1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

The reproductive and developmental toxicity of DINP was evaluated by Waterman et al. (1999; 2000). The reports describe both a one generation and a two generation reproductive toxicity study.

 

In the one generation study which was a range-finding test for the subsequent two-generation study, groups of 30 male or female Crl:CDBR, VAF Plus rats were administered DINP in the feed at doses of 0, 0.5, 1.0, or 1.5% w/w for 10 weeks prior to mating. The females were exposed throughout mating, gestation, and lactation until post natal day (PND) 21. The males were killed immediately after the mating period.

 

Parental effects included a statistically significant lower mean body weight, as well as suppression in body weight gain, primarily observed in the mid and high-dose groups. The greatest decrease from controls was observed during the postpartum period. Similarly statistically significant lower mean food consumption was observed primarily in the mid and high-dose groups. Statistically significant increases in the mean and absolute and/or mean relative liver and kidney weights of both male and female animals as all dose levels tested were observed. Males in the high dose group exhibited a statistically significant increase in the mean absolute and relative right testis weight, left testis and right epididymis weights and the mean relative left epididymis and seminal vesical weights. High dose females showed a significant decrease in the mean absolute and relative right ovarian and mean absolute left ovarian weights.

 

No significant differences in male mating, male fertility, female fertility, female fecundity, or female gestational indices were noted. Mean days of gestation were unaffected by treatment as well as the mean sex ratio of the treated offspring when compared with controls.

 

Offspring effects were noted for a number of parameters. The mean live birth index, day 4 survival-index, day 14 survival index and lactation index of the high-dose offspring were statistically significantly decreased. Dose related decreases in mean offspring body weight were observed during the postnatal period (PND 0-21). There were statistically significant lower mean body weights in the high-dose males and females, mid dose females at all weighing intervals and in mean offspring body weight of the mid dose males on PND 0, 1, 7, 14 and 21. Statistically significant lower mean body weights in the low-dose males on PND 0, 1, 14, and 21 and low-dose females at all weighing intervals were also observed.

 

No effect was observed on fertility parameters indicating a reproductive NOAEL of 1000 mg/kg/day; however, a decrease of live birth and survival indices occurred at 1.5% which led to a developmental NOAEL of 1% (622 mg/kg/day for parental males during pre-mating).

 

A two generation study was designed based on the results of the one generation range finding study. Crl:CDBR VAF Plus rats (30/group) were fed DINP in the diet at 0.2, 0.4, or 0.8% (w/w) for 10 weeks prior to mating, and through gestation and lactation.

 

There were no treatment-related deaths and no clinical signs which were judged to be directly related to treatment with DINP in P1 and P2 animals.

 

During gestation, significantly lower mean food consumption in the P2 high-dose females compared with controls was noted without an associated decrease of the body weight change during gestation days 0-21. During the postpartum period, parental toxicity was limited to a lower mean body weight in the high dose P1 females on post partum days 14 and 21 which corresponded to significant body weight gain suppression during the overall postpartum interval and was associated with decreased mean food consumption. Lower mean body weights were observed in the P2 high-dose females with an associated decrease of mean food consumption but without an associated decrease of the body weight gain.

 

Statistically significant increases in the mean absolute and mean relative liver weights in P1 and P2 in both sexes at 0.4% and 0.8% were observed. Microscopic hepatic changes were noted from 0.2% in P1 and P2 animals. High-dose males exhibited a statistically significant increase of relative right and left epididymal weights in P2 animals with a concurrent increase (not statistically significant) of absolute epididymis weight.

 

There were no statistically significant differences in male mating, male fertility, female fertility, female fecundity or female gestational indices in P1 generation. A slight decrease, not statistically significant, of male mating, male fertility, female fertility, and female fecundity indices was observed in P2 generation. Mean days of gestation of the P1/P2 treated and control animals were equivalent.

 

No treatment-related clinical findings and no biologically significant differences in the F1 or F2 offspring survival indices were observed between the treated and control offspring or gross post-mortem findings. There were statistically significant, dose-related, lower mean offspring bodyweights in all treatment groups compared with controls during the F1 or F2 generations. However, when the litter size was taken into account (Waterman et al., 2000), effects were only significant in high-dose males on PND 0, in males and females of the mid and high-dose levels on PND 7 and 14 and in all treated animals on PND 21. In addition, the weights of all F1 and F2 treated offspring were within the historical control range of the laboratory with the exception of the F2 high-dose males and females on PND 0 and the F2 high-dose males on PND 1 (considering litter size). These findings were considered by the laboratory to be a result of maternal stress and/or direct effects of DINP via exposure through lactation. Studies with other phthalates concluded that these decreases were apparently due to decreased food consumption by the dams and changes in the quality or quantity of milk (Dostal et al., 1987). Thus the laboratory concluded that the lower body weights in the pups might have resulted from decreased milk consumption.

