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Toxicity to reproduction: other studies

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toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Testicular toxicity of DEHP, but not DEHA, is elevated under conditions of thioacetamide-induced liver damage
Jin Seok Kang, Keiichirou Morimura, Chitose Toda, Hideki Wanibuchi, Min Wei, Nakao Kojima, Shoji Fukushima
Bibliographic source:
Reproductive Toxicology 21 (2006) 253–259

Materials and methods

Principles of method if other than guideline:
As part of an investigation of possible enhancement by liver disease of testicular toxicity caused by phthalates, the effects of di(2-ethylhexyl)phthalate (DEHP) and di(2-ethylhexyl)adipate (DEHA) in a thioacetamide (TAA)-induced rat liver damage model were tested. Male, 6-week-old, F344 rats (n = 60) were divided into ten groups. Animals of groups 1–5 received TAA (200 mg/kg, intraperitoneal, three times per week) for 4 weeks, and groups 6–10 served as controls without TAA. After a 1 week interval, at week 5, powder diet containing DEHP or DEHA was provided to the animals of groups 1 and 6 (DEHP 25 000 ppm), groups 2 and 7 (DEHP 6000 ppm), groups 3 and 8 (DEHA 25 000 ppm) and groups 4 and 9 (DEHA 6000 ppm), while groups 5 and 10 received basal diet. All animals were sacrificed at week 9.
GLP compliance:
not specified
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
Molecular formula:
bis(2-ethylhexyl) adipate
Specific details on test material used for the study:
DEHP (>99.5%), purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
Sixty, 5-week-old, male F344 rats were obtained from Charles River Japan Inc. (Atsugi, Japan), and housed in rooms maintained on a 12 h light/dark cycle, at constant temperature and humidity. They were allowed free access to MF powder diets (Oriental Yeast Co., Tokyo, Japan) during the experiment. All procedures were approved by the Institutional Animal Care and Use Committee of Osaka City University Medical School.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
At 6 weeks of age, the rats were divided into ten groups (six rats per group). Animals of groups 1–5 were treated with TAA (200 mg/kg, intraperitoneal, three times per week) dissolved in phosphate buffered saline (PBS) for 4 weeks, and those of groups 6–10 received PBS alone without TAA. After a 1 week interval, at week 5, powder diet containing DEHP or DEHA was provided to the animals of groups 1 and 6 (DEHP 25 000 ppm), groups 2 and 7 (DEHP 6000 ppm), groups 3 and 8 (DEHA 25 000 ppm) and groups 4 and 9 (DEHA 6000 ppm), while groups 5 and 10 received basal diet.
Details on study design:
Body weights were measured every week and all animals were sacrificed at week 9 under ether anesthesia. At necropsy, blood samples were collected from the abdominal vein for measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (g-GTP). The organs of interest (liver,testes, epididymes, prostates, seminal vesicles) were measured, resected and processed.For the investigation of spermatogenesis, the testes and epididymes were removed. Sperm head counts were determined by methods as a previous report with some modification. In briefly, the right testes were homogenized (Politoron-TP20, Politron, Switzerland) for 1 min in 10mL water and counts of sperm heads were made in 1 mL chambers (Kova® Glasstic® Slide 10 with grids, Hycor Biomedical Inc., USA) under a microscope at 200× magnification. Sperm collected from the caudal epididymes were incubated in Medium 199 (with Hank’s salt, l-glutamine; Invitrogen) containing 1% bovine serum albumin (Calbiochem) at 37 ◦C for 5 min. Subsequently more than 100 sperms were assessment for sperm motility and for sperm morphology abnormalities by manual counting under a microscope at 200× and 400× magnification, respectively.
Statistical analyses were performed using the JMP program (SAS Institute, Cary, NC). For all comparisons, probability values less than 5% (p < 0.05) were considered to be statistically significant. The body weights, organ weights, sperm data, and metabolites data were analyzed by Bartlett’s test (significance concluded at the 5% level) for homogeneity of variance for mean values of each group following the decision tree mentioned below. Homogenous data in Bartlett’s test were subjected to the Tukey–Kramer test. Heterogenous data in Bartlett’s test were subjected to the Wilcoxon/Kruskal–Wallis and subjected to Dunnett test.

Results and discussion

Observed effects

Relative liver weights of DEHA 25000 group without TAA was significantly increased compared to control group. There were no differences in relative prostate and seminal vesicles weights among the groups. There were no differences in AST and g-GTP values among the groups. No significant change for sperm number, motility and morphology abnormality. The animals treated with DEHA did not exhibit evident testicular toxicity. However, hypertrophy of hepatocytes was evident in livers.

Applicant's summary and conclusion

DEHA did not show testicular toxicity in any group. The, hypertrophy of hepatocytes in livers has also been observed in other repeated dose studies with DEHA at high doses (e.g. Xu et al., 2019). However, this change was completely reversible with a recovery phase in other studies. It is therefore considered as non adverse.