weeks of age and exploratory behavior at 8 weeks of age. Statistical analyses were performed using ANOVA or Kruskal-Wallis test followed by Bonferroni multiple comparison test. Proportions were evaluated using chi-squared or Fisher test. [It is not stated whether litter was considered in the analysis of the preweaning neurobehavioral tests.] Based on measured feed consumption, mean DEHP intake by treated males [rounded by CERHR] was 46 mg/kg bw/day. Treated females received 53–57 mg/kg bw/day during the preconception period, ~43 mg/kg bw/day during mating, 46–49 mg/kg bw/day during gestation, and 154–171 mg/kg bw/day during lactation. DEHP had no effect on feed consumption or dam body weight. As repeated in Section 4.2.3, there were no significant treatment effects on the number of pregnant females, number of litters, number of offspring, average litter size or weight, or offspring sex ratio. Offspring body weight during the lactation period was similar between groups except for an 8% decrease in body weight on PND 14 in female offspring when the parents both had received DEHP. The author did not consider this isolated alteration to be treatment related. Swimming ability was accelerated in PND 4 female offspring when the dam received DEHP. The number of movements in the test of exploratory behavior was decreased in male offspring the parents of which both received DEHP. There were isolated differences in T-maze performance by sex, trial, and treatment group that were not considered to represent treatment-related alterations in maze-learning. None of the other behavioral tests revealed effects of DEHP treatment. The author concluded that “few adverse effects on several behavioral parameters were produced at the dose level of DEHP in the present study.”

Price et al.(1988a)conducted a study in CD-1 mice of the postnatal effects of prenatal exposure to DEHP. The design is similar to Tyl et al. except that developmental toxicity was evaluated postnatally rather than at the end of pregnancy. Pregnant mice, 28–29 per group, were fed a diet containing 0, 0.01, 0.025, or 0.05% DEHP, resulting in mean doses of 0, 19, 48, or 95 mg/kg bw/day (estimated by authors), respectively, from gd 0 to 17. Dams were evaluated for maternal toxicity and their offspring were evaluated for viability, growth, and development. The F1 pups were then mated within dose groups and their F2 offspring were evaluated for viability and growth through pnd 4. There were no observed adverse effects on the (F0) females during pregnancy. The only indication of a maternal DEHP effect was a trend for decreasing body weight gain on pnd 4 and 7 that was not significant in a pairwise comparison with controls. For F1 litters the percent prenatal mortality was significantly increased at the 95 mg/kg bw/day dose with a concomitant decrease in live litter size at pnd 1. On pnd 4, the percentage of viable pups was also decreased in litters exposed to 95 mg/kg bw/day. No other effects of DEHP upon growth, viability, age at acquisition of landmarks, or spontaneous locomotor activity were seen in any dose groups on pnd 14, 21, or 50. The NOAEL for developmental toxicity was 48 mg/kg bw/day and the maternal NOAEL was 95 mg/kg bw/day. No adverse effects were consistently observed upon the reproductive performance of the F1 generation or the F2 offspring through pnd 4.

CD-1 Swiss mice, 11 weeks old at the start of exposure, were used for continuous breeding phase and cross-over mating studies(Lamb et al., 1987). There were 20 breeding pairs in each treated dose group, and 40 pairs in the control group. DEHP was mixed with feed to levels of 0, 0.01, 0.1, and 0.3% (w/w); this yielded calculated doses of 0, 14, 141, and 425 mg/kg bw/day. Following a 7-day premating period, the mice were housed as breeding pairs for 98 days. Litters were removed immediately after birth. Endpoints in-life were clinical signs, parental body weight and food consumption, fertility (numbers of pairs producing a litter/total number of breeding pairs), number of litters/pair, number of live pups/litter, proportion of pups born alive, sex ratio, pup body weights within 24 hours of birth, and water consumption. There was clear indication that DEHP affected fertility when administered in the diet. At 425 mg/kg bw/day no breeding pairs delivered a litter. At 141 mg/kg bw/day, fertility was significantly reduced as evidenced by fewer litters, fewer pups/litter, and fewer pups born alive. A cross-over mating study was conducted between the 425 mg/kg bw/day treatment group and the controls. Fertility was significantly reduced in the groups of treated males and control females and the groups of treated females and control males. The treated females produced no litters and only 4/20 treated males sired a litter. Only the control and high-dose groups were necropsied. High-dose males had reduced testicular and epididymal weights and histologic evidence of seminiferous tubular destruction accompanied by major changes in epididymal sperm number, morphology, and motility. In addition, the males had decreased prostate weight, reduced serum testosterone, and elevated LH and FSH. There were no histopathologic effects in the reproductive tracts of high dose females, but the weight of the reproductive tract was lower than controls (probably because the animals were not pregnant). The high-dose group was infertile, the mid-dose group was affected, and the low-dose group was unaffected. Thus, the NOAEL was at a calculated dose of ~14 mg/kg bw/day. The LOAEL was at ~141 mg/kg bw/day, based on reductions in litter size and in proportions of pairs having litters.

Subcutaneous

Agarwal et al., (1989)dosed adult male ICR mice in groups of 25 (control) or 10-13 (treated) were treated subcutaneously with undiluted DEHP at volumes of 1, 2, 5, 10, 15, 120, 40, 60, 80, and 100 mL/kg, on days 1, 5, 10, a regimen used in the previous study (Agarwal et al., 1985). Doses in g/kg were not provided; in the previous report, a dose of 10 mL/kg was equivalent to 9.86 g/kg. The animals were mated from day 21-28, and then sacrificed. Other animals were sacrificed on day 21; one testis and epididymis from 10 mice was fixed in formalin and evaluated histologically. The other testis was used for biochemical measures. The disposition of the animals was not clear from the Methods description in the prescribed study. The data were analyzed by ANOVA and then compared pairwise by t-test; this is an incorrect comparison, as it produces false positives. The authors noted that doses of 20 mL/kg or more were incompletely absorbed, and the animals had fluid filled pouches containing some DEHP and some apparent lymph. Testis weights (unclear when during the study these were collected) were reduced at and above 20 mL/kg. There were various biochemical changes (whose meaning is difficult to determine) at 10 mL/kg and higher. Testicular histology was affected at and above 10 mL/kg, with inflammation being most common at 10 mL/kg, and tubular changes appearing at 40 mL/kg (which might be calculated to be an administered dose of 39.4 g/kg). The testicular pathology (description of the changes and the doses at which they began to appear) was not tabulated, and the text description was cursory. The effect on fertility was limited to determining how many females per group were pregnant; this was 76% in the controls, and as the dose increased, the proportions of pregnant females were: 50, 25, 33, 42, 25, 8, 20, 0, 8, and 0%. The authors did not perform statistical analyses on these data, and it can be seen that even at the lowest dose (which can be calculated to be 0.99 g/kg) there may be a decrease in percent pregnant females, and the next highest dose(2 mL/kg, wherein 25% of the females were pregnant), gave the fourth lowest pregnancy value, the same as that found with 15 mL/kg. Pregnancy and histology were evaluated in this study, which is informative. However, the methods used for histologic evaluation were suboptimal; the time between treatment and mating and necropsy meant that there might have been some degree of recovery from any DEHP effect; the concentration and volume of the dosing solution meant that residual neat DEHP was in a depot at the site of injection, complicating the kinetics in vivo; the numbers of animals for the mating trials (10) were relatively small, and the measures of fertility (the % of females getting pregnant) are gross; the apparently most sensitive period of male development was not evaluated.

Short description of key information:

There are very limited human data examining the reproductive effects of DEHP. These studies largely examine the relationship between urine levels of the DEHP metabolite, MEHP, and differing measurements.

There are many experimental animal studies, largely oral, using rats or mice. The most sensitive effects, perturbations in testicular structure and function, have been consistently observed in several reproductive toxicity studies in rats and mice by both oral and parenteral routes of exposure. In vivo and in vitro assays have demonstrated that the Sertoli cell is the most sensitive target of toxicity, causing subsequent germ cell depletion. Rats appear to be the more sensitive species than mice for testicular effects.

The NOAEL for fertility toxicity in a multigeneration study (Wolfe & Layton (2003) was 1,000-ppm (equivalent to approximately 77 mg DEHP/kg bw/day in the F0 animals, and 48 and 46 mg DEHP/kg bw/day in the F1 and F2 animals respectively) and was based on impaired fertility and litter parameters noted at 7,500-ppm and above, and decreased various sperm end-points noted at 7500 (F1-, F2-, F3 males) and 10,000-ppm (F0-, F1 males).

The consistent finding of testicular effects in rats and mice is in contrast to studies in marmosets. No treatment-related changes in testicular histology or more gross parameters were observed at the highest dose used, 2500 mg/kg bw/d.

Justification for selection of Effect on fertility via oral route:

Wolfe and Layton (2004): Lowest reliable NOAEL

A number of human studies have attempted to link maternal MEHP levels with gestation length, onset of puberty and AGD. However, these studies, which were largely negative, are considered inadequate as they generally lacked an adequate control group and were of small sample size. Developmental studies in experimental animals comprise single and multiple generation exposure largely by the oral route and predominantly in rodents.

There are no dermal studies, only a single inhalation study and few studies using parenteral routes of exposure. There are no developmental studies in primates.

Numerous studies have shown that DEHP is embryotoxic in rats at doses close to maternally toxic levels. In mice, several studies have shown that DEHP is embryotoxic and teratogenic at dose levels below those producing observable evidence of toxicity to the dams. Developmentally induced effects are salso een at low doses in multigenerational studies. A well conducted oral 3-generational study in rats derived a NOAEL for developmental toxicity of 4.8 mg/kg bw/d based on the finding of small male reproductive organs at 14 mg/kg bw/d (Wolfe & Layton, 2003). At higher levels of exposure, effects on in utero survival, reduced AGD, undescended testes, retained nipples/areolae, incomplete preputial separation and disruption of spermatogenesis in offspring were evident. In this study, testicular abnormalities in the F1 and F2 generations were much more severe than in F0, indicating the developmental phases were more sensitive to the testicular toxicity of DEHP. The critical study for developmental toxicity is considered to be Wolfe & Layton (2004). For developmental effects, the NOAEL is 100 ppm (4.8 mg/kg bw/d) and the LOAEL is 1000 ppm (14 mg/kg bw/d), based on effects on the male reproductive organs.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4.8 mg/kg bw/day

Species:

rat

Quality of whole database:

high

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

300 mg/m³

Species:

rat

Endpoint conclusion:

no study available

Numerous studies have shown that DEHP is embryotoxic in rats at doses close to maternally toxic dose levels. In mice, several studies have shown that DEHP is embryotoxic and teratogenic at dose levels below those producing observable evidence of toxicity to the dams.

Standard developmental toxicity/teratogenicity studies

Inhalation

Twenty-five pregnant Wistar rats per dose group were used to study the teratogenicity of DEHP by inhalation as liquid aerosol at dose levels of 0, 0.01, 0.05 or 0.3 mg/litre (0, 10, 50 or 300 mg/m3) (Merkle et al., 1988, BASF, 1986). The particle MMAD was < 1.2 ± 7.3, ± 16.8 and ± 5.8 μm for the low, middle and high dose group, respectively. The study was performed according to OECD Guideline 414 and GLP principles. The dams were exposed by head-nose exposure for 6 hours per day from gestation day 6 through 15 (the period of male sexual differentiation between days 16-19 is not included in this study). Twenty rats per group were sacrificed on day 20 of pregnancy and five rats per group were allowed to litter. The offspring was raised and observed for postnatal signs of toxicity. In a range-finding study, “exposure-related” peroxisome proliferation was observed in dams from 200 to 1,000 mg/m3. The number of live foetuses/dam was slightly, but statistically significantly decreased in the 50 mg/m3 group and the percentage of resorptions/dam was elevated. These effects, however, were not seen at the next dose level. That effects were only seen in the middle dose group may reflect the large standard deviation of the particle MMAD. The effects reported were not regarded as exposure related, since no dose dependency was observed. The number of corpora lutea, uterine weights, body weights, living and death implants, early and late resorptions, dead foetuses, pre- and post-implantation losses were unchanged compared to controls. The validity of this study is questioned, as the systemic dose was not determined. By comparison with another inhalation dose study there may be problems with delivering the expected dose of DEHP. Hence, this study is considered inadequate for use in risk characterisation.

Oral

Rats

In a study comparable to a guideline study and performed according to GLP principles, dietary levels of 0, 0.5, 1.0, 1.5 or 2% of DEHP (no information on mg/kg bw/day is given) were given to groups of F344/CrlBr rats (34-25) throughout gestation (days 0-20) (Tyl et al., 1988). The rats were sacrificed on day 20. Food intake was significantly decreased at all dose levels. Reduced maternal body weight gain and increased absolute and relative liver weights were observed at a dietary level of 1.0%. Reduced foetal body weights per litter were observed at the same dietary level. There were no treatment-related differences in the number of corpora lutea or implantation sites per dam, nor in the percent pre-implantation loss. At a dietary level of 2% the number and percent of resorptions, non-live and affected implants per litter were significantly increased and the number of live foetuses per litter was significantly decreased. Mean foetal body weight was significantly reduced at all dose levels. The number and percentage of malformed foetuses per litter was not significantly different from control. The NOAEL for maternal and developmental toxicity was 0.5% DEHP (approximately 357 mg/kg bw/day).

In a study performed according to OECD Guideline 414 and GLP principles, DEHP was tested for its prenatal toxicity in Wistar rats (Hellwig et al., 1997). DEHP (99.8% pure) was administered as an oily solution to 9-10 pregnant female rats/group by stomach tube at doses of 40, 200 or 1,000 mg/kg bw on day 6 through 15 of gestation. On day 20 of pregnancy, all females were sacrificed and assessed by gross pathology. Maternal toxicity at 1,000 mg/kg bw was reported: Slightly reduced maternal food consumption was noted. Reduced uterus weight was assessed as to be associated with the high embryolethality. The corrected body weight gain did not show any differences of biological relevance. Statistically increased relative kidney and liver weights was observed. Developmental toxicity at 1,000 mg/kg bw: Severe developmental effects were observed: statistically significantly increased implantation loss (about 40%). There also was a statistically significant lower number of live foetuses/dam, decreased foetal body weights, a drastically increased incidence of of external, soft tissue, and skeletal malformed foetuses/litter (in total approximately 70% of the foetuses/litter), predominantly of the tail, brain, urinary tract, gonads, vertebral column, and sternum. There also were an increased percentage of foetuses/litter with soft tissue and skeletal variations and skeletal retardations. The NOAEL for maternal and developmental toxicity was 200-mg/kg bw/day.

Pregnant female Wistar rats were exposed toMEHPby gavage daily on the day 6 to the day 15 of gestation, at the dose of 0, 225, 450 or 900 mg/kg bw (Ruddick 1981). The dams were necropsied at day 22 of gestation. MEHP was lethal to some mothers at dosages of 225, 450 and 900 mg/kg bw therefore the others groups was exposed to new doses of 0, 50, 100 and 200 mg/kg bw. Dosages of 100 and 200 mg/kg reduced the maternal weight gain of dams in the treatment when compared to the control group (p<0.05). Treatment with 450 mg/kg statistically affected the mean litter weight of the live pups (p<0.05). Examination of the fetal skeleton did not reveal any pronounced alterations between treated and control groups other than disturbances in the placement of the sternebrae plates, a 14th and wavy ribs which were observed in both experimental and control fetuses. Visceral anomalies were not seen.