 

No statistically significant differences were observed in reproduction indices indicating a reproductive NOAEL of 0.8% (1000 mg/kg/day).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Dose descriptor:

LOAEL

159 mg/kg bw/day

Additional information

Using Crl:CDBR mated female rats, DINP was administered by gavage at doses of 0, 40, 200, 500 or 1000 mg/kg/day on gestation day 6 through day 15 (Waterman et al., 1999). Overt signs of maternal toxicity were not apparent at any dose level. Similarly, there were no significantly elevated fetal observations or body weight changes at any dose level. Therefore, the maternal and fetal NOAELs were determined to be 1000 mg/kg/day.

 

The comparative developmental toxicity of a number of phthalates including three DINP compounds was evaluated by Hellwig et al., 1997. DINP was administered by gavage at 0, 40, 200, and 1000 mg/kg/day to 8-10 sperm-positive Wistar females/group on gestation day 6 through day 15. The dams were sacrificed on day 20 and implantation sites were examined. Fetuses were weighed and examined for external malformations; half of the fetuses were examined for skeletal malformations and the other half for visceral malformations.

 

Maternal toxicity at the high dose consisted of reduced food consumption and increased relative liver and kidney weights. There were no treatment-related effects on the number of live fetuses/dam or fetal weight. There was an increase in percent fetuses per litter with variations at the highest dose. These variations consisted of rudimentary cervical and/or accessory 14th ribs. A modest increase in dilated renal pelves in the high-dose group was also noted. There were no maternal or developmental effects at 40 or 200 mg/kg/day. A maternal and developmental NOAEL of 200 and LOAEL of 1000 mg/kg/day were determined.

 

The two-generation reproductive study by Waterman et al. (2000) suggests an adverse effect on weight gain in pups during the perinatal and pre-weaning period of life. F1 mean pup body weight was significantly reduced on PND 0 in males at 0.8% DINP (555 and 1,026 mg/kg bw/day during gestation and lactation, respectively, as calculated by study sponsors). On PND 7 and 14, mean male and female pup body weights were significantly reduced at 0.4% (287 and 539 mg/kg bw/day during gestation and lactation, respectively) and 0.8%, and by PND 21, mean male and female body weights were reduced at all dose levels. In the F2 generation, mean female pup body weights were significantly reduced at 0.4 and 0.8% on PND 4, 7, 14, and 21 and at 0.2% (143 and 285 mg/kg bw/day during gestation and lactation, respectively) at PND 7. Mean male pup body weights were significantly reduced at 0.4 and 0.8% at PND 7, 14, and 21. The LOAEL for developmental effects was therefore identified as 0.2% (143–285 mg/kg bw/day during gestation through lactation for changes in body weight). 

 

 

Toxicity to reproduction: other studies

Additional information

In a three-day uterotrophic assay similar to the OECD 440 test guideline, groups of six immature female Wistar rats were dosed orally by gavage with 0 (control), 0 (vehicle control; corn oil), 276 or 1380 mg/kg day DINP, once daily for three days (Sedha et al., 2015). Body weight and clinical signs were recorded daily. On Day 4 all females were sacrificed and uterus and ovaries were removed and weighed wet. There were no deaths or clinical signs. Females given DINP (276 or 1380 mg/kg day) gained significantly less weight than Controls in a dose-related manner. DINP had no effect on group mean relative uterine wet weight at either dose level.

The same authors (Sedha et al., 2015) then dosed groups of six immature female Wistar rats orally by gavage with 0 (control), 0 (vehicle control; corn oil), 276 or 1380 mg/kg day DINP, once daily for 20 days in a 20-day pubertal female assay from post-natal day 21 to PND 41. Body weight and vaginal opening were monitored daily. On PND 41, females were sacrificed, and the vagina of each animal was removed and weighed along with the uterus, laminal fluid and ovaries. Precocious vaginal opening was not observed on PND 41 in any treatment group, including controls. DINP (276 or 1380 mg/kg day) did not affect group mean relative vagina weight. Group mean relative ovary weight for females given 1380 mg/kg day DINP was significantly lower than Controls, although this difference was marginal and in the opposite direction to suggest an estrogenic effect of DINP. The results from both assays demonstrated that DINP had no estrogenic effect in the female Wistar rat at a dose level up to 1380 mg/kg day, and since no significant toxicity was observed at this dose level, the No Observed Adverse Effect Level (NOAEL) can be considered to be ≥ 1380 mg/kg day.

In another study, ven den Driesche et al. (2020) assessed and compared the effects of DINP and DBP exposure on the male rat reproductive tract by exposing pregnant wistar rats to corn oil (vehicle control), DINP (125 or 750 mg/kg/day) or DBP (positive control; 750 mg/kg/day) during the male masculinisation programming window (MPW) (embryonic days 15.5 to 18.5). Assessments were made on foetal testes sampