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Effects on fertility

Effect on fertility: via oral route
Dose descriptor:
NOAEL
600 mg/kg bw/day
Additional information

Short description of key information:


The reproductive toxicity of di-isodecyl phthalate (DIDP) has been evaluated in Sprague Dawley rats using a preliminary one-generation reproductive toxicity study, as well as two multigeneration reproductive toxicity studies. All studies were performed in accordance with applicable regulatory guidelines (EC Dangerous Substances Directive 67/548/EEC; USEPA OPPTS 870.3800) and in compliance with OECD Principles of Good Laboratory Practice (GLP).


 


The preliminary one-generation study administered 0%, 0.25%, 0.5%, 0.75% or 1.0% DIDP in the diet to P1 male and female rats beginning at least ten weeks prior to mating through gestation and lactation periods for P1 females, until weaning of F1 offspring on postnatal day (PND) 21 (EBSI, 1997). Adverse effects were limited to decreased body weights in both sexes in the 0.75% and 1.0% dose groups and decreased food consumption in 1.0% DIDP-treated males and females through gestation. During lactation, the 0.75% and 1.0% P1 females had reduced body weight gain, and 0.5%, 0.75% and 1.0% P1 females had lower food consumption. In offspring, no treatment-related clinical findings or gross toxicity findings were found in any dose group. Significant decreases in body weights were found in male and female F1 offspring in the 0.75% and 1.0% dose groups. Slight decreases in mean body weights were observed in the 0.5% dose group at PNDs 14 and 21, although body weights were within the historical control range of the laboratory. Based on signs of toxicity at 0.75% and 1.0% DIDP in both parental animals and offspring and decreased food consumption, 0.8% was selected as the high dose for the two-generation reproduction toxicity study.


 


In a modified two-generation reproductive toxicity study, P1 and P2 male and female rats were administered 0%, 0.2%, 0.4% or 0.8% DIDP in feed following the same exposure duration described for the one-generation study (EBSI, 1998; Hushka et al., 2001). In addition, two satellite groups were treated with either control diet or 0.8% DIDP in feed during the P1 generation. In one of the satellite experiments, litters were switched after birth (cross-fostered) between controls and 0.8% groups. In another satellite experiment, control and 0.8% P1 offspring raised by their biological dams were switched to the opposite diet at weaning to assess recovery. No adverse changes in either parental generation for reproductive indices, gonadal function, or histopathology in reproductive tissues were observed. The NOAEL (no-observed-adverse-effect level) for overall reproductive effects was 0.8% DIDP, the highest dose tested. Parental findings were limited to decreased body weight gain and/or food consumption in P1 and P2 0.8% DIDP-treated females during the lactation period and in P2 males during the pre-mating period. No treatment-related clinical signs or deaths occurred in DIDP-treated parental animals. No abnormalities occurred in offspring or parental animals. Both parental generations exhibited increased absolute and relative weights and/or histopathological changes in the liver and kidneys. These effects were not considered adverse, because they were consistent with peroxisome proliferation and male rat-specific nephropathy, neither of which is relevant to humans (IARC, 1995; EPA, 1991). Hepatomegaly and induction of hepatic peroxisome activity has been measured previously in rats fed a 1.0% DIDP diet (Lake et al., 1991). Treatment-related lower offspring body weight gain observed in the high-dose group (0.8% DIDP) at most postnatal intervals in both generations was consistent with results from the preliminary one-generation study (EBSI, 1997). Findings from the satellite studies indicated that offspring exposed to 0.8% DIDP in utero and during lactation regained body weight once switched to the control diet. It is not possible to discriminate if reduced pup body weight is due to changed maternal behavior, alteration of maternal metabolism affecting milk quality, effect on milk production or palatability of the milk for offspring during lactation. Although the live-birth index was unaffected, lower pup survival was noted in F1 offspring at the high dose and in F2 offspring at all doses on PNDs 1 and 4. Due to inconsistencies in survival between the preliminary one-generation study and the F1 pups of this two-generation study, which used lower DIDP doses, and the finding that most of the decreased survival occurred in ten specific F2 litters across low-, mid-, and high-dose groups, these differences in survival were considered to be the result of biological variability and not treatment-related. As a result, the offspring NOAEL, based on treatment-related effects on pup body weight at 0.8% DIDP, was determined to be 0.4% DIDP.


 


A follow-up, two-generation study was conducted to clarify conflicting evidence on offspring survival between the one-generation (ESBI, 1997) and two-generation (EBSI, 1998) studies. In this follow-up, two-generation study, P1 and P2 male and female rats were administered 0%, 0.02%, 0.06%, 0.2%, or 0.4% DIDP in the diet following the same study design and exposure duration as the initial two-generation study (EBSI, 1998, 2000; Hushka et al., 2001). No adverse changes in either parental generation for reproductive indices—including mating, fertility, fecundity, or gestational indices—were observed, so the fertility NOAEL was 0.4%. No treatment-related clinical signs, deaths, or effects on body weight or food consumption occurred in DIDP-treated parental animals. Most P1 and P2 animals were free from abnormalities throughout the study, and the only notable findings were increased absolute and relative liver and kidney weights in both DIDP-treated parental generations, consistent with previous findings (EBSI, 1998). Fl offspring showed no biologically significant effects for any measure of fertility, survival, or body weight. However, F2 offspring had significantly lower body weights in 0.4% DIDP-treated male pups on PND 14, in 0.2% and 0.4% DIDP-treated female pups on PND 14, and in 0.4% DIDP-treated female pups on PND 21. While no effect on the live-birth index was observed in either generation, significantly decreased survival occurred at PND 1 and PND 4 in the 0.2% and 0.4% DIDP groups, consistent with the previous two-generation study (EBSI, 1998). Due to the decreased survival in the 0.2% and 0.4% DIDP groups, the offspring NOAEL was equal to 0.06% DIDP.


 


In summary, these data provide no evidence of adverse effects on reproductive parameters following DIDP exposure (up to 0.8% DIDP in diet) in rats (EBSI, 1997, 1998, 2000; Hushka et al., 2001). No statistically significant differences in absolute reproductive organ weights were observed, including testicular weight in both P1 and P2 generations (EBSI, 1998; Hushka et al., 2001). Although a decrease in the relative weight of some reproductive organs was observed in the highest dose group (0.8% DIDP) of P2 males in first two-generation study, these relative weight decreases reflect the decrease in body weight that was also observed in this group. Further, no correlating histopathology or morphological changes were observed (EBSI, 1998; Hushka et al., 2001). Treatment-related effects in offspring in the two-generation studies (decreased body weight and survival at 0.2% DIDP or above in the diet) are observed concurrently with parental toxicity at this dose-level  (ie., liver changes). However, it is not possible to discriminate whether observed survival effects are related to the development/vitality of the offspring, maternal behavior or ability to produce enough milk.


The EU Risk Assessment on DIDP reviewed this data and concluded that no effect was observed on development landmarks assessed at any dose tested.


Considerations for classification: 


The decrease in pup viability indices in F2 (day 1 and day 4) in the DIDP two-generation study (Hushka et al. 2001) as well as skeletal variations in developmental studies on DIDP (EBSI 1997; Hellwig et al. 1997) are not sufficient to justify classification for fertility, developmental or lactational effects. This opinion on fertility, developmental and lactational effects for DIDP was concluded during the May 2000 meeting of the EU CMR and Pesticides Working Groups on Classification and Labelling (European Commission Working Group. 2000a; 2000b) and included in the EU Risk Assessment for DIDP. This conclusion is quoted by ECHA (2013) in their report “Evaluation of new scientific evidence concerning DINP and DIDP – In relation to entry 52 of Annex XVII to REACH regulation (EC) No. 1907/2006 – Final Review Report”. This interpretation is consistent with the RAC (2018) opinion on similar skeletal variations induced by diisononyl phthalate (DINP), where the overall conclusion was that no classification was warranted for DINP (reference: https://echa.europa.eu/documents/10162/56980740-fcb6-6755-d7bb-bfe797c36ee7). 

Effects on developmental toxicity

Description of key information
Developmental toxicity studies of DIDP conducted at doses of 100, 500, and 1000 mg/kg provided evidence of slight and transient signs of maternal toxicity at 1,000 mg/kg/d (significant reversible decrease of body weight gain and food consumption) suggesting a conservative NOAEL of 500 mg/kg/d for maternal toxicity. The only statistically significant changes were skeletal variations (supernumerary cervical and rudimentary lumbar ribs) on a per litter basis at the high dose. Rudimentary ribs are a common finding in rat fetuses and should not be regarded as associated with malformations, but may only be related to transient maternal stress. It should be noted that supernumerary ribs were located in the cervical region which is less common (Waterman et al., 1999), but the biological significance of cervical supernumerary ribs remains uncertain. A NOAEL of 500 mg/kg/d may be assumed for skeletal variations.

 

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
500 mg/kg bw/day
Additional information

The developmental toxicity of di-isodecyl phthalate (DIDP) has been evaluated in rats and mice generally following OECD Principles of Good Laboratory Practice (GLP) according to EC Commission Directive 87/302/EEC, OECD TG 414, and/or USEPA 40 CFR Part 798 Toxic Substances Control Act Guidelines.


 


Pregnant Sprague Dawley rats received 0, 100, 500, and 1000 mg/kg-d DIDP via oral gavage from gestation day (GD) 6 through 15 in both the range finder and main studies (EBSI, 1994; EBSI, 1995). No maternal toxicity was observed in the range finder, but decreased maternal weight gain and food consumption occurred during dosing at 1000 mg/kg DIDP in the main study. No differences in body weight or food consumption were observed in any treatment group compared to controls by study termination (GD 21), suggesting a recovery effect. No maternal mortality or adverse clinical effects were seen in either study. No treatment-related effects on implantation, fetal development, or growth were observed. There were no significant increases in external, visceral, or skeletal malformations in DIDP treated groups. A significant increase in skeletal variations on a per-fetus basis in the 500 mg/kg and 1000 mg/kg groups, and increased skeletal variations (rudimentary lumbar and cervical ribs) per litter in the 1000 mg/kg group were observed in the main study (EBSI, 1995). These incidences were within the historical control range of the laboratory or were regarded as alternative normal patterns that were not considered to be adverse (Harris and DeSesso, 1994). The maternal NOAEL (no-observed-adverse-effect level) was 500 mg/kg, and the developmental NOAEL was 1000 mg/kg DIDP.


 


The EBSI (1995) study report findings were published by Waterman et al. (1999). In this publication, the authors considered 500 mg/kg-d to be the NOAEL for both maternal and developmental toxicity based on the rudimentary lumbar and cervical ribs. Further, the National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction Phthalates Expert Panel (CERHR) chose 100 mg/kg-d as the developmental NOAEL based on cervical and rudimentary lumbar ribs at 500 mg/kg/d, and advised the study sponsor of improved statistical approaches, which the study sponsor used to reanalyze the data. This reanalysis resulted in a developmental NOAEL of 100 mg/kg-d, which was in agreement with the review panel (NTP-CERHR, 2003, and citations therein).


 


Hellwig et al. (1997) administered pregnant Wistar rats DIDP by oral gavage at 0, 40, 200, or 1000 mg/kg-d DIDP on GD 6–15. Decreased maternal food consumption was observed at 1000 mg/kg-d, and increased fetal skeletal variations were observed at 200 and 1000 mg/kg-d. Hellwig et al. considered 200 mg/kg-d to be the maternal and developmental NOAEL. The NTP CERHR review panel chose 40 mg/kg-d as the developmental NOAEL, based on total fetal variations at 200 mg/kg/d (NTP-CERHR, 2003).


 


A developmental toxicity screening study in CD-1 mice that did not include fetal examinations showed no maternal toxicity or effects on postnatal survival or growth up to PND 3 following exposure to approximately 10,000 mg/kg-d DIDP on GD 6–13 (Hardin et al., 1987).


 


Based on the findings of Waterman et al. (1999), Hellwig et al., (1997), and Hardin et al. (1987), the NTP-CERHR panel concluded that there was clear evidence of adverse developmental effects from exposure to high levels of DIDP in laboratory animals. Considering these studies and human exposure estimates, the NTP agreed with the CERHR panel that there was minimal concern for developmental effects in fetuses and children.


  


In a two-generation reproductive toxicity study in Sprague Dawley rats administered 0%, 0.2%, 0.4%, or 0.8% DIDP in feed, there were no effects on the parental (P1) animals. Lower postnatal survival of F1 pups was observed through postnatal day 4 in the high-dose group. In F2 pups, postnatal survival on postnatal days 1 and 4 was lower at all doses. No effect on the day of onset of preputial separation was observed, but vaginal patency was delayed in the 0.4% and 0.8% dose groups of the F1 generation (developmental landmarks not examined in F2 generation). This small delay (1–2 days) co-occurred with lower body weight in the high-dose group (ESBI, 1998; Hushka et al., 2001). A follow-up two-generation study in rats administered 0%, 0.02%, 0.06%, 0.2%, or 0.4% DIDP in feed showed no differences in anogenital distance, male nipple retention, or age at vaginal patency in either generation. Age at preputial separation was one day later than controls in the 0.4% group of the F2, which coincided with lower body weights (ESBI, 2000; Hushka et al., 2001). Decreased body weight and survival through postnatal day 4 of the F2 pups were seen at DIDP concentrations of 0.2% and above in feed. The effects on postnatal survival were more pronounced in the F2 offspring in both the original and follow-up studies.


 


To evaluate effects on the development of the male reproductive tract, pregnant rats were dosed orally with 0, 500, 750, 1000, or 1500 mg/kg-d DIDP from GD 14 through 18 (Hannas et al., 2012). Maternal body-weight gain and fetal survival were not affected at any dose. DIDP had no effect on fetal testicular ex vivo testosterone production or on fetal testes expression of genes related to sexual determination and differentiation, steroidogenesis, gubernaculum development, and androgen signaling.


 


Effects of DIDP on fetal testicular development were examined in a study in which pregnant rats were treated with 0, 10, 100, or 1000 mg/kg-d DIDP via oral gavage daily from GD 14 through 21 (Zhang et al., 2020). Maternal body weights, birth weight, litter size, and sex ratio were not affected by DIDP. The 1000-mg/kg-d group had lower serum testosterone levels, increased incidence of multinucleated gonocytes, increased fetal Leydig cell aggregation, and decreased specific steroidogenesis and differentiation-related gene (Star, Cyp11a1, Hsd17b3, Insl3, Sox9) and protein (Star, Cyp11a1, Hsd17b3, Insl3, Sox9) expression in pup testes.


 


In summary, exposure to high levels of DIDP (i.e., >0.2% in diet or >200 mg/kg-d) in rats resulted in increased incidence of skeletal variations, and reduced pup weight gain and survival (EBSI, 1994, 1995, 1998, 2000; Hushka et al., 2001; Waterman et al., 1999). Effects of DIDP on male reproductive development are absent or observed at very high exposures (i.e., 1000 mg/kg-d) during the masculinization programming window in rats (Hannas et al., 2012; Zhang et al., 2020). However, no adverse effects on male reproductive endpoints were observed in two-generation reproductive toxicity studies in rats fed up to 0.8% DIDP in their diet (EBSI, 1998, 2000). Therefore, the high-dose effects observed by Zhang et al. (2020) are likely the result of developmental delay from the longer high dose gestational exposure than was used by Hannas et al. (2012) and appear to have no lasting effects in developing rat pups as no adverse effects on male reproductive endpoints have been observed. The DIDP exposure levels used in rodent studies are orders of magnitude higher than environmental exposure levels that may occur in humans (NTP-CERHR, 2003).


Considerations for classification: 


Skeletal variations were observed in the developmental studies at 1,000 mg/kg/d concurrently with slight signs of maternal toxicity and lead to a NOAEL of 500 mg/kg/d (European Commission, 2003). The decrease in survival indices mainly in F2 (day 1 and day 4) in the two-generation study as well as skeletal variations in developmental studies are not sufficient to justify classification for fertility, developmental or lactational effects (ECBI/51/00 – Rev.2 23.11.00). This opinion on fertility, developmental and lactational effects was concluded during the May 2000 meeting of the EU CMR and Pesticides Working Groups on Classification and Labelling (European Commission Working Group, 2000a; 2000b). This interpretation is consistent with the RAC (2018) opinion on skeletal variations induced by diisononyl phthalate (DINP), where the overall conclusion was that no classification was warranted for DINP (reference: https://echa.europa.eu/documents/10162/56980740-fcb6-6755-d7bb-bfe797c36ee7). 

Justification for classification or non-classification

Fertility: A 2-generation reproductive toxicity test of DIDP conducted in rats showed there were no changes in reproductive indices (Exxon Biomedical Sciences, 1997 and 2000; key data published in Hushka et al., 2001). Up to the highest dose tested, no overt signs of reproductive toxicity were reported and no effects were observed on fertility parameters.  Additionally, there were no effects on the testis weights from male rats fed DIDP for 21 days and the testicular histology was normal (BIBRA, 1986). Accordingly, the overall conclusion from these studies was that DIDP has no effect on fertility and does not meet the classification criterion for reproductive toxicity. 


Developmental toxicity: DIDP did produce a developmental effect in the 2-generation reproductive toxicity studies (Exxon Biomedical Sciences, 1997 and 2000; key data published in Hushka et al., 2001) as evidenced by the decrease in postnatal survival indices which was observed in both two-generation studies leading to the NOAEL of 0.06% (33-76 mg/kg/d). Results from the cross-fostering and switched diet satellite groups suggest that lactation exposure may contribute to the toxicity of DIDP (EU RAR on DIDP, 2006), however it is not possible to distinguish if reduced pup body weight is due to changed maternal behavior, alteration of maternal metabolism affecting milk quality, effect on milk production or palatability of the milk for offspring during lactation. The results of a developmental toxicity study in rats (Waterman et al., 1999) indicate that DIDP did not induce severe developmental effects. Skeletal variations were observed at 1,000 mg/kg/d concurrently with slight signs of maternal toxicity which led to a NOAEL of 500 mg/kg/d (EU RAR on DIDP, 2006).


 


The decreases in survival indices mainly in F2 (day 1 and day 4) in the two-generation study as well as skeletal variations in developmental studies are not severe enough to justify a classification (EU RAR on DIDP (2006). This opinion was concluded during the May 2000 meeting of the EU CMR and Pesticides Working Groups on Classification and Labeling for adverse effects on development (ECBI/56/02 Add. 12, Rev. 6, Table 1c).


 

Additional information