Mice

In a study comparable to a guideline study and performed according to GLP principles, dietary levels of 0, 0.025, 0.05, 0.10, or 0.15% of DEHP (0, 44, 91, 190.6 or 292.5 mg/kg bw/day; > 99% pure) were administered to groups of 1-CR outbred mice (30-31 per group) throughout gestation (days 0-17) (Tyl et al., 1988). Maternal toxicity, indicated by reduced maternal body weight gain, was noted in the two highest dose groups, mainly due to reduced gravid uterine weight. There were no treatment-related effects on the number of corpora lutea, implantation sites per dam, the percent pre-implantation loss, and sex ratio of live pups. The number and percent of resorptions, late foetal deaths, and dead and malformed foetuses were all significantly increased from 0.1%. Foetal weight and the number of live foetuses per litter were significantly reduced from the same dose level. Both the percentage of foetuses with malformations and the percentage of malformed foetuses per litter were significantly increased from 0.05%. The observed external malformations included unilateral and bilateral open eyes, exophthalmia, exencephaly, and short, constricted, or no tail. Visceral malformations were localised predominantly in the major arteries. Skeletal defects included fused and branched ribs and misalignment and fused thoracic vertebral centra. The NOAEL for maternal toxicity was concluded to be 0.05% (91 mg/kg bw/day) and for developmental toxicity 0.025% (44 mg/kg bw/day).

Reader (1996)performed a GLP study of the embryo-foetal toxicity in the CD-1 mice by oral gavage administration. Doses of 0, 40, 200 or 1,000 mg DEHP/kg bw/day were administered to groups of 15 pregnant mice from day 6 to 15 of gestation. A control group of 30 pregnant mice received a vehicle (0.5% carboxymethylcellulose containing 0.1% Tween 80). Litter parameters following necropsy of the females on day 17 of gestation revealed low numbers of viable young, high numbers of resorptions, and a greater extent of post-implantation loss for females given 1,000 mg/kg bw/day than in the control group. Cardiovascular abnormalities, tri-lobed left lungs, fused ribs, fused thoracic vertebral centres and arches, immature livers, and kidney anomalies were observed. At 200-mg/kg bw/day, there was a slightly higher incidence of foetuses with intra-muscular or nasal haemorrhage or dilated orbital sinuses. There also was a small number of foetuses with anomalous innominate or azygous blood vessels. From this study a NOAEL of 200-mg/kg bw/day can be derived for maternal toxicity and a NOAEL of 40-mg/kg bw/day for developmental toxicity.

DEHP was given to female ICR mice (8 to 16 weeks old) at dietary levels of 0, 0.05, 0.1, 0.2, 0.4 or 1.0% (equivalent to 0, 70, 190, 400, 830 and 2,200 mg/kg bw, purity not specified) from day 1 to 18 of gestation (Shiota and Nishimura, 1982). On day 18 the animals were killed. The average weight of live foetuses was decreased and the incidence of malformed foetuses was significantly higher from 400 mg/kg bw. The most common malformations were neural tube defects (exencephaly and spina bifida), malformed tail, gastroschisis and club foot. The NOAEL for maternal and developmental toxicity was 70 mg/kg bw of DEHP.

NTP (1991)evaluated the developmental toxicity ofMEHPin CD-1 mice fed a diet containing the chemical on gd 0–17. Groups of 25–27 mice received doses of 0, 0.017, 0.035, 0.07, or 0.14% MEHP in feed. Average doses were reported as 0, 35, 73, 134, or 269 mg/kg bw/day MEHP. Doses were selected to be approximate molar equivalents to the DEHP doses studied by Tyl et al. (1988) in the same mouse strain using a similar protocol. Maternal body weights and food and water consumption were recorded throughout the treatment period. At scheduled sacrifice on gd 17, the numbers of resorptions and dead or live fetuses were recorded. All fetuses were weighed, and live fetuses were sexed and examined for external, visceral, and skeletal malformations. MEHP-exposed females exhibited no clinical signs of maternal toxicity, and food and water consumption were similar to those of controls. There was a decrease in the adjusted body weight gain of mice in the 269 mg/kg bw/day dose group. The relative liver weights of mice fed a diet containing 134 and 269 mg/kg bw/day increased. The maternal NOAEL was stated to be 134 mg/kg bw/day. The percent litters with resorptions increased at all dose levels. The numerical values increased in a dose-related manner, reaching 77% in the high-dose group. A significant linear decrease in average number of live fetuses per litter was observed with increasing dose level; values for the 73, 134, and 269 mg/kg bw/day dose groups were significantly different from controls by pair wise comparison. Fetal malformations were observed in a significantly higher percentage of litters at dose levels of 73 mg/kg bw/day and greater, and in a significantly higher percentage of fetuses at doses of 134 mg/kg bw/day and higher. MEHP exposure was associated with an increase in skeletal and visceral malformations, with the latter increase primarily due to cardiovascular malformations. A NOAEL for developmental toxicity was not observed in this study. The LOAEL (based on incidence of litters with resorptions) was 35 mg/kg bw/day MEHP. The Panel has high confidence in the quality of this study and its ability to identify the developmental LOAEL for oral exposure.

Rabbits

No developmental studies have been performed in rabbits given DEHP.

Other developmental toxicity studies

Oral

Rat

Two multi-generation studies (Schilling et al., 2001 and Wolfe et al., 2003) in rats give important information on development as well. Based on Wolfe et al. (2003) a NOAEL of 4.8 mg/kg/day for developmental effects on the testis is deduced.

The reproductive effects of in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP) in adult male offspring rats were investigated (Andrade et al., 2006b). The selected endpoints included reproductive organ weights, testicular function, hormonal status, sexual behaviour and fertility. Two wide ranges of doses, low and high, were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 21. The low-doses were 0.015, 0.045, 0.135, 0.405 and 1.215 mg DEHP/kg body weight (bw) /day, and the high-doses were 5, 15, 45, 135 and 405 mg DEHP/kg bw/day. A reduction in daily sperm production of 19-25% in relation to control was observed in animals exposed to 15, 45, 135 and 405 mg/kg/day. Quantitation of specific cell types shows that the observed effects in daily sperm production are not related to changes in the number of Sertoli cells or their capability to support early stages spermatocytes. A low incidence of cryptorchidism was observed in DEHP exposed groups with a lowest observed adverse effect level of 5mg/kg/day. Serum testosterone concentration was similar to control at most doses but was significantly increased at 0.045, 0.405 and 405 mg DEHP/kg/day. In spite of this effect, the weight of seminal vesicle with coagulating glands was significantly reduced at 405 mg/kg/day. Testis, epididymis and prostate weights were similar among groups. Fertility and sexual behaviour were not affected by DEHP treatment at any dose. Overall, our results show that in utero and lactational DEHP exposure reduces daily sperm production and has the potential to induce reproductive tract abnormalities (of which cryptorchidism seems to be the most sensitive in our rat strain) in male offspring rats. The lowest observed adverse effect levels (LOAELs) for these effects were 15 and 5 mg/kg/day, respectively. Therefore, the no observed adverse effect level (NOAEL) for this study can be set at 1.215 mg/kg/day.

Time-mated Wistar rats were gavaged from GD7 to PND 16 with doses of DEHP from 3 to 900 mg/kg bw/d (Christiansen et al., 2010). Male pups were investigated besides others for anti-androgenic effects like anogenital distance (AGD), nipple retention (NR), external genital development, organ weights and histopathology, gene expression of androgen-regulated genes. At doses >/= 10 mg/kg bw/d adverse effects like reduced AGD, increased NR, reduced weight of levator ani/bulbocarvernosus muscles and prostate as well as mild external genitalia dysgenesis were observed. At higher doses histopathological effects on the testes, reduced testis weight and expression of androgen-regulated genes in the prostate occurred. The NOAEL and LOAEL for male reproductive toxic effects were 3 and 10 mg/kg bw/d, respectively.

Carbone et al. (2010) exposed Wistar rats (3 dams per group) to 0, 3, 30 mg DEHP/kg bw/d via drinking water from GD1 till PND21. Male offspring (10 per group) were examined on PND30. Whereas no adverse effects were observed at 3 mg/kg bw/d undescendent testis, decreased testis weight, increased hypothalamic GABA and reduced serum FSH were observed in the 30 mg/kg bw/d dose group. Body weight, serum LH and hypothalamic aspartate were not affected.

Akingbemi at al. (2001)studied effects of oral exposure to DEHP on male steroidogenesis in Long-Evans rats using several different exposure regimes. Hormone levels (testosterone and LH) were determined in vivo in serum, and Leydig cells were isolated and cultured for analyses of in vitro androgen biosynthesis. Pregnant dams (n=7) were administered 100 mg/kg and day of DEHP by gavage during gestation day 12 to 21. Serum levels of testosterone and LH were significantly reduced in the offspring at 21 and 35 days of age (approximately to 70 and 40% of controls levels, respectively), but not at 90 days, as measured in 9-18 randomly selected male pups per group. In Leydig cells isolated from 18 pups, the testosterone production was reduced at day 21 (by approximately 50%), but not later. After exposure of lactating dams to 100 mg/kg and day (n = 7) during postnatal day (PND) 1 to 21 by gavage, serum concentrations of testosterone in the offspring were reduced at day 21, but not at day 35 and 90 post-exposure. No effects were seen on LH. Prepubertal rats (n=10) were gavaged with 0, 1, 10, 100 or 200 mg/kg and day for 14 days during either PND 21-34 or 35-49. No effects were observed on serum hormone levels, but the Leydig cells were affected by DEHP as indicated by decreased in vitro testosterone production and inhibited steroidogenic enzymes (measured in isolated Leydig cells) after exposure to 100 or 200 mg/kg and day (exposure PDN 21-34), or 10, 100 or 200 mg/kg and day (exposure PND 35-48). When prepubertal rats were exposed as above, but for 28 days (PND 21-48), increased concentrations of serum testosterone, interstitial fluid testosterone, and serum LH were observed (30-40 % at 10, 100 and 200 mg/kg and day). Similarly, the testosterone production was dosedependently increased in isolated Leydig cells obtained from these rats. When young adult rats were exposed as above for 28 days (PND 62-89), no effects were observed on any parameter. This study shows that the younger the rats are, the more sensitive they are to the effects of DEHP. Exposure of the dams during pregnancy or at the first postnatal weeks to 100 mg/kg and day reduced the serum levels of testosterone in male offspring. Effects on the Leydig cells were indicated at even lower exposure (10 mg/kg and day), but the relevance of the in vitro assay is not clear. The LOAEL for effects of DEHP on the serum concentration of testosterone in very young rats is 100 mg/kg and day.

Moore et al.(2001)studied the effects of DEHP (0, 375, 750 or 1,500 mg/kg and day, by gavage) on male reproductive system development and sexual behaviour in Sprague-Dawley rats (n = 5-8/group). The exposure started at gestation day 3, ended at postnatal day 21, and male pups were examined at PND 21, 63 and 105. Numerous effects, including those normally observed after high doses of DEHP, such as malformations and reduced weights of organs related to the male sexual system, were observed in the pups at the highest doses. The lowest dose was a LOAEL (375 mg/kg and day), with findings of adverse effects on aerola and nipple retention, as well as testis and anterior prostate weight (reductions). Although not statistically significant, there were indications of effects on sexual behaviour at PND 105 in all dose groups (inactivity in 3 of 7 low dose males when kept together with females).

Parks et al.(2000)studied the effects of DEHP on male reproductive parameters in Sprague-Dawley rats. Sprague-Dawley rats were randomly assigned to groups that were gavage dosed with 0 (corn oil vehicle) or 750 mg/kg bw/day DEHP from GD 14 (GD 1 = day after mating) until necropsy. Rats were killed and necropsied on GD 17, 18, or 20 or PND 2 (PND 1 = day after birth). The study was conducted in 2 blocks, and a total of 4–5 litters per group were examined at each necrospy period. At GD 17, 18, and 20 and PND 2, 1 testis from 2 or 3 males/litter was incubated in media for 3 hours to determine ex vivo testosterone production, and the other testis was used to measure testosterone content. In GD 17, 18, and 20 males, testosterone levels were also measured in the carcasses from which testes were removed (n = 18–20 per group). Testosterone levels were measured by RIA. One testis from each of 4 DEHP-treated and 6 control PND 2 males was fixed in 5% glutaraldehyde for histopathological examination. One testis from each of 4 control and 5 DEHP-exposed PND 20 males and an unspecified number of DEHP-exposed PND 3 males from a parallel study was stained for 3β-hydroxysteroid dehydrogenase, which is specific for Leydig cells. Anogenital distance was measured in all male and female offspring on PND 2. Litter means were used in statistical analyses. Data were analyzed by ANOVA followed by 2-tailedt-tests if ANOVA resulted in significant findings. Testicular histopathological findings were analyzed by Fisher exact test. Maternal weight gain during gestation was significantly reduced in the DEHP-treated group. Number of live pups at birth was not significantly affected by DEHP treatment. Ex vivo testicular testosterone production in GD 17, 18, and 20 and PND 2 offspring from DEHP-exposed groups was significantly lower compared to control groups. Testicular testosterone content in DEHP-exposed offspring and pups was reduced by 60–85% compared to controls examined at each necropsy period; the effect was statistically significant at all time points except GD 20. [It appears a footnote regarding GD 20 is missing in Table 1 of the study.]Whole body testosterone levels were significantly lower in DEHP-exposed fetuses on GD 17 (71% lower than controls) and 18 (47% lower than controls), but the reduction on GD 20 was not significant. Significant reductions in testis weight were noted in the DEHP group on GD 20 (18% lower than controls) and PND 2 (49% lower than controls). Body weights of DEHP-exposed pups were described as 23% lower than controls on PND 2, but statistical significance was not achieved. Testis weights adjusted for body weights were significantly decreased in PND 2 pups exposed to DEHP. Anogenital distance was significantly reduced by 36% in PND 2 males compared to controls but was not affected in female pups exposed to DEHP. Histopathological examination of PND 2 testes of DEHP-treated rats revealed an increased number of enlarged and multinucleated gonocytes and aggregates of hyperplastic Leydig cells. 3β-Hydroxysteroid dehydrogenase staining confirmed the presence of Leydig cell aggregates in DEHP-exposed males on GD 20 and PND 2. In contrast, 3β-hydroxysteroid dehydrogenase staining revealed an even dispersion of Leydig cells and less intense staining in testes of control fetuses and pups. The study authors concluded that treatment with 750 mg/kg bw/day DEHP inhibited testosterone production in male pups during the period of sexual differentiation, and this inhibition was a likely cause of malformations observed in other studies

Gray et al.(1999)investigated the reproductive effects of ten known or suspected antiandrogens, including flutamide, Vinclozolin, dibuthyl phthalate (DBP) and DEHP. Eight pregnant Sprague Dawley dams were administered DEHP (750 mg/kg bw/day; > 99% pure) in corn oil by gavage from gestation day 14 to day 3 of lactation. The male offspring was examined for abnormalities (retained nipples, cleft phallus, vaginal pouch, and hypospadias). The animals were also examined internally (ectopic or atrophic testes, agenesis of the gubernaculum, epididymides, sex accessory glands, and ventral prostate, epididymal granulomas, hydronephrosis, and enlarged bladder with stones). Weights measured included body, pituitary, adrenal, kidney, liver, ventral prostate, seminal vesicle (with coagulating gland and fluid), testis, and epididymis. Gonads and sex accessory tissues were examined microscopically.DEHP was considerably more toxic than was DBP to the reproductive system of the male offspring. The gestational and lactational exposure induced a statistically significantly increased incidence of both reproductive and non-reproductive malformations including decreased anogenital distance, areolas (88%), hypospadias (67%), vaginal pouch (45%), ventral prostate agenesis (14%), testicular and epididymal atrophy or agenesis (90%), and retained nipples in examined pups. In addition, several 8-day old pups displayed haemorrhagic testes by gross examination. In adult offspring (5 months old) the weight of the gonads, accessory sex organs, and the Levator ani-bulbocavernosus were statistically significantly decreased. Gray and coworkers found that the chemicals investigated could be clustered into three or four separate groups, based on the resulting profiles of reproductive effects. DBP and DEHP induced a higher incidence of testicular and epididymal abnormalities, including atrophy and agenesis, which is not generally found with flutamide or Vinclozolin even at high dose levels. A LOAEL of 750-mg/kg bw/day is derived from this study.

In order to define the dose-response relationship between di(2-ethylhexyl) phthalate (DEHP) and the Phthalate Syndrome of reproductive alterations in F1 male rats,Gray et al. (2009)dosedSprague-Dawley (SD) rat dams by gavage from gestational day 8 to day 17 of lactation with 0, 11, 33, 100, or 300 mg/kg/day DEHP (71–93 males per dose from 12 to 14 litters per dose). Some of the male offspring continued to be exposed to DEHP via gavage from 18 days of age to necropsy at 63–65 days of age (PUB cohort; 16–20/dose). Remaining males were not exposed after postnatal day 17 (in utero-lactational [IUL] cohort) and were necropsied after reaching full maturity. Anogenital distance, sperm counts and reproductive organ weights were reduced in F1 males in the 300 mg/kg/day group and they displayed retained nipples. In the IUL cohort, seminal vesicle weight also was reduced at 100 mg/ kg/day. In contrast, serum testosterone and estradiol levels were unaffected in either the PUB or IUL cohorts at necropsy. A significant percentage of F1 males displayed one or more Phthalate Syndrome lesions at 11 mg/kg/day DEHP and above. The study was able to detect effects in the lower dose groups only because it examined all the males in each litter rather than only one male per litter. Power calculations demonstrate how using multiple males versus one male/litter enhances the detection of the effects of DEHP. The results at 11 mg/kg/day confirm those reported from a National Toxicology Program multigenerational study which reported no observed adverse effect levels-lowest observed adverse effect levels of 5 and 10 mg/kg/day DEHP, respectively, via the diet.

Srivastava et al.(1989) dosed groups of 21 pregnant albino rats (strain not specified) on day 6-15 of gestation with 0 or 1,000 mg DEHP/kg bw by gavage. On day 20 of gestation all pregnant rats were killed and seven litters from each group were used for standard teratology studies, the remaining 14 litters were used for a study of liver enzyme activities and determination of DEHP in liver tissue. There was no significant difference in the number of total live foetuses between control and treated animals. No gross or skeletal abnormalities were observed in the foetuses of the control or DEHP-exposed animals (no data were shown). Significant amounts of DEHP were, however, found in foetal livers and foetal relative liver weights were increased, whereas the activity of mitochondrial succinate dehydrogenase, ATPase, malate dehydrogenase and cytochrome c oxidase was decreased. The authors concluded that maternal exposure to DEHP during pregnancy could adversely affect the foetal livers. These results also indicate that DEHP can cross the placental barrier.

The effects on the testicular development in the offspring exposed to DEHP in utero were studied by Tandon et al. (1991). Groups of six pregnant rats were given vehicle (ground nut oil) or DEHP (1,000 mg/kg bw/day; purity not specified) by gavage, during the entire gestation period. Birth weight of all pups and body weight gain of two randomly selected male pups from each litter were recorded at day 7, 15, 31, 61 and 91 days of age. Absolute and relative testes weights were significantly reduced at day 31, but normalised at day 61 and 91. The offspring of rats exposed to DEHP during the gestational period exhibited a significant increase in the activities of testicular lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase and a decrease in sorbitol dehydrogenase at the age of 31 days, which was persistent up to the age of 61 days. The concentration of epididymal spermatozoa was significantly reduced day 91, the only day it was measured (5.04 ± 0.24 million in DEHP-treated versus 6.48 ± 0.35 in controls).

The Wilson (2007) study was designed to test the hypothesis that gubernacular lesions would be more prevalent in the DEHP-treated (750 mg/kg/day, gestational days 14–18) Wistar male than in the SD rat offspring, whereas the SD rat would display a higher incidence of epididymal agenesis. As hypothesized, striking differences were seen in the incidences of epididymal (67% in SD versus 8% in Wistar) and gubernacular lesions (0% in SD versus 64% in Wistar) among the two strains. In addition, fetal androgen and insl3 mRNA levels differed among the strains. SD fetal males had higher insl3 mRNA and lower T levels than Wistar males. The ratio of ins l3 mRNA to T differed among DEHP-treated SD and Wistar fetal males, indicating that the steroidogenic pathway was more affected in the SD strain than in the Wistar strain. Taken together, these results suggest that the different malformation profiles produced by in utero phthalate treatment arise, at least in part, from strain differences in fetal Leydig cell function and the manner in which these cells respond to DEHP treatment.

Pregnant Sprague Dawley rats were gavaged on gestation days (GD) 14–18 with vehicle control, 500 mg/kg DBP, 500 mg/kg DEHP, or a combination of DBP and DEHP (500 mg/kg each chemical; DBP + DEHP) (Howdeshell 2007); the dose of each individual phthalate was one-half of the effective dose predicted to cause a 50% incidence of epididymal agenesis. In experiment one, adult male offspring were necropsied, and reproductive malformations and androgen-dependent organ weights were recorded. In experiment two, GD18 testes were incubated for T production and processed for gene expression by quantitative real-time PCR. The DBP + DEHP dose increased the incidence of many reproductive malformations by > 50%, including epididymal agenesis, and reduced androgen-dependent organ weights in cumulative, dose-additive manner. Fetal T and expression of insl3 and cyp11a were cumulatively decreased by the DBP + DEHP dose. These data indicate that individual phthalates with a similar mechanism of action, but with different active metabolites (monobutyl phthalate versus monoethylhexyl phthalate), can elicit dose-additive effects when administered as a mixture.

Howdeshell (2008)characterized the dose-response effects of six individual phthalates (BBP, DBP, DEHP, diethyl phthalate [DEP], diisobutyl phthalate [DiBP], and dipentyl phthalate [DPP]) on gestation day (GD) 18 testicular testosterone production following exposure of Sprague-Dawley rats on GD 8–18. BBP, DBP, DEHP, and DiBP were equipotent (ED50 of 440 ± 16 mg/kg/day), DPP was about threefold more potent (ED50 5 130 mg/kg/day) and DEP had no effect on fetal testosterone production. they hypothesized that coadministration of these five antiandrogenic phthalates would reduce testosterone production in a dose-additive fashion because they act via a common mode of toxicity. In a second study, dams were dosed at 100, 80, 60, 40, 20, 10, 5, or 0% of the mixture. The top dose contained 1300 mg of total phthalates/kg/day including BBP, DBP, DEHP, DiBP (300 mg/kg/day per chemical), and DPP (100 mg DPP/kg/day). This mixture ratio was selected such that each phthalate would contribute equally to the reduction in testosterone. As hypothesized, testosterone production was reduced in a dose-additive manner. Several of the individual phthalates and the mixture also induced fetal mortality, due to pregnancy loss. These data demonstrate that individual phthalates with a similar mechanism of action can elicit cumulative, dose additive effects on fetal testosterone production and pregnancy when administered as a mixture.

Influence of di-(2-ethylhexyl) phthalate (DEHP) on testicular development was studied by oral administration of DEHP at doses of 500 and 1000 mg/kg/day to pregnant rats on gestational days (G) 7 to 18 (Shirota 2005). Ethinyl estradiol (EE) at dose levels of 0.25 and 0.5 mg/kg/day was used as a reference substance. Each 5-6 pregnant rats were sacrificed and their fetuses were examined on G12, 14, 16, 18 and 20. Fetal deaths averaging 20-36% were observed at every examination in the group receiving 1000 mg/ kg of DEHP. Increases of fetal deaths over 50% were also observed in the reference group that received 0.5 mg/kg of EE. Microscopic examination of the fetal testis in groups treated with DEHP revealed degeneration of germ cells in G16 fetuses and localized proliferation or hyperplasia of interstitial cells in G18 and 20 fetuses. Germ cells having more than two nuclei were observed in a few cases including the control testes of G14 fetuses. These multinucleated cells were observed frequently in G20 fetuses treated with DEHP. Examination of testes of naturally delivered offspring of dams treated with 1000 mg/kg of DEHP at 7 weeks of age revealed scattered atrophy or dilatation of seminiferous tubules. Another experiment was carried out to confirm the dose of DEHP affecting testicular development and spermatogenesis. DEHP was given to pregnant rats at doses of 125, 250 and 500 mg/kg/day during G7-18. Similar histopathological changes were observed in fetal testes of the group exposed to 500 and 250 mg/kg of DEHP, but not in those exposed to 125 mg/kg. In postnatal examinations, however, no abnormality was found in the testes at 5 and 10 weeks alter birth in any of the treated groups. Furthermore, no abnormal findings were observed in the function of sperm, sperm counts and sperm morphology in the offspring of the group treated with DEHP during the fetal period at 10 weeks of age. Thus, 125 mg/kg/ day is considered the no-observed-effect-level of DEHP on testicular development of rats by exposure in utero during the period of organogenesis.

Gray et al.(2000) examined the effect of perinatal phthalate exposure in rats. Sprague-Dawley rats were gavage dosed with 0 (corn oil vehicle) or 750 mg/kg bw/day DEHP (99% purity) from GD 14 (GD 1 = day sperm detected) to PND 3 (PND 1 = postcoital day 23). The experiment was repeated with a second block of animals. In each block of the experiment, there were 7–9 treated dams and 9–10 control dams. Parameters examined in pups (period examined) included body weight (PND 2), anogenital distance (PND 2), testicular histology (PND 2, 9–10, and 13, 3–5 months, and 4–7 months), areolas/nipples (PND 13), preputial separation (beginning on PND 28), mating behavior (adulthood), abnormalities of reproductive organs (3–5 months and 4–7 months), and sperm counts. Statistical analyses were based on litters, and blocks were pooled in cases of identical results. Analyses included 1-way ANOVA followed by post hoct-tests when statistical significance was obtained. Anogenital distance and organ weight data were covaried with body weight. Categorical data were analyzed by Fisher exact test or chi-squared test. DEHP treatment resulted in a small reduction in maternal body weight gain. Litter weight at birth was significantly reduced by 15% in the DEHP group, but there was no effect on number of live pups at birth. In DEHP-treated males on PND 2, anogenital distance was significantly decreased by ~30%, with or without adjustment for body weight, and paired testis weights were significantly decreased by 35%. There was no effect on anogenital distance in female pups. Histological examination of testes from DEHP-treated rats on PND 2–3 revealed focal interstitial hemorrhage and multinucleated gonocytes containing 3–5 nuclei or undergoing degenerative changes. Hemorrhagic testes were observed in 7 DEHP-treated males from 3 litters at PND 8–9. Histological examination of testes on PND 9–10 revealed evidence of focal hemorrhage in some testes and extensive coagulative necrosis in other testes of DEHP-treated rats; loss of seminiferous epithelium was observed in areas with hemorrhage or necrosis. Areolas were observed in 87% of DEHP-treated male pups versus none in control pups. DEHP treatment did not delay the age of preputial separation, but preputial separation was incomplete due to malformations in 19 of 56 treated pups. DEHP did not appear to affect sexual behavior in adult rats, except that males with malformed penises were unable to achieve intromission. At necropsy, 45 DEHP-treated adult rats from 15 litters were assessed for malformations of reproductive organs, which were observed in 82% of DEHP-treated males. The types of malformations included permanent nipples, clefting of phallus and hypospadias, vaginal pouches, agenesis of prostate, seminal vesicles, or coagulating glands. Sperm production and numbers were said to be unaffected by DEHP treatment [data not shown]. Testicular defects included hemorrhage, granuloma, fibrosis, reduced or atrophy, and non-descent associated with abnormal gubernacula or ligaments. Significant reductions in weight were observed for all male reproductive organs including testis, levator ani plus bulbocavernosus muscle, seminal vesicle, prostate, penis, and epididymis. Liver, pituitary, kidney, and adrenal weights were not affected by DEHP treatment. Serum testosterone levels were unaffected in DEHP-treated rats. The study authors concluded that 750 mg/kg bw/day DEHP severely alters sexual differentiation in an anti-androgenic manner.

Culty (2008)examined the effects of fetal exposure to a wide range of di-(2-ethylhexyl) phthalate (DEHP) doses on fetal, neonatal, and adult testosterone production. Pregnant rats were administered DEHP from Gestational Day (GD) 14 to the day of parturition (Postnatal Day 0). Exposure to between 234 and 1250 mg/kg/day of DEHP resulted in increases in the absolute volumes of Leydig cells per adult testis. Despite this, adult serum testosterone levels were reduced significantly compared to those of controls at all DEHP doses. Organ cultures of testes from GD20 rats exposed in utero to DEHP showed dose-dependent reductions in basal testosterone production. Surprisingly, however, no significant effect of DEHP was found on hCG-induced testosterone production by GD20 testes, suggesting that the inhibition of basal steroidogenesis resulted from the alteration of molecular events upstream of the steroidogenic enzymes. Reduced fetal and adult testosterone production in response to in utero DEHP exposure appeared to be unrelated to changes in testosterone metabolism. In view of the DEHP-induced reductions in adult testosterone levels, a decrease in the expression of steroidogenesis-related genes was anticipated. Surprisingly, however, significant increases were seen in the expression of Cyp11a1, Cy17a1, Star, and Tspo transcripts, suggesting that decreased testosterone production after birth could not be explained by decreases in steroidogenic enzymes as seen at GD20. These changes may reflect an increased number of Leydig cells in adult testes exposed in utero to DEHP rather than increased gene expression in individual Leydig cells, but this remains uncertain. Taken together, these results demonstrate that in utero DEHP exposure exerts both short-term and longlasting effects on testicular steroidogenesis that might involve distinct molecular targets in fetal and adult Leydig cells.

Borch et al.(2005)evaluated early testicular effects of perinatal exposure to DEHP with or without diethylhexyl adipate in Wistar rats. In the first experiment, pregnant females were treated by gavage with vehicle, DEHP 750 mg/kg bw/day or DEHP 750 mg/kg bw/day + diethylhexyl adipate 400 mg/kg bw/day beginning on GD 7 (plug = GD 0; n = 18/dose group). Chemicals were of 99% purity. On GD 21, 8 dams/group were killed and fetal testes were harvested. The remaining 8 dams/group continued to receive treatment until PND 17. These animals were permitted to litter. Male offspring were killed on PND 26 (birth = PND 0), and testes were harvested. A second experiment used 20 pregnant animals in each of 4 dose groups: vehicle control, DEHP 300 mg/kg bw/day, DEHP 750 mg/kg bw/day, and DEHP 750 mg/kg bw/day + diethylhexyl adipate 400 mg/kg bw/day. Treatment was from GD 7 through PND 17. On PND 22, 3 males/litter were killed and testes were harvested. On PND 190, 1 or 2 males/litter were killed and testes harvested. Of the testes collected on GD 21, 14–19/dose group (2–4/litter) were fixed in formalin, embedded in paraffin, and sections were stained with hematoxylin and eosin for light microscopy. Ten testes/dose group (1 or 2/litter) from PND 22 and PND 26 animals were processed in the same manner. Another 10 testes from these age groups as well as 16 testes/dose group (1 or 2/litter) were fixed in Bouin fluid, and stained with hematoxylin and eosin for light microscopy [embedding material not specified]. Tubule diameters were measured, and a 10% increase over the control maximum was defined as enlarged. Terminal deoxynucleotidal transferase- mediated dUTP nick-end labeling (TUNEL) staining was performed using a commercial kit, and immunostaining was performed for caspase-3, proliferating cell nuclear antigen (PCNA), histone H3, anti-Müllerian hormone, 3β-hydroxysteroid dehydrogenase, vimentin, and smooth muscle actin. Caspase-3 activity was measured in 10 testes/dose group (from 5–10 litters/group) at GD 21, PND 22, and PND 26. [The method was described only by reference to another paper.] DNA laddering was assessed based on relative fluorescence of DNA ladders on gels. Statistical analysis was by ANOVA with post hoc Dunnett test or by Kruskall-Wallis test. Litter was included as a factor in the ANOVA. In testes evaluated on GD 21, vacuolization of Sertoli cells, shedding of gonocytes, reduced interstitial cell cytoplasm, and enlarged tubules were identified in offspring of all dams exposed to DEHP 750 mg/kg bw/day, regardless of diethylhexyl adipate co-exposure, compared to 0–14% of dams exposed to vehicle. Leydig cell hyperplasia was identified in offspring of more dams with DEHP treatment than control dams [statistical analysis not shown]. The number of histone H3-positive cells per testis section was not altered by treatment. [Other immunohistochemistry results were not quantified but were not reported as affected by treatment.] Staining for anti-Müllerian hormone to identify Sertoli cells showed positive cells within Leydig cell clusters, outside the tubules. DNA laddering was increased by DEHP treatment, although TUNEL-positive cells and caspase-3-positive cells were not increased by maternal DEHP 750 mg/kg bw/day. On PND 26, tubules without spermatocytes were found in all litters exposed to DEHP compared to 29% of control litters [statistical analysis not shown]. Malformed tubules were identified in 17–29% of DEHP exposed litters compared to none of the control litters. There were no effects of DEHP treatment on any of the measures of apoptosis on PND 22, 26, or 190, although the authors indicated that “a few animals in the treated groups had very high numbers of TUNEL positive cells, presumably spermatocytes.” The authors concluded that the development of dysgenic tubules in response to DEHP exposure was related to interstitial changes occurring during gestation, including the presence of Sertoli cells in the interstitium. They believed that Sertoli cell dysfunction in the fetal period might underlie the focal testicular dysgenesis seen in older animals. The authors proposed that the lack of alteration in Sertoli cell structure in prepubertal rats in this study might reflect recovery from DEHP, which was last administered on PND 17.

Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 21 (Andrade 2006a and b,Grande 2006,Grande 2007). The low doses were 0.015, 0.045, 0.135, 0.405 and 1.215 mg/kg/day, and the high doses were 5, 15, 45, 135 and 405 mg/kg/day.At the dose levels tested, DEHP had no statistically significant effect on prenatal and postnatal body weight gain of dams. Litter size, sex ratio, postimplantation losses, and number of viable pups were also unaffected by treatment. Pup birth and weaning weight were similar in all groups, and no signs of toxicity were observed in dams and offspring. DEHP had no effect on brain, spleen, thymus, ovary, and thyroid weights of dams. A significant increase in liver and kidney weights was detected at the highest dose level (405 mg/kg/day).In the female offsprings, a significant delay in the age at vaginal opening at 15 mg DEHP/kg/day and above, as well as a trend for a delay in the age at first estrus at 135 and 405 mg/ DEHP/kg/day, was observed. Anogenital distance and nipple development were unaffected.A normal pattern of estrous cyclicity with no hormonal alterations (serum estradiol and progesterone) was observed. A statistically significant increase in tertiary atretic follicles was observed at the highest dose (405 mg DEHP/kg/day). Morphometric analysis indicated that uterus and vagina luminal epithelial cell height were unaffected by treatment. An increase in the number of ovarian atretic tertiary follicles was the only effect observed in adult female offspring exposedin uteroand during lactation to DEHP.The NOAEL for female reproductive development may be set at 5 mg/kg/day.In male offsprings, nipple retention and reduced anogenotal distance, both sensitive markers of anti-androgenic effects during development, were only seen in males exposed to the highest dose (405 mg/kg/day). Delayed preputial separation was observed in animals exposed to 15 mg/kg/day and higher doses. Histopathological examination of the testis on postnatal days (PND) 1 and 22 revealed changes at 135 and 405 mg DEHP/kg/day. The most prominent finding on PND 1 was the presence of bi and multinucleated gonocytes. On PND 22 signs of reduced germ cell differentiation in seminiferous tubules of exposed animals were observed. The current results show that DEHP acts as an anti-androgen at a high dose exposure (405 mg/kg/day). At the adult age, a reduction in daily sperm production of 19-25% in relation to control was observed in animals exposed to 15, 45, 135 and 405 mg/kg/day. Quantitation of specific cell types shows that the observed effects in daily sperm production are not related to changes in the number of Sertoli cells or their capability to support early stages spermatocytes. A low incidence of cryptorchidism was observed in DEHP exposed groups with a lowest observed adverse effect level of 5 mg/kg/day. Serum testosterone concentration was similar to control at most doses but was significantly increased at 0.045, 0.405 and 405 mg DEHP/kg/day. In spite of this effect, the weight of seminal vesicle with coagulating glands was significantly reduced at 405 mg/kg/day. Testis, epididymis and prostate weights were similar among groups. Fertility and sexual behaviour were not affected by DEHP treatment at any dose. Overall, the results show that in utero and lactational DEHP exposure reduces daily sperm production and has the potential to induce reproductive tract abnormalities (of which cryptorchidism seems to be the most sensitive in our rat strain) in male offspring rats. The lowest observed adverse effect levels (LOAELs) for these effects were 15 and 5 mg/kg/day, respectively. Therefore, the NOAEL For male reproductive development can be set at 1.215 mg/kg/day.

However, these results also indicate that other subtle developmental effects occur at lower DEHP doses. In males on PND 1, aromatase activity was inhibited at low doses and increased at high doses resulting in a non-monotonic dose-response. Inhibition was statistically significant at 0.135 and 0.405 mg DEHP/kg/day, while increased activity was observed at 15, 45, and 405 mg/kg/day. In contrast to findings on PND 1, aromatase activity at weaning (PND 22) was more affected in females than in males. An increase in aromatase activity was observed at only one dose in males (0.405 mg/kg/day) while an increase in activity was observed at all doses in the females except regard to the age at which effects are manifested.

Pregnant Wistar rats were orally (gavage) exposed to 0, 0.25 or 6.25 mg DEHP/kg bw/d throughout gestation and lactation (Wei et al., 2012). Offspring were investigated for effects on renal histology, renal function and blood pressure (week 3, 15, 21, 33). DEHP exposure affected renal histology (e.g. number of nephrons, glomerular volume) and function (creatinine clearance) in all dose groups. Effects on kidney weight and blood pressure were inconsistent. Renal protein expression of renin and angiotensin II was reduced at birth and increased a weaning, without clear dose-response. DEHP-exposure also affected the expression of some genes involved in renal development. This is the first study reporting kidney toxicity of DEHP at very low doses which needs confirmation from further studies as other studies on developmental toxicity did not observe kidney toxicity in this low dose range. Additionally the partially observed differences between sexes and different findings at the investigated time points need further clarification.

Pregnant Wistar rats were orally (gavage) exposed to 0, 1.25 or 6.25 mg DEHP/kg bw/d throughout gestation and lactation (Lin et al., 2011). Offspring (n</=6 male and 6 female per dose group) were investigated for effects on body weight, glucose and insulin tolerance, beta cell morphometry and function. In all treatment groups effects on beta cells ultrastructure, beta cells mass and pancreatic insulin content were observed as well as alterations in the expression of genes involved in pancreas development and beta cells function at weaning. Adult female offspring revealed elevated blood glucose, reduced serum insulin, impaired glucose tolerance and insulin secretion. Male offspring had increased serum insulin, but there were no significant differences in blood glucose at fasting and during glucose tolerance test. Additionally body weight was reduced during weaning and till adulthood. These preliminary findings in a reduced number of offspring need confirmation from further studies.

Mice

In a continuous breeding study, comparable to a guideline study and performed according to GLP principles, DEHP (> 99% pure) was given to CD-1 mice (20 animals of each sex per dose group and 40 control animals of each sex) at dietary levels of 0, 0.01, 0.1, or 0.3% (equivalent to 0,14, 141, and 425mg/kg bw/day, respectively) (Lamb et al., 1987). Both male and female mice were exposed during a 7-day premating period and were then randomly grouped as mating pairs. The dosing continued during the 98-day cohabitation period and thereafter for 21 days during which final litters were delivered and kept for at least 21 days. Reproductive function was evaluated by measuring the number of litters per breeding pair, number of pups per litter, proportion of pups born alive, and mean pup weight. Exposure to 0.1% DEHP produced a dose-dependent and significant decrease in the number of litters as well as the number and proportion of pups born alive. No pairs were fertile at 0.3%. At a diet of 0.3% DEHP caused an increased liver weight (both absolute and relative) and significantly reduced weights of the reproductive organs in parental animals of both sexes (testes, epididymis, prostate, and seminal vesicles in males and ovaries, oviducts, and uterus in females). All but one of the high-dose males showed some degree of bilateral atrophy of the seminiferous tubules. In addition, this dose level also caused decreased sperm motility and sperm concentration and an increased incidence of abnormal sperm forms. DEHP did not significantly decrease body weight gain in the high-dose group. A crossover mating trial conducted with F0 mice showed a decrease in fertility both for treated males and for treated females, with a complete loss of fertility in the females. Four litters out of twenty were born to treated males mated to control females; in addition, the proportion of pups born alive was decreased. No pups were born when dosed females were mated to control males.The NOAEL for maternal and developmental toxicity was equivalent to 600 and 20-mg/kg bw/per day, respectively.

In a dietary 2-generation study (comparable to a guideline study and performed according to GLP principles) in CD-1 mice, DEHP was given in the diet at 0.0, 0.01, 0.025, and 0.05% (equivalent to 0, 19, 48 and 95 mg/kg bw, respectively) to CD-1 mice (NTIS, 1988). DEHP treatment did not affect the number of implantation sites per dam, the percent fertile matings, the pregnancies with live litters on pregnancy day 1, or the percent viable litters through gestation to postnatal day 4. The F1 generation was mated within dose groups at sexual maturity and F2-offsprings were evaluated for viability and growth at postnatal day 4. For F1-litters, the percentage of prenatal mortality was increased at the high dose (9% versus 26.4%). During the neo-natal period, the percent of viable pups was significantly decreased at 0.05% DEHP. No other effects of DEHP were observed upon growth, viability, age of acquisition for developmental landmarks (incisor eruption, wire grasping, eye opening, testes decent or vaginal opening or spontaneous locomotor activity) on postnatal days 14, 21 or 50/day. Treatment-related lesions were not observed in the dams and no maternal LOAEL was established. The NOAEL for parental toxicity and for F2-offspring was 0.05% DEHP (95 mg/kg bw/day), the highest dose tested. The LOAEL for F1 offspring was 0.05% (95 mg/kg bw/day) (NTIS, 1988).

Tanaka (2002)examined neurobehavioral toxicity in mice exposed to DEHP (> 97% purity) during prenatal development. At 5 weeks of age, 10 CD-1 mice/sex/group were fed diets containing 0, 0.01, 0.03, or 0.09% DEHP for 4 weeks prior to mating and during a 5-day mating period that began at 9 weeks of age. Females continued to receive the control or DEHP-containing diets throughout the gestation and lactation periods. The authors converted DEHP doses to a mg/kg bw/day basis. Each female was mated to 1 male, and the females were allowed to litter and rear their offspring. At birth (PND 0), litter size, litter weight, and sex ratio were determined. Offspring were individually weighed, and postnatal survival was monitored during the lactation period. Neurobehavioral parameters examined in all offspring during the lactation period included surface righting (PND 4 and 7), negative geotaxis (PND 4 and 7), cliff avoidance (PND 7), swimming behavior (PND 4 and 14), and olfactory orientation (PND 14). Weaning occurred at 4 weeks of age, and 1 male and 1 female per litter were selected to continue receiving treatment until 9 weeks of age. [Though not specified, it is assumed that the offspring from each treatment group received the same doses as their parents. ]. At 7 weeks of age, the F1mice were tested using a Biel type water T-maze. Exploratory behavior was assessed using an animal movement analyzing system in 3-week-old mice from the F1generation and 8-week-old mice from the F0and F1generations. Statistical analyses included Bonferroni multiple comparison, ANOVA, Kruskall-Wallis test, chi-squared test, Fisher exact test, Wilcoxon sign test, and/or Jonckheere test. [It does not appear that statistical analyses were conducted on a per litter basis. ]. In F0mice, DEHP treatment had no effect on body weight gain, movement, or exploratory activity. As a result of non dose-related failures to become pregnant or abortions in 1–2 dams of the low- and mid-dose groups, 8–10 litters were available for evaluation in each treatment group. There were no significant effects on sex ratio or litter size or weight at birth. A 7% decrease in body weight in male offspring of the low-dose group compared to control males on PND 0 was the only significant body weight effect observed in offspring. Significant reductions in survival were noted in the high-dose group for female offspring from PND 4 to 14 and for total offspring from PND 4 to 21. Percentages of total surviving offspring at PND 21 were 98.4% in the control group and 92.8% in the high-dose group. Time for surface righting was significantly delayed in females of the low- and mid-dose groups on PND 4, in males of the high-dose group on PND 7, and in females of the low-dose group on PND 7. There were no other significant findings in neurobehavioral parameters examined during the lactation period [data not shown]. Compared to controls, there were no adverse effects in water T-maze performance in treated animals at 7 weeks of age, and movement and exploratory behavior were not affected by treatment at 3 or 8 weeks of age. The study authors concluded that “few adverse effects on several behavioral parameters were produced at the high-dose level of DEHP in the present study. ”

Female C3H/N mice were exposed via diet to DEHP (0, 0.05, 5, 500 mg/kg bw/d) for 8 weeks (Schmidt et al., 2012). In study one the mice were mated one week after start of exposure and exposure continued till the end of lactation, offspring were grown up till PND 84 and mated with untreated males. In study two the mice were mated with untreated males at the end of the 8-weeks exposure period. DEHP intake lead to an increase in food intake, body weight and visceral fat tissue in female C3H/N mice. Similar effects were observed in offspring. However, these effects did not always clearly correspond with dose. Early embryo development was impaired, even if the mothers were only exposed before mating: DEHP-treated animals revealed an increased number of degenerated blastocytes, however without clear dose response. The abortion rate was 100% in the 500 mg/kg bw/d group, but not affected at lower doses. Opposite findings on body weight and fat tissue in mice were reported by Pocar et al. (2012). Pregnant CD-1 mice were orally (gavage) exposed to 0, 0.05, 5.0 or 500 mg DEHP/kg bw/d throughout gestation and lactation (Pocar et al., 2012). Offspring were investigated for effects on pituitary-gonadal axis. At the highest dose group fertility was dramatically reduced, only 1 out of 10 dams was able to deliver, post-implantational losses were significantly increasesd. DEHP exposure at 0.05 and 5.0 mg/kg/d resulted in decreased body weight at PND21 and 42 in males and females and reduced abdominal fat in females but not in males. These effects did not reveal a clear dose-response relation. Sperm count and sperm viability was decreased in treated animals without clear dose-response relationship.DEHP affected in vitro oocyte maturation and evelopmental competence in adult female offspring. Expression of steroidogenesis-related genes in the gonads and of gonadotropin mRNA in the pituitary were also affected in treated animals. Due to these contradictory findings in mice further investigations are necessary to evaluate the possible correlation of gestational/lactational DEHP exposure and body weight and fat tissue development.

In vitro studies

In theBowden study (2000), DEHP was administered by the oral route (gavage) to pregnant CD rats on Days 6-11 of gestation at a generally recognised limit dosage of 1000 mg/kg/day. On Day 11 of gestation serum was obtained via the dorsal aorta approximately 1.5 hours after the final administration of DEHP. The influence of this serum upon growth and development of embryos in vitro was assessed in Day 9 embryos from rats of the CD strain. Embryos were cultured for a period of approximately 48 hours at a temperature of 38.0 ± 1.0 °C. It was observed in this exploratory investigation that treatment with di-(2-ethylhexyl) phthalate at a dosage of 1000mg/kg/day produced no adverse effects in pregnant female rats or any apparent reduction in the level of zinc in their serum. Culture of embryos in serum from these treated rats, however, produced adverse effects on embryonic growth, development and morphology in vitro. It was concluded, therefore, that the rat whole embryo culture test system is sensitive to detecting teratogenic agent(s) present in serum from DEHP-treated pregnant rats.

The influence of the main metabolites of di-(2-ethylhexyl) phthalate (DEHP) upon growth and development in vitro, was assessed in Day 9 embryos from rats of the CD strain (Bowden 2001). The materials tested were mono-2-ethyl-1-hexylphthalate (MEHP), metabolites I, V, VI and IX. In part 1 of the main study MEHP was added to the culture medium at nominal concentrations of 0.01, 0.04, 0.2, 1, 5 and 10 µmole/ml, in both the presence and absence of metabolic activation. S-9 metabolic activation mixes were prepared from animals treated with Arochlor 1254 or DEHP. In part 2 of the main study metabolites I, V, VI and IX were each added to the culture medium at nominal concentrations of 0.002, 0.01, 0.04, 0.2, 1 and 5 µmole/ml in the absence of metabolic activation. In Part 3 of the study DEHP and the metabolites 2-ethyl hexanol and 2-ethyl hexanoic acid were investigated. All embryos were evaluated alter approximately 48 hours in culture. Data was compiled according to the achieved concentrations for discussion and interpretation of the results. It was observed in this exploratory investigation that embryos cultured in the presence of MEHP at concentrations in the region of 0.6 - 1.0µmole/ml exhibited adverse effects on embryonic growth, development and morphology in vitro both in the presence and absence of metabolic activation; slight effects were apparent at lower concentrations in the presence of DEHP-derived S-9. Generally more effects were apparent in the presence of metabolic activation, the DEHP-derived S-9 producing slightly greater effects than the Arochlor-derived S-9. At concentrations of 4imole/ml and above embryos had failed to survive to the end of the treatment period, both in the presence and absence of metabolic activation. Embryos cultured in the presence of Metabolite I exhibited only marginal adverse effects on embryonic development at concentrations of 0.023-0.029µmole/ml, adverse effects on morphology were apparent at 0.023µmole/ml and above. Embryos cultured in the presence of Metabolite V exhibited no adverse effects on embryonic growth or development in vitro at concentrations up to 0.408 µmole/ml. Adverse effects on embryonic morphology were apparent at 0.399µmole/ml and above.Embryos cultured in the presence of Metabolite VI exhibited no adverse effects on embryonic growth, development or morphology in vitro at concentrations up to 0.091 µmole/ml. At 0.3 µmole/ml only a marginal effect on embryonic morphology was observed. Embryos cultured in the presence of Metabolite IX exhibited no adverse effects on embryonic growth or development in vitro at concentrations up to 0.05. tmole/m1, slight adverse effects were observed at 0.17µmole/m1. Adverse effects on morphology were apparent at 0.0004µmole/ml and above. The types of malformations observed in this study were also seen in a previous study using DEHP-treated rats in particular abnormalities of the otic system and fore limb buds. A comparison of the data for each metabolite suggests the following order of potential toxicity: IX >I>VI>V>MEHP. Embryos cultured in the presence of DEHP exhibited no adverse effects on growth, development or morphology at concentrations up to 0.19µmole/ml. Adverse effects on embryonic growth and morphology were apparent at 0.85µmole/ml and above, whilst adverse effects on development were apparent at 2.01. tmole/ml and above. Embryos cultured in the presence of 2-ethyl hexanol at concentrations up to 0.09 µmole/ml exhibited no adverse effects on growth, development or morphology. A slight adverse effect on development was apparent at 0.16 - 0.23 µmole/ml. Adverse effects on embryonic growth, development and morphology were apparent at 0.991µmole/ml, in addition half of the embryos had failed to survive to the end of the treatment period. Embryos cultured in the presence of 2-ethyl hexanoic acid at concentrations up to 1.1 µmole/ml exhibited no adverse effects. At 4.364 - 5.131.1mole/ml adverse effects on growth, development and morphology were apparent. A comparison of these data suggest the following order of potential toxicity: DEHP = 2-ethyl hexanol > 2-ethyl hexanoic acid.

Justification for selection of Effect on developmental toxicity: via oral route:

Wolfe and Layton (2004): Lowest reliable NOAEL

Justification for selection of Effect on developmental toxicity: via inhalation route:

Key study

MALE REPRODUCTIVE TRACT TOXICITY

Testicular toxicity in young and adult animals

Testicular effects have been observed in several repeated dose toxicity studies in rats (Poon et al., 1997; NTP, 1982c; Gray et al., 1977; Ganning et al., 1987, 1990; David et al., 2000c). These studies are described in more detail, including systemic effects, in the section on Repeated dose toxicity.

Oral

Rat

Young and adult rats (1, 2, 3, 6, and 12 weeks of age) were given five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (0, 10, 100, 1000, 2000 mg/kg) and histological changes in the testes were examined 24 hr after the last dose (Dostal et al., 1988). Relative testis weights were reduced at doses of 1000 mg/kg in 1, 2, 3, and 6-week-old but not in 12-week-old rats, while doses of 2000 mg/kg were fatal to suckling rats and caused decreased relative testis weight but not death in 6- and 12-week-old rats. In neonatal rats (1 week old), DEHP (1000 mg/kg) caused a 35% decrease in Sertoli cell numbers while 2- and 3-week-old rats showed losses of spermatocytes but not of Sertoli cells. The 6- and 12-week-old rats showed loss of both spermatids and spermatocytes at 1000 and/or 2000 mg/kg. Total testicular zinc concentrations were decreased in 12-week-old but not in suckling (3-week) or weaned (6-week) rats. The results support the hypothesis that the Sertoli cell is the primary testicular target of phthalate ester toxicity since effects were observed at an age when only Sertoli cells were present. Fertility was assessed in mating trials in adult male rats after neonatal exposure to DEHP on Days 6-10. Although Sertoli cell number was reduced 24 hr after the last dose, the numbers were normal at 6 and 13 weeks of age. However, at 6 weeks there was a dose-related decrease in maturation of the spermatids in the tubules. There were no consistent changes in fertility, implantation rate, or numbers of live fetuses in untreated females mated with the DEHP-treated males. However, there were decreases in testis weight and testicular spermatid numbers at 13 and 19 weeks but not at 11, 12, 16, or 23 weeks of age. Therefore, a loss of Sertoli cells due to DEHP exposure neonatally did not affect the fertility of the rats as adults, but may have caused subtle effects on sperm production.

Male Sprague-Dawley rats (4, 10, or 15 weeks of age, 8 animals per group) were used to study the age-dependent effects on male reproductive organs (Gray and Gangolli, 1986). The rats were given 2,800 mg/kg bw of DEHP (purity not specified) orally for 10 days. Administration to 4-week-old rats produced a marked reduction in absolute weights of the testes, seminal vesicles, and prostate. There was only a slight reduction in testis weight in 10-week-old rats but the seminal vesicle and prostate weights were significantly reduced. DEHP had no effect in 15-week-old rats. Histologically, the testes of the 4-week-old rats showed severe atrophy affecting virtually all the tubules. These were populated only by Sertoli cells, spermatogonia, and occasional primary spermatocytes. In the 10-week-old rats, these histological changes were evident in 5 to 50% of the tubules, the remainders appearing essentially normal. No histological abnormalities were seen in testes from the 15-week-old rats. In the same study, the effects of MEHP on Sertoli cell function were studied in immature rats by measuring the secretion of seminiferous tubule fluid and androgen binding protein. A single dose of 1,000 mg/kg bw of MEHP reduced fluid and protein production to around 50% of the concurrent control group and to 25% after three repeated doses.

To study the differing response between immature and mature male rats,Sjöberg et al. (1985)carried out a series of experiments. Groups of 8 male Sprague-Dawley rats (25, 40 or 60 days old) were dosed with 0 or 1,000 mg/kg bw of DEHP in corn oil by gavage for 14 days. After sacrifice liver, testes, ventral prostate, and seminal vesicles were removed, cleaned from fat and weighed. The left testis and epididymis were fixed in Bouin’s fluid for histopathological examination. The liver weight was significantly increased in all three age groups. The absolute testicular weight was significantly decreased; histopathological examination showed severe testicular damage in the 25-day-old rats, whereas the older animals were unaffected. In the youngest age group, there was a marked reduction in the number of germ cells, a high occurrence of degenerating cells, and a reduction of the tubular diameter. There also was a marked reduction in the number of spermatogonia.

Sjöberg et al.(1986)studied the age-dependent testis toxicity of DEHP (1,000 and 1,700 mg/kg bw in the diet for 14 days) in rats at 25, 40 and 60 days of age. Body weight gain was retarded in all dosed groups and testicular weight was markedly reduced in 25- and 40-day-old rats given 1,700 mg/kg bw. Severe testicular damage was shown for the 25-day and 40-day-old rats at both dose levels. No changes were found in the 60-day-old rats. The authors propose that the difference in response to DEHP to male rats of different age may be due to a higher oral absorption of the DEHP-derived metabolite MEHP in younger animals (Sjöberg et al., 1985).

Parmar et al.(1986) found that DEHP affects spermatogenesis in adult male albino rats. Groups of 6 adult male rats were administered 0, 250, 500, 1,000 or 2,000 mg DEHP/kg bw (purity not specified) in groundnut oil by gavage for 15 days. Body weight, testicular weight, sperm concentration, and activity of several testicular enzymes were determined. In the 2,000-mg/kg bw group, both absolute and relative weights of the testes were significantly reduced. In all dosed groups, sperm counts were significantly reduced in a dose dependent manner from 250 mg/kg bw. The activities of γ-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH) were significantly increased at doses from 500 mg/kg bw, sorbitol dehydrogenase (SDH) was decreased from 1,000 mg/kg bw, and acid phosphatase was reduced at 2,000 mg/kg bw. The activity of β-glucuronidase was significantly increased at 2,000 mg/kg bw. The authors suggested that DEHP can affect spermatogenesis in adult rats by altering the activities of these enzymes responsible for the maturation of sperms and that the reduced number of sperms may be responsible for the antifertilic effects of DEHP. The authors also concluded that even at 250 mg/kg bw DEHP causes a decrease in testicular function after short-term dosing. A LOAEL of 250 mg/kg/day DEHP is derived from this study.

To further understand the mechanisms responsible for the enhanced sensitivity of the testes of developing animals to DEHP, the activities of the testicular enzymes associated with spermatogenesis including LDH, GGT, SDH, β-glucuronidase, and acid phosphatase were studied in a similar study investigating the oral effect of DEHP on 25 day old male Wistar rats (Parmar et al., 1995). Doses of 0, 50, 100, 250 or 500 mg/kg bw of DEHP (purity not specified) in groundnut oil were given for 30 consecutive days to 6 male rats per dose group. There was an exposure-related and significant decrease of absolute and relative testicular weight at all dose levels. From 50 mg/kg also a dose-dependent and significant increase in the activities of LDH and GGT was noted while that of SDH decreased. β-glucuronidase increased at 250 or 500 mg DEHP/kg, while acid phosphatase decreased at the same dose levels. The administration also resulted in marked destructive changes in the advanced germ cell layers and marked degrees of vacuolar degeneration in the testes at 250 and 500 mg/kg bw. The significant alterations in the activities of SDH, LDH, and GGT occurred thus at much lower DEHP levels and prior to the histopathological changes. The Leydig cells and the fibroblasts appeared normal. A LOAEL for young rats of 50 mg/kg bw/day is derived from this study for effects on absolute and relative testis weight, and reduced testicular enzyme activities.

In a 90-day study performed according to OECD Guidelines and the principles of GLP mild to moderate seminiferous tubular atrophy and Sertoli cell vacuolation were observed in the testes of young male Sprague-Dawley rats (Poon et al., 1997). Groups of 10 young males per dose level were given 0, 5, 50, 500 or 5,000-ppm (0, 0.4, 3.7, 37.6 or 375.2 mg/kg bw) per day in the diet for 13 weeks. The rats were 105-130 g (approximately 32-37 days) at initiation of dosing and reached sexual maturity at 70 days (Charles River, 2000). The method for preparing testicular tissue included Zenker’s fluid fixation, paraffin embedding and haematoxylin and eosin staining., The histopathological slides were controlled blindly. No clinical signs of toxicity were observed. Feed consumtion and body weight gain were not affected. At 5,000-ppm, rats of both sexes had significantly increased absolute and relative liver weights and relative kidney weight. In the 500-ppm dose group, a high incidence of minimal to mild Sertoli cell vacuolation was observed in 7 out of 10 rats. No other effects were noted at this dose level. At 5,000-ppm, the absolute and relative testis weights were significantly reduced. Microscopic examination revealed a mild to moderate, bilateral, multifocal, or complete atrophy of the seminiferous tubules with complete loss of spermatogenesis and cytoplasmic vacuolation of the Sertoli cells lining the tubules in 9 out of 10 rats. The incidence and severity of seminiferous tubular atrophy were similar to those found in a following study on di-n-octyl phthalate with a positive control group fed a diet containing 5,000-ppm DEHP. The progressive increase in vacuolation of Sertoli cells plus injury and loss of germinal epithelium and spermiogenesis in a treatment-related fashion is regarded as a powerful evidence that the changes observed were not artifactual and that the conclusions were not compromised by the technology employed. A NOAEL of 50-ppm DEHP in the diet (3.7 mg/kg bw/day) is derived from the study.

Fischer 344 rats (10 animals/sex/group) were given 0, 1,600, 3,100, 6,300, 12,500 or 25,000-ppm (0, 80, 160, 320, 630 or 1,250 mg/kg/day) of DEHP (> 99.5% pure) in the diet for 13 weeks prior to an oncogenicity study (NTP, 1982). The mean body weight gain of male rats was depressed (29%) in males at 25,000-ppm relative to controls. Testicular atrophy was observed in all males fed 25,000-ppm and was present, but less pronounced in males fed 12,500-ppm (630 mg/kg/day). No other compound-related histopathological findings were observed. A NOAEL of 320 mg/kg/day DEHP is derived from this study.

In a study reported byGray et al. (1977),groups of 15 male and female Sprague-Dawley rats were exposed to DEHP via incorporation in the diet at concentrations of 0, 0.2, 1.0 or 2.0% (0, 143, 737 or 1,440 mg/kg bw/day in males) from 1 up to 365 days. The absolute testicular weight in mid- and high-dose rats was lower than compared to control rats while the relative weights were increased. Histological examination revealed a severe seminiferous tubular atrophy and cessation of spermatogenesis related to the dietary level of DEHP. These changes were demonstrated from week 2. A LOAEL of 143 mg/kg/day DEHP is derived from this study.

In an oncogenicity study, performed according to EPA guidelines and in conformity with the principles of GLP, F-344 rats (70 males and females/group; approx. 6 weeks old at initiation of dosing) were administered DEHP (99% pure) at dietary concentrations of 0, 100, 500, 2,500 or 12,500-ppm (0, 5.8, 28.9, 146.6 or 789.0 mg/kg/day) for at least 104 weeks (David et al., 2000c, 2001). An additional group (55/sex) was administered 12,500-ppm DEHP for 78 weeks, followed by a recovery period of 26 weeks. An increased incidence of clinical abnormalities was observed in males from the two highest dose groups and the recovery group, significant at the highest dose level. There also was a decreased survival and decreased body weight gain in both sexes at these dose levels, significant only at the highest dose level. In males that died or were sacrificed in extremis during the study, there was an increased (not statistically significant) incidence of small and/or soft testis, small epididymis and/or seminal vesicle. At study termination a dose-related increase of small and/or soft testis was observed in the 2,500 and 12,500-ppm group. Also the recovery group had an increased incidence of small or soft testis. An increased incidence (not significant) of aspermatogenesis was present at 2,500-ppm in unscheduled deaths, at interim sacrifice, and at study termination. At 12,500-ppm, the absolute and relative testis weights were significantly decreased with associated increased incidence of bilateral aspermatogenesis in all males accompanied by hypospermia in the epididymis and decreased incidence of interstitial cell neoplasms (3/10 compared to 9/10 in control group). In the pituitary, an increased number of castration cells were observed in 30/60 males compared to 1/60 of the control males. There was no indication in rats killed at study termination that DEHP-related changes in the testes and pituitary were reversible upon cessation of DEHP-exposure. Due to the dose-related serious effects on the testicles, a NOAEL of 500-ppm corresponding to 28.9 mg/kg/day can be derived for testicular effects.

In a 102 week-study, adult male Sprague-Dawley rats were exposed to DEHP via incorporation in the diet at dose levels of 0, 0.02, 0.2 or 2% (0, 7, 70 or 700 mg/kg bw/day) (Ganning et al., 1987, 1990). In all dose-groups, DEHP exerted a pronounced effect on the function of the testes after prolonged treatment, consisting of inhibition of spermatogenesis and general tubular atrophy. The LOAEL was 0.02% in the diet (7 mg/kg bw/day), the lowest dose administered. The study was, however, designed to study the effects of phthalates on the liver and the information on testicular effects is very limited. Therefore, the study results cannot be included in the risk assessment.

In the following oncogenicity study, Fischer 344 rats (50 animals/sex/group; initial body weight just above 200 mg for males and around 150 mg for females) were given 0, 6,000 or 12,000-ppm (0, 322 or 674 mg/kg/day for males) DEHP (> 99.5% pure) in the diet for 103 weeks (NTP, 1982). Mean daily doses of DEHP were 322 and 674 mg/kg body weight per day for low- and high-dose male rats, respectively. The survival rate was unaffected. At the end of the study, mean body weights of dosed male rats and high-dose female rats were marginally to moderately lower than those of the corresponding controls. Food consumption was slightly reduced in rats of either sex. Interstitial-cell tumours of the testis were observed in a statistically significant negative relation to dose. There was a statistically significant increase in bilateral tubular degeneration of the seminiferous tubules and atrophy in the testes. The incidences were 1/49 (2.0%) in the control, 2/44 (5%) in the low-dose, and 43/48 (90%) in the high-dose group. Histologically, the seminiferous tubules were devoid of germinal epithelium and spermatocytes (tissues had been preserved in 10% buffered formalin and embedded in paraffin). Only Sertoli cells were seen on tubular basement membranes. Interstitial cells were somewhat prominent. In high-dose males, the incidence of hypertrophy of the anterior pituitary was significantly increased (45% compared with 2% of controls). A LOAEL of 322 mg/kg/day DEHP is derived from this study. No other toxic lesions were associated with compound administration.

Mouse

Swiss (CD-1) mice (20 animals of each sex) were dosed with 0.30% DEHP (150 mg/kg bw/day; purity not specified) in the diet (Morrissey et al., 1988). Continuous breeding studies were used to evaluate reproductive performance over a 98-day cohabitant period. Mice were separated by sex during the first 7 days of DEHP treatment. After detection of an adverse effect of DEHP treatment, a 1-week crossover mating trial was carried out between previously treated males and control females. Reproductive ability was assessed at 10 weeks of age in a single breeding trial over a 7-day period. Necropsy for the treated males included organ weights, percentage motile sperm, sperm concentration, and percentage abnormal sperm. In DEHP treated mice, there was a reduction in epididymal and testicular weights, sperm motility, and sperm concentration and an increased number of abnormal sperm cells. No further details are given.

In a study performed according to EPA guidelines and the principles of GLP, B6C3F1 mice (70-85 of each sex/dose group, about 6 weeks of age at the initiation of the study) were administered DEHP daily in the diet at concentrations of 0, 100, 500, 1,500 and 6,000-ppm for 104 weeks (0, 19.2, 98.5, 292.2 or 1,266 mg/kg/day) (David et al., 2000c; Moore, 1997). One additional group (55 males) were administered 6,000-ppm DEHP for 78 weeks, followed by a 26-week recovery period. At 1,500-ppm, there was a significant decrease in testicular weight, with an increased incidence and severity of bilateral hypospermia and an associated increased incidence of immature/abnormal sperm forms and hypospermia in the epididymis. At the highest dose level, there was a statistically significant decrease in survival, treatment-related clinical signs and a significantly reduced body weight gain. In the recovery group, the effects of DEHP in the kidney and testis were at least partially reversible following cessation of exposure. The NOAEL for testicular effects in this study is 500-ppm corresponding to 98.5 mg/kg.

In an oncogenicity study performed according to GLP principles, B6C3F1 mice (50 animals/sex/group) were given 0, 3,000 or 6,000-ppm of DEHP (> 99.5% pure) in the diet for 103 weeks (NTP, 1982). Mean daily ingestion of DEHP was calculated to 672 and 1,325 mg/kg bw for low- and high-dose males, respectively. The survival rate was unaffected. In 14% of the high-dose males bilateral seminiferous tubular degeneration and testicular atrophy were observed. This lesion was also found in one control male mouse and in two low-dose males. A NOAEL of 672 mg/kg/day DEHP is derived from this study.

Primate

Male marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo) (Tomonari 2006, Kurata 2003c). No treatment-related changes were observed in male organ weights, and no microscopic changes were found in male gonads or secondary sex organs. Sperm head counts, zinc levels, glutathione levels, and testicular enzyme activities were comparable between groups. Electron microscopic examination revealed no treatment-related abnormalities in Leydig, Sertoli, or spermatogenic cells. Histochemical examination of the testis after 3β-hydroxysteroid dehydrogenase (3β-HSD) staining did not reveal any alterations in steroid synthesis in the Leydig cells. Thus, although marmoset monkeys were treated with 2500 mg/kg DEHP, throughout the pre- and periadolescent period, no histological changes were noted in the testes. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6β-hydroxylase, and lauric acid ω-1ω-hydroxylase activities) were increased in males of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

In a 13-week oral study performed according to GLP principles, mature male marmosets (4/sex/group, from 13 or 14 months of age) were given daily doses of 0 (corn oil), 100, 500 or 2,500 mg/kg DEHP (purity not specified) in corn oil (Kurata et al., 1998). The body weight gain was significantly suppressed in males administered 2,500 mg/kg. Dose-related decreases in spleen weight were observed in all dosed males. Other organ weights, including liver, testes, and pancreas, were not different from the control weights. In the DEHP dosed groups there was a significant rise in the total and free cholesterol and phospholipid levels in administration week 4. In week 13, only the total cholesterol value in the 500 mg/kg males was different from the control value. A clear rise in blood testosterone and oestradiol concentrations in all groups, including controls, were concluded to be hormonal changes accompanying sexual maturity occurring at the age of about 12 months. CLEA Japan, Inc, the animal supplier, have communicated that male marmosets are sexually mature at around 7 months and mate at around 12 months (CLEA, 2000).

Inhalation

Rat

Kurahashi (2005) researched the effects of inhalation of DEHP on testes of pre-pubertal rats. Our results showed that inhalation of DEHP by 4-wk-old male Wistar rats at doses of 5 or 25 mg/m3, 6 h per day, for 4 and 8 wk significantly increased the concentration of plasma testosterone and weight of seminal vesicles. However, the concentration of luteinizing hormones (LH), follicular stimulating hormone (FSH) and the expression of mRNAs of androgene biosynthesis enzyme, cytochrome P450 cholesterol side-chain-cleavage enzyme (P450scc), 3beta-hydroxysteroid deshydrogenase (3beta-HSD), cytochrome P450 17aplha-hydroxylase/17, 20 lyase (CYP17) and aromatase (CYP19) did not change. Rats with precocious testes did not increase in any of the DEHP groups. The estimated effective dose in this study was less than those reported in previous studies using oral dosing. This study showed that inhaled DEHP increased plasma testosterone concentrations in pre-pubertal rats and suggested that their effects were more sensitive to inhalation than oral dosing.

Testicular toxicity in neonatal animals

Oral

Rat

The effects of DEHP, MEHP and 2-ethylhexanol (2-EH) were determined on gonocytes and Sertoli cell morphology, Sertoli cell proliferation, and expression of cell cycle markers in neonatal rats (three-day old, CD Sprague-Dawley) (Li et al, 2000). A single bolus dose of DEHP (20, 100, 200 and 500 mg/kg) was given in corn oil to five pups per group. Diethyl phthalate (DEP: 500 mg/kg) served as the non-toxic control. MEHP (393 mg/kg), 2-EH (167 mg/kg), or vehicle was administered by gavage to 4 pups per group. The doses of MEHP and 2-EH were molar equivalent with 500 mg/kg DEHP. In this dose-response study, all pups were killed 24 hours after dosing. A time-course study was conducted following a single dose DEHP (200 mg/kg), where the pups were killed after 6, 9, 12, 24 or 48 hours. Biochemical analyses was performed for serum FSH levels, Sertoli cell proliferation (as BrdU labelling; BrdU administered 3 hours before euthanasia), cell cycle regulators cyclin D1, D2, D3, p27kip1 proteins and cyclin D2 mRNA in the testes. Morphological examination revealed a dose-dependent presence of abnormally large, multi-nucleated germ cells (gonocytes) by 24 hours post-treatment with DEHP (100-500 mg/kg). With 200 mg/kg DEHP these effects were first determined 12 h after treatment, and persisted for 48 hours. Effects on Sertoli cell morphology were not detailed in the report. MEHP (single dose group) induced effects on gonocytes similar to DEHP. BrdU-labelled Sertoli cells were dose-dependently decreased from 100-500 mg/kg DEHP. No marked difference in BrdU-labelled Sertoli cells was marked with 20 mg/kg DEHP, DEP and vehicle controls. Serum levels of FSH were not affected by DEHP treatment (200 and 500 mg/kg). MEHP also caused a significant decrease in BrdU-labelled Sertoli cells. D2 mRNA was specifically down-regulated by DEHP in a dose-dependent manner (200 and 500 mg/kg only doses reported), and this decrease was manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein with 200 mg/kg DEHP (only dose reported). The effects of MEHP and 2-EH were not determined. 2-EH was without effect on testicular cell morphology, or Sertoli cell proliferation. A NOAEL for young pups of 20 mg/kg is derived for effects on altered gonocyte morphology and decreased Sertoli cell proliferation by a single oral dose of DEHP.

Di-(2-ethylhexyl) phthalate (DEHP) was administered to 3- to 5-day-old male Sprague-Dawley rats by daily oral gavage of 300 or 600 mg/kg/day for 21 days (Cammack et al., 2003). Histopathological evaluation and organ weight measurements were performed on some animals after 21 days of dosing (primary group) and later on the recovery group animals that were held without further treatment until sexual maturity at approximately 90 days of age. Testicular changes, consisting of a partial depletion of the germinal epithelium and/or decrease in diameter of seminiferous tubules, were present in all animals of the 300- and 600-mg/kg/day groups after the 21-day dosing period. Testes weight decreased and liver weight increased in these animals. In the recovery animals, a residual DEHP-induced decrease in seminiferous tubule diameter was present in the testis of several animals dosed orally at 300 and 600 mg/kg/day. There was no germinal cell depletion or Sertoli cell alteration observed in any dose group at any time. Notably, no effects on sperm count, sperm morphology, or sperm motility were observed at 90 days of age in any of the groups.

Other routes

Di-(2-ethylhexyl) phthalate (DEHP) was administered to 3- to 5-day-old male Sprague-Dawley rats by daily intravenous injections of 60, 300, or 600 mg/kg/day or by daily oral gavage of 300 or 600 mg/kg/day for 21 days (Cammack 2003). Histopathological evaluation and organ weight measurements were performed on some animals after 21 days of dosing (primary group) and later on the recovery group animals that were held without further treatment until sexual maturity at approximately 90 days of age. No effects of any type were observed in animals treated intravenously with 60 mg/kg/day. Testicular changes, consisting of a partial depletion of the germinal epithelium and/or decrease in diameter of seminiferous tubules, were present in all animals of the 300- and 600-mg/kg/day groups after the 21-day dosing period. Testes weight decreased and liver weight increased in these animals. Testes changes were dose-related and generally more severe among animals dosed orally versus intravenously. In the recovery animals, a residual DEHP-induced decrease in seminiferous tubule diameter was present in the testis of several animals dosed orally at 300 and 600 mg/kg/day, but not in animals dosed intravenously. There was no germinal cell depletion or Sertoli cell alteration observed in any dose group at any time. Notably, no effects on sperm count, sperm morphology, or sperm motility were observed at 90 days of age in any of the groups.

Effects on the development of the male reproductive tract

Rat

In theGe study (2007),the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 +/- 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 +/- 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 +/- 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 +/- 0.37 ng/mL) and seminal vesicle weights (0.33 +/- 0.02 g) compared with control serum T (1.98 +/- 0.20 ng/mL) and seminal vesicle weight (0.26 +/- 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 +/- 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 mM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels.

The Noriega study (2009) was designed to determine if the dose response to DEHP was non-monotonic, as hypothesized. Pubertal administration of DEHP delayed the onset of puberty and reduced androgen-dependent tissue weights in both Long-Evans (LE) and Sprague-Dawley (SD) male rats 300 and 900 mg DEHP/kg/day. These effects were generally of greater magnitude in LE than SD rats. By contrast, alterations in testis histopathology (300 and 900 mg/kg/day) were more severe in SD than in LE rats. Taken together, these results suggest that DEHP may be acting on the pubertal male rat testis via two modes of action; one via the Leydig cells and the other via the Sertoli cells. Treatment with DEHP generally reduced serum testosterone and increased serum luteinizing hormone (LH) levels, demonstrating that the reduction in testosterone was due to the effect of DEHP on the testis and not via an inhibition of LH from hypothalamic-pituitary axis. Testosterone production ex vivo (with and without human chorionic gonadotropin stimulation) was consistently reduced in males at the time of puberty and shortly thereafter. DEHP treatment did not accelerate the age at puberty or enhance testosterone levels at 10 or 100 mg/kg/day in either LE or SD rats, as some have hypothesized. Taken together, these results do not provide any evidence of a non-monotonic dose response to DEHP during puberty.

Non-human primates

McKinnell et al. (2009) exposed marmosets in utero (weeks 7 -15 of gestation) or for 14 days after birth to 500 mg/kg bw MBP. Male offspring exposed in utero were investigated at birth (days 1 -5) or in adulthood (weeks 18 -21) for testicular development and function. Additionally 5 pairs of co-twins (n=5 exposed and n=5 controls) exposed for 14 days after birth were investigated immediately after end of exposure for testicular development and function. No effects on testis development/function or testicular dysgenesis were measurable. Some effects on germ cell development were found, but these effects were inconsistent and of uncertain significance.

FEMALE REPRODUCTIVE TRACT TOXICITY

Ovarian toxicity

Rat

In a study comparable to a guideline study, regularly cycling Sprague-Dawley rats (6-9 in each study group) were dosed daily with DEHP (> 99% pure) at 2,000 mg/kg bw in corn oil by gavage for 1-12 days (Davis et al., 1994). Ovarian morphology and serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestradiol, and progesterone levels were analysed. DEHP treatment resulted in prolonged oestrus cycles compared to a control group. DEHP also suppressed or delayed ovulation by the first prooestrus/oestrous after the metoestrus-initiated dosing. Histopathological evaluation of the ovaries showed that 7 out of 10 DEHP-exposed rats had not ovulated by vaginal oestrous in contrast to 13 out of 13 control rats which ovulated by vaginal oestrous. Pre-ovulatory follicles were quantitatively smaller in DEHP-exposed rats than in controls due to smaller granulosa cells. Suppressed serum oestradiol levels caused a secondary increase in FSH levels and did not stimulate the LH surge necessary for ovulation. According to the authors, these results suggest that DEHP-treatment causes hypo-oestrogenic anovulatory cycles and polycystic ovaries in adult female rats.

The effects of in vivo administered DEHP (1,500 mg/kg bw orally for 10 consecutive days) on in vitro ovarian steroid profiles in immature and cycling female rats have been studied byLaskey and Berman (1993).Groups of 20 and 21 mature female Sprague-Dawley rats were administered 0 or 1,500 mg DEHP/kg bw in corn oil for 10 days. On day 5 before dosing and daily during dosing, the stage of the oestrus cycle was determined for all animals. The day after the final dosing the animals were killed and ovaries, adrenals and serum were used to determine rates of steroid production. No ovary weight differences were noted in the control cycling animals or between the control and DEHP-treated rats. The alterations caused by DEHP in the in vitro ovarian steroidogenic profile were most apparent in rats during dioestrus and oestrous. In the DEHP dosed animals the incidence of animals in prooestrus was clearly reduced from day seven to day ten of dosing. In cultures of adrenals and serum no significant differences in rates of steroid production were observed. In the ovary cultures, di-oestrus rats dosed with DEHP had significantly higher testosterone and oestradiol production, and in rats in oestrus the oestradiol production was significantly lower in DEHP-dosed females. There were no significant differences in the steroid production of rats in prooestrus (only two dosed animals). The authors conclude that DEHP treatment alters the oestrus cycle and causes concentration changes of testosterone and oestradiol in rats in dioestrus.

Whole ovary cultures from cycling Sprague-Dawley rats fed 1,500 mg/kg bw/day of DEHP (purity not specified; in corn oil) by gavage for 10 days were used to evaluate if DEHP altered steroidogenic profiles (Berman and Laskey, 1993). Ovaries were removed and cultured for one hour. Steroidogenic profiles of progesterone, testosterone, and oestradiol release into the medium were measured using radioimmunoassay techniques. Dioestrous ovaries produced more oestradiol after DEHP administration and oestrus ovaries significantly less oestradiol; proestrous ovary production was not significantly changed. Testosterone production was significantly increased only in dioestrous. DEHP had no significant impact on progesterone production or serum levels of progesterone and oestradiol in treated rats.

Monkey

Female marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo) (Tomonari 2006, Kurata 2003). Increased ovarian and uterine weights and elevated blood estradiol level were observed in higher dosage groups, 500 and 2500 mg/kg. These increased weights were associated with the presence of large corpus luteum, a common finding in older female marmosets. Although an effect on the ovary cannot be completely ruled out, no abnormal histological changes were observed in the ovaries or uteri in comparison to controls. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6β-hydroxylase, and lauric acid ω-1ω-hydroxylase activities) were increased in females of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

In vitro

The female reproductive toxicity of di-(2-ethylhexyl) phthalate and its active metabolite mono-(2-ethylhexyl) phthalate (MEHP) is attributed to suppression of ovarian granulosa cell estradiol production. In these studies, several structurally related phthalates (0-200 microM) and Wy-14,643 (0-100 microM) were compared to MEHP for their effects on granulosa cell estradiol production and transcript levels of cytochrome P450 enzyme CYP 19, also known as aromatase (P450arom), the rate-limiting enzyme in the conversion of androgens to estrogens (Lovekamp and Davis, 2001). Granulosa cells were obtained from 28-day-old Fisher 344 rats and were cultured for 48 h. Test chemical or DMSO was added at the time of culture, along with testosterone as a substrate for aromatase. 17beta-Estradiol production was measured by standard radioimmunoassay, mRNA was measured by fluorescent RT-PCR, and protein was measured by Western blot analysis. MEHP was unique among the phthalates in its ability to decrease estradiol production, while Wy-14,643 had effects similar to MEHP at 100 microM. MEHP and Wy-14,643 also significantly decreased aromatase mRNA levels. The decrease in mRNA was concentration dependent and was paralleled by a decrease in aromatase protein. MEHP did not alter levels of CYP 11A1, the cholesterol side-chain cleavage enzyme (P450scc). Treatment with a cAMP analogue increased expression of P450scc in the presence of MEHP (100 to 200 microM) while the decrease in aromatase remained. Thus, these studies suggest that MEHP is distinct from several structurally related phthalates but similar to the peroxisome proliferator Wy-14,643 in its action on granulosa cell estradiol production. Moreover, the suppression of estradiol by MEHP is likely mediated through its action on aromatase transcript levels independent of cAMP-stimulated regulation.

Effects on the development of the female reproductive tract

Rat

Ma (2006)evaluated the effects of inhaled di(2-ethylhexyl) phthalate (DEHP) on the onset of puberty and on post-pubertal reproductive functions in pre-pubertal female rats. DEHP was administered by inhalation at doses of 0, 5, and 25 mg/m3 to groups of female rats for 6 h/day, 5 contiguous days/week from postnatal days (PNDs) 22 to 41 and to PND 84. The onset of puberty was determined by daily examination for vaginal opening (VO) and first estrous cycle. Reproductive function was evaluated by observing estrous cyclicity from PNDs 49 to 84. Upon completion of exposure, the rats were sacrificed at PND 42 and PNDs 85–88 during the diestrous stage. DEHP exposure advanced the age of VO and first estrous cycle, and serum cholesterol, luteinizing hormone, and estradiol levels were significantly elevated in the 25-mg/m3 DEHP group. Irregular estrous cycles were observed more frequently in DEHP exposed rats, and serum cholesterol decreased in DEHP-exposed rats in adulthood; RT-PCR showed that the expression of aromatase mRNA, encoding a rate-limiting enzyme that catalyzes the conversion of testosterone to estradiol, was elevated in the 25-mg/m3 DEHP group. These data suggest that inhaled DEHP may advance the onset of puberty and alter post-pubertal reproductive functions.

MECHANISTIC STUDIES

Role of zinc, testosterone and vitamin B12

Seven immature male Crj: Wistar rats (30 days, 75-90 g) per group were orally dosed with DEHP (2,000 mg/kg bw/day) for 0, 1, 3, 6 and 10 days (Oishi, 1986). Organ weights were significant decreased: testes by day 3; Seminal vesicle by day10; ventral prostrate by day 3. Testicular morphology was normal on day 1 but changes occurred for longer exposures. By day 3: number of spermatocytes and spermatids were decreased in some seminiferous tubules; day 6: active spermatogenesis was rarely found, seminiferous tubules contained necrotic debris and variable numbers of multinucleated gaint cells. By day 10, all seminiferous tubules had shrunken. Zinc concentration in the testes significantly decreased by by day 6 and 10, and by day 10 in the ventral prostrate. The zinc content was not affected in the seminal vesicle and serum. Specific activities of some zinc containing enzymes such as carbonic anhydrase, alcohol dehydrogenase and aldolase significantly decreased by day 10. The author concludes that several testicular cell-specific enzymes appear to be useful biochemical markers of testicular injury. However, these changes occurred after or simultaneously with massive histological or morphological changes rather than prior to such changes.

In a study comparable to a guideline study, groups of 48 male F344 rats were maintained on synthetic diets containing 2-ppm (low), 20-ppm (normal) or 200-ppm (high) zinc (Agarwal et al., 1986b). After one week of acclimation to the various diets, groups of 12 rats from each dietary regimen were gavaged for 13 consecutive days with 0, 330, 1,000 or 3,000 mg/kg bw of DEHP (> 99% pure). Organ weights of testis, epididymis, prostate, and seminal vesicles were not affected by DEHP in rats at normal- and high zinc diet, but were significantly and dose-dependently reduced in rats on low-zinc diet. The combination of low-zinc diet plus 1,000 or 3,000 mg/kg bw of DEHP caused dose-dependent tubular degeneration and atrophy. Seven young male Crj: Wistar rats (115 g) per group were orally dosed with DEHP (2,000 mg/kg bw/day) for 10 days (Oishi, 1986). Zinc sulphate (9 mg/kg) was co-administered intraperitoneally. DEHP caused a significant reduction in testes weight and testicular zinc concentration. Co-administration of zinc did not significantly prevent the DEHP-induced effects.

The involvement of testosterone in the testicular atrophy caused by DEHP was examined by co-administration of testosterone (1 mg/kg bw) subcutaneously along with 2,000 mg/kg bw of DEHP (purity not specified) in groundnut oil to adult male Wistar rats (150-200 g) for 15 days (Parmar et al., 1987). Administration of DEHP was found to significantly reduce relative and absolute testes weights and the sperm count (approximately 75%), and also significantly increase the activity of GGT, LDH, and β-glucuronidase and to decrease the activity of SDH and acid phosphatase. Co-administration of testosterone seemed to normalise the testes weights, sperm count and the activity of testicular enzymes. The role of testosterone in testis toxicity of DEHP is not fully elucidated. Several reports refer to increased or decreased testosterone levels in plasma and testicular tissue.

Curto and Thomas (1982)examined changes in testes and sex accessory weight as well as gonadal zinc in sexually mature rats and mice injected with various doses of DEHP or MEHP (purities not specified). Intraperitoneal and subcutaneous routes of administration were used to avoid hydrolyzation in the gastrointestinal tract and to exclude phthalate-induced reduction in the gastrointestinal absorption of zinc. Male Swiss-Webster mice (number not stated) received one of the following dose regimens: a) daily sc injections of 1, 5 or 10 mg/kg MEHP for 5 days; b) daily sc injections of 5, 10 or 20 mg/kg MEHP for 10 days; c) daily ip injections of 50 or 100 mg/kg MEHP or DEHP for 5 days; or d) alternate daily ip injections of 50 or 100 mg/kg MEHP or DEHP for 20 days (10 injections). Male Sprague-Dawley rats (number not specified) received daily ip injections of 50 or 100 mg/kg MEHP or DEHP for 20 days (10 injections). In mice, no significant alterations in testicular weight, seminal vesicle or anterior prostate weight or zinc levels occurred. Rats revealed significant reductions in both gonadal and prostate gland zinc. Rats injected with MEHP (50 mg/kg) showed a 37% decrease in prostate zinc; DEHP (100 mg/kg) caused a 33% decrease in prostatic zinc and a significant loss of testicular zinc (31%). The results indicated that the male rat is more sensitive to DEHP- or MEHP-induced effects on male gonads than the male mouse. It was also shown that sc or ip injected DEHP or MEHP caused gonadal zinc depletion, thus eliminating altered intestinal absorption as the cause for species differences.

Two strains of mice (Jcl: ICR and Crj: CD-1, four weeks old), were fed diets containing 0, 0.1, 0.2, 0.4 or 0.8% (approximately 300, 600, 1,200 mg/kg bw/day) of DEHP (purity not specified) for two weeks (Oishi, 1993). In ICR-mice, testicular weights were unchanged by DEHP treatment at all concentrations when compared to controls. In CD-1 mice, testicular weights were significantly reduced from a dose level of 0.2%. The testicular zinc content was statistically significantly reduced in both strains at dose levels of 0.4 and 0.8%. Testicular activities of lactate dehydrogenase isoenzymes (LDH-X) were significantly reduced in CD-1 mice from a dose level of 0.2%, while a significant reduction of testicular LDH-X activity in ICR mice was observed only at a dose level of 0.8%. The primary metabolite, MEHP, was significantly increased in blood samples of CD-1 mice at 0.8% when compared to ICR mice suggesting that toxicokinetics differences may explain some of the shown strain differences in susceptibility to DEHP testicular toxicity.

A study of the influence of the vitamin B12 derivative adenosylcobalamin on testicular toxicity of DEHP was performed in young male Crj: Wistar rats (30 days; 86-100 g) (Oishi, 1994). Groups of 8 animals each were treated for 7 days. DEHP (0 and 2,000 mg/kg bw/day) was given orally and co-administration of adenosylcobalamin or methylcobalamin, both 0.5 mg/kg, was administered intraperitoneally. DEHP significantly reduced body, testis and prostate weights, inhibited active spermatogenesis, reduced the activity of testicular specific lactate dehydrogenase, and decreased the levels of testicular zinc, magnesium and potassium. Co-administration of adenosylcobalamin, but not methylcobalamin, prevented the DEHP-induced effects.

Apoptosis induction

Expression of apoptosis-related proteins FasL, Fas and Caspase-3, as well as DNA fragmentation were examined in mouse testis 12 h after exposure to 4–0.004 mg/g bw di(2-ethylhexyl) phthalate (DEHP) (Ichimura 2003). Immunocytochemical examination of the highest dose (4 mg/g DEHP) mouse revealed a distribution of FasL in Sertoli cell and Fas in nearby spermatocyte, and Fas and Caspase-3 in the same spermatocyte. Fas-positive spermatocytes had a DNA-fragmented nucleus detectable by terminal deoxynucleotidyl transferase-mediated fruorescein-dUTP nick end labeling (TUNEL) method. After exposure to 4, 0.4, 0.04 or 0.004 mg/g DEHP, the maximum number of nuclei with fragmented DNA per 0.5µm testis section was 22, 7, 5 and 3, respectively. In unexposed control the maximum number was 3. To further estimate total amount of the fragmented DNA in testis of the exposed mouse, the extracted DNA fragments were analyzed by agarose gel electrophoresis. The amount of fragments in the first three steps of the DNA ladder was estimated by a photo-densitometry. In the highest dose mouse (4 mg/g DEHP), the fragmented DNA was 2.2 times as much in the control. In lower dose mouse (0.4, 0.04 or 0.004 mg/g DEHP), it was 1.1 times as much in the control. Taken together, these observations suggest that a single oral exposure to DEHP as low as 0.04 mg/g might be effective to testicular DNA fragmentation and apoptosis.

Age-dependency

Dostal et al. (1988)studied the age-dependency of testicular effects (study comparable to a guideline study) in rats of different age. Oral doses of 0, 10, 100, 1,000 or 2,000 mg/kg bw of DEHP (> 99% pure) were given daily by gavage in corn oil for 5 days to Sprague-Dawley rats (7-10 animals per group) at 1, 2, 3, 6 and 12 weeks of age. Absolute and relative testis weights were significantly reduced at doses of 1,000 mg/kg bw/day in 1, 2, 3 and 6- week-old but not in 12-week-old rats compared to controls of the same age. Doses of 2,000 mg/kg bw/day were fatal to suckling rats and caused decreased relative testis weight but no lethality in 6- and 12-week-old rats. The number of Sertoli cell nuclei per tubule was reduced by 35% at 1,000 mg/kg in neonatal rats; two- and three-week old rats showed loss of spermatocytes but not of Sertoli cells. At 1,000 and 2,000 mg/kg also loss of spermatids and spermatocytes was shown in 6- and 12-week old rats.

Metabolites of DEHP

The formation of the monoester is an important step in the intestinal absorption of the orally ingested phthalates. In pubertal and adult animals, the testicular toxicity of DEHP appears to be mediated by the monoester MEHP. Studies mainly focusing on this or other metabolites are therefore compiled separately.

To establish the compound or compounds responsible for the testicular damage after oral administration of DEHP,Sjöberg et al. (1986)administered DEHP and five of its major metabolites (MEHP, 2-ethylhexanol and three identified metabolites (V, VI, or IX) of MEHP) for five days. Groups of 6 male Sprague-Dawley rats (35 days old at the start of the experiment) were given 2.7 mmol kg bw of DEHP (1,055 mg /kg bw) or one of the metabolites. A counting of degenerated cells per tubular cross section was carried out. No testicular damage was observed following oral doses of DEHP or 2-EH. The number of degenerated spermatocytes and spermatids was increased in animals which received MEHP; no such effects were seen in animals given the MEHP-derived metabolites.

Dalgaard et al.(2001)studied the effects of MEHP on 28-day old male rats, by looking at testicular morphology and apoptosis, and expression of some cellular markers (vimentin filaments, the androgen receptor, and a gene coding a Sertoli cell secretory product) 3, 6 or 12 hours (n=12) after a single oral dose of 400 mg/kg MEHP. At 3-12 hours, vimentin filaments in Sertoli cells had collapsed, and the expression of the apoptosis gene Caspase-3 was increased. However, there were no other indications of apoptosis as measured by DNA ladder analyses or tunel staining. The expression of TRPM-2 (coding a Sertoli cell secretory product) was transiently increased at 3 hours. At 3 hours there were no histological signs of toxicity, but at 6 and 12 hours the tubuli were disorganised and germ cells detached and sloughed into the lumen of the seminiferous tubules. The results support the Sertoli cells being early targets for MEHP toxicity.

The testicular toxicity of DEHP (> 98% pure) was studied in male Wistar rats (26 days old, 6 animals per group) after a single oral dose of 2,800 mg/kg bw (Teirlynck et al., 1988). In the same experiment, MEHP was given in doses of 400 or 800 mg/kg bw. The doses were selected in accordance to previous data showing that oral administration of 2,800 mg/kg bw of DEHP and 400 mg/kg bw of MEHP leads to similar MEHP plasma levels. Seven days after dosing the rats were killed and the testicular zinc concentration was measured. The severity of the histopathological lesions was graded on the basis of the percentage of seminiferous tubules affected. The diameter of the seminiferous tubules was also measured. The rats showed testicular atrophy 7 days after dosing, as indicated by a significant reduction in relative testicular weight. Histological examination revealed a “dose-dependent” increase in the number of atrophic seminiferous tubules with decreased diameters of the seminiferous tubules and loss of spermatids and spermatocytes. The study suggests that MEHP is more toxic to the testes than DEHP. A significant reduction of the testicular zinc concentration was observed in DEHP treated rats and in rats given MEHP doses of 800 mg/kg bw, but not at doses of 400 mg/kg bw. The concentration of the follicle-stimulating hormone (FSH) in serum was determined but no treatment-related alteration was observed. The authors suggest that the toxic effects of MEHP are not secondary to inhibition of pituitary gonadotropin secretion and that the absence of an elevation of FSH suggests that the function of the Sertoli cells is preserved.

Prepubertal male Fischer rats (28-day-old; number not stated) were given a single 2,000-mg/kg dose of MEHP (95% pure) in corn oil by gavage at a volume equal to 4 ml/kg (control rats received a similar volume of corn oil) to study the effect on germ cell apoptosis in testes (Richburg and Boekelheide, 1996). Preliminary experiments had also suggested that phthalates may cause alterations in the rat Sertoli cell cytoskeleton, particularly the intermediate filament vimentin. The rats were killed at 3, 6 and 12 hours after treatment. From each rat, one testis was rapidly frozen in liquid nitrogen and the other was cut in halves for immersion fixation in Bouin´s fixative and in neutral buffered formalin. Cryosections were stained with a monoclonal antibody to bovine vimentin. In situ Tunel staining was used to stain for DNA. The number of apoptotic germ cells was counted in 100 randomly selected seminiferous tubules of testis cross-sections from each of four different rats. MEHP induced collapse of Sertoli cell vimentin filament 3 hours after MEHP-administration. Six and 12 hours after MEHP exposure, intense vimentin staining surrounding the nucleus was seen, suggesting that vimentin filament had collapsed toward the Sertoli cell nucleus. The incidence of apoptotic events observed in 100 seminiferous tubule cross sections of testes from each of four rats was counted and tabulated into categories. In control testes, 44.5% of the seminiferous tubule cross-sections did not contain any apoptotic cells. However, 3 hours after MEHP treatment, the number of tubule cross sections with no incidence of apoptosis significantly increased to 63.3%. This shift was reflected by a significant decrease in the incidence of tubules containing 1-3 apoptotic cells per cross section at 3 hours. Cross sections of the seminiferous tubules from the 6- and 12-hours groups showed a dramatic increase in the number of apoptotic events as evident by the increased incidence of seminiferous tubules which contained high categories of apoptotic germ cells and a decrease in the incidence of seminiferous tubule cross sections that contained no apoptosis. The authors suggest that the MEHP-induced collapse in vimentin filaments may lead to alterations in germ cell apoptosis by a disruption in contact-mediated communication between the Sertoli cells and germ cells and that the normal physiological incidence of germ cell apoptosis decreased as early as 3 hours after exposure to MEHP.

Four phthalate diesters, DEHP, DPP (di-n-pentyl phthalate), DOP (di-n-octyl phthalate), and DEP (diethyl phthalate) were investigated in vivo for effects on Leydig cell structure and function (Jones et al., 1993). The study was performed due to earlier study results indicating that communication and control exists between Sertoli and Leydig cells which appear to be of a paracrine nature. The corresponding monoesters were investigated in vitro (MEHP, MPP, MOP, and MEP). The in vivo study was performed by giving 2,000 mg/kg bw by oral gavage on two consecutive days to 3 male Wistar rats (6-8 weeks old, 200-300 g bw) per phthalate. The rats were sacrificed 24 hours after the final dose. Testicular tissues were studied by light and electron microscopy after glutaraldehyde perfusion fixation, Taab embedding and toluidine blue staining (a highly reliable technique in preparing testis tissue for identifying testicular toxicity). The in vitro study was performed with primary cultures of Leydig cells incubated with 1,000-μM monoester for 2 hours. Phthalate esters exerted a direct effect on Leydig cell structure and function as determined by testosterone output with correlation of the in vitro and in vivo effects of MEHP and DEHP, respectively. The changes observed in vivo were present in all animals in each group. Leydig cells stained more densely than other cell types, generally displaying an elongate profile often with thin lamellar processes. In Leydig cell cytoplasmatic ultrastructure, several subtle but highly significant alterations were produced. DEHP administration also resulted in slight rarefaction or vacuolation of a few Sertoli cells in seminiferous tubules, while treatment with DOP or DEP produced no change in seminiferous tubular structure or Leydig cell morphology. Exposure to DPP produced the most severe changes in Sertoli cells but no changes in Leydig cells. In the in vitro study, MEHP and MPP produced marked effects on structure and function including decreased LH-stimulated secretion of testosterone from Leydig cells incubated with MEHP while MOP caused decreased secretion and MEP was without effect. The results show that DEHP may exert a direct effect on Leydig cell structure and function and that DEHP and MEHP produce similar changes both in vivo and in vitro both in Leydig cells and in Sertoli cells. The authors concluded that a malfunction of Leydig cells likely affects the physiology of adjacent Sertoli cells. The authors also concluded that different phthalates may exert changes that are unique to one or common to both cell types.

The effects of DEHP, MEHP and 2-ethylhexanol (2-EH) were determined on gonocytes and Sertoli cell morphology, Sertoli cell proliferation, and expression of cell cycle markers in neonatal rats (three-day old, CD Sprague-Dawley) (Li, 2000). A single bolus dose of DEHP (20, 100, 200 and 500 mg/kg) was given in corn oil to five pups per group. Diethyl phthalate (DEP: 500 mg/kg) served as the non-toxic control. MEHP (393 mg/kg), 2-EH (167 mg/kg), or vehicle was administered by gavage to 4 pups per group. The doses of MEHP and 2-EH were molar equivalent with 500 mg/kg DEHP. In this dose-response study, all pups were killed 24 hours after dosing. A time-course study was conducted following a single dose DEHP (200 mg/kg), where the pups were killed after 6, 9, 12, 24 or 48 hours. Biochemical analyses was performed for serum FSH levels, Sertoli cell proliferation (as BrdU labelling; BrdU administered 3 hours before euthanasia), cell cycle regulators cyclin D1, D2, D3, p27kip1 proteins and cyclin D2 mRNA in the testes. Morphological examination revealed a dose-dependent presence of abnormally large, multi-nucleated germ cells (gonocytes) by 24 hours post-treatment with DEHP (100-500 mg/kg). With 200 mg/kg DEHP these effects were first determined 12 h after treatment, and persisted for 48 hours. Effects on Sertoli cell morphology were not detailed in the report. MEHP (single dose group) induced effects on gonocytes similar to DEHP. BrdU-labelled Sertoli cells were dose-dependently decreased from 100-500 mg/kg DEHP. No marked difference in BrdU-labelled Sertoli cells was marked with 20 mg/kg DEHP, DEP and vehicle controls. Serum levels of FSH were not affected by DEHP treatment (200 and 500 mg/kg). MEHP also caused a significant decrease in BrdU-labelled Sertoli cells. D2 mRNA was specifically down-regulated by DEHP in a dose-dependent manner (200 and 500 mg/kg only doses reported), and this decrease was manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein with 200 mg/kg DEHP (only dose reported). The effects of MEHP and 2-EH were not determined. 2-EH was without effect on testicular cell morphology, or Sertoli cell proliferation. A NOAEL for young pups of 20 mg/kg is derived for effects on altered gonocyte morphology and decreased Sertoli cell proliferation by a single oral dose of DEHP.

In vitro studies

Grasso et al. (1993)studied the effects of DEHP and MEHP on cultured rat Sertoli cells. The Sertoli cells were obtained from Fischer 344 rats (13-82 days of age) and cultured in the presence of MEHP at concentrations ranging from 0.001 to 100 μM. MEHP was found to specifically reduce the ability of FSH to stimulate cAMP accumulation in rat Sertoli cells. This inhibition by MEHP of FSH-stimulated cAMP accumulation had a lag period of 6 hours and reached a maximal inhibition of 40-60% after 24-hours. Preincubation of Sertoli cells for 24 hours with 100 μM DEHP had no effect on FSH binding. The authors concluded that the ability of certain phthalate esters to disrupt the FSH-linked signal transduction pathway in primary Sertoli cell cultures by a mechanism, located at the cell membrane, is likely to be a part of the mechanism responsible for their testicular toxicity.

Lehraki (2009)used organ culture of fetal testes at different stages of development to analyze the direct effects of phthalates on both steroidogenesis and gonocyte development and to determine if the effects of MEHP on these functions reported in the rat can be extended to other mammalian species. We defined specific periods of sensitivity of the fetal mouse testis to MEHP for these two functions and showed that the effects of phthalates on steroidogenesis vary with the developmental stage. Conversely, the strong deleterious effects of phthalates on germ cells were constantly present during the active phases of gonocyte development and thus share no relationship with the steroidogenic status. Moreover, all the effects of phthalates were unchanged in testes from mice deficient for estrogen (ERaKO or ERbKO) or androgen (Tfm) receptors. In conclusion, these results demonstrate that phthalates impair mouse fetal germ cell number similarly to other mammalian species, but are neither estrogenic nor antiandrogenic molecules because their effects do not involve, directly or indirectly, ER or AR.

Sjöberg et al.(1986)investigated the ability of DEHP, 2-EH, MEHP and metabolites V, VI, and IX to induce germ cell detachment from mixed primary cultures of Sertoli and germ cells. Only exposure to MEHP (10 μM for 24 hours or 1-200 μM for 48 hours) caused a significantly higher degree of germ cell detachment.

Grasso et al.(1993)studied the effects of DEHP and MEHP on cultured rat Sertoli cells. The Sertoli cells were obtained from Fischer 344 rats (13-82 days of age) and cultured in the presence of MEHP at concentrations ranging from 0.001 to 100 μM. MEHP was found to specifically reduce the ability of FSH to stimulate cAMP accumulation in rat Sertoli cells. This inhibition by MEHP of FSH-stimulated cAMP accumulation had a lag period of 6 hours and reached a maximal inhibition of 40-60% after 24-hours. Preincubation of Sertoli cells for 24 hours with 100 μM DEHP had no effect on FSH binding. The authors concluded that the ability of certain phthalate esters to disrupt the FSH-linked signal transduction pathway in primary Sertoli cell cultures by a mechanism, located at the cell membrane, is likely to be a part of the mechanism responsible for their testicular toxicity.

Cell cultures of Sertoli cells were also used to study lactate and pyruvate production after adding MEHP or DEHP (Moss et al., 1988). MEHP (0.1-200 μM) produced a concentration-dependent stimulation of lactate, but not pyruvate production over a 24-hour treatment period and an increase in the ratio of lactate/pyruvate concentration in the culture medium. DEHP had no such effects. The developing germ cells cannot utilise glucose to maintain ATP levels and are apparently dependent on a supply of lactate and pyruvate produced by Sertoli cells under control by FSH. The authors conclude that loss of germ cell in phthalate-induced testicular atrophy is not due to inhibition of energy substrate production by the Sertoli cells and that stimulation of lactate production may be a useful in vitro marker for phthalate esters that cause testicular injury.

Li et al.(1998)studied the effects of MEHP and DEHP on neonatal Sertoli cells and gonocytes (primitive spermatogonia) maintained in hormone- and serum-free coculture. They found that MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time and dose-dependent manner. The cocultures of Sertoli cells and gonocytes were prepared from testes of 2-day-old male rat pups. Final concentrations of 0.01, 0.1, or 1.0 μM MEHP (≥ 99% pure) was added to the cocultures. DEHP (≥ 99% pure) was used as a negative control. At a dose of 0.1 μM MEHP, gonocytes rounded up and started to detach from cocultured Sertoli cells after 24 hour of exposure. At 1.0 μM, MEHP caused a rapid detachment of gonocytes detectable after 12 hours of exposure. No morphological changes were found in cultures treated with vehicle alone or with DEHP, added at a 10-fold higher concentration than the maximal dose of MEHP. When cultures were labelled with BrdU (5-bromo-2’-deoxyuridine) few labelled cells could be found in the cultures treated with 1.0 μM MEHP compared to controls. No visually detectable increase in labelling could be observed in cultures simultaneously treated with FSH and 1.0 μM MEHP. Labelling indices in cultures treated with 0.1 or 1.0 μM MEHP were significantly lower than that in the vehicle-treated controls, reflecting decreases in Sertoli cell proliferation of 33.6 and 83.6%, respectively, over controls. The labelling indices of cultures treated with 10 μM MEHP was, however, significantly higher than that of the vehicle-treated controls. The study results show that MEHP directly targets the Sertoli cells and impairs their proliferation and that MEHP also may affect the interaction of gonocytes with Sertoli cells and/or target gonocytes directly. The findings also show that phthalate-induced changes in germ cell-Sertoli cell adhesion may occur during early postnatal development in rats.

In this study (Li 2003), organ cultures of fetal and neonatal rat testes were used to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of El 3 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest chat MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.

The in vitro effects of a 24 hours exposure of a Leydig cell line to MEHP were studied by means of electron microscopy (EM) and by measuring progesterone production and cell viability (Dees et al., 2001). At a concentration of 1 μM MEHP, the first signs of toxicity appeared, as indicated by morphological changes involving nuclei (heterochromatin, euchromatin and large nucleoli), mitochondria (generally condensed, but some were swollen or had an abnormal form and contained degenerated christae) and presence of moderate numbers of lipid droplets. At 10 μM MEHP, the cell shape was affected (large and round to oval), SER was lost, large vacuoles and numerous lipid droplets were present in the cytoplasm, and some mitochondria were seen in close apposition to the lipid droplets. At 100 μM MEHP, the effects were more severe and some apoptotic cells were seen. A more general cell death was observed at 1-3 mM, as determined both by structure and a cell viability assay (MTT). Progesterone production was reduced in the 1-10 μM range (by approximately 50%), returned to normal values at 100 μM, and ceased when cell started to die. The authors discuss a mechanism where the mitochondria are the first targets of the toxicity, they then fuse with lipid droplets and degrade. The study conduct seems proper, and the study pinpoints Leydig cells as potential sensitive targets for the DEHP-metabolite MEHP. However, the relevance for DEHP toxicity is not clear.

Lovekamp and Davis (2001)studied the in vitro effects of a 48 hours exposure period to 0-200 μM MEHP on primary rat granulosa cells. The authors find dose-dependent effects of MEHP on the levels of aromatase RNA, which is decreased as from exposure to 50 μM MEHP, and on the amount of aromatase protein and estradiol production (as from 100 μM MEHP). The relevance to DEHP toxicity is not clear.

Lague (2008)reports that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. Testosterone activated the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. The testosterone-responsive element to the proximal Insl3 promoter region has also been mapped. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Mono-(2-ethylhexyl) phthalate (MEHP) represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Ins13 transcription in Leydig cells and the mode of action of phthalates.

The effects of MEHP on granulosa cell function were studied in vitro (Treinen and Heindel, 1992). It was shown that MEHP inhibited FSH- but not forskolin-, isoproterenol-, or cholera toxin-stimulated granulosa cell cAMP accumulation in vitro. MEHP also inhibited FSH-stimulated progesterone production, a cAMP-dependent process. Similar to MEHP, the protein kinase C activator (TPA) has been shown to inhibit rat granulosa cell cAMP accumulation in a FSH specific manner, and decrease FSH-stimulated progesterone production.According to the authors, these data indicate that the inhibitory effects of MEHP on granulosa cell function are independent of phorbol ester-sensitive PKC activation.

Classification according to:

Regulation (EC) No 1272/2008 Annex VI Table 3.1: Reprotoxic 1B

Regulation (EC) No 1272/2008 Annex VI Table 3.2: Repr. Cat. 2; R60-61

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Bis(2-ethylhexyl) phthalate

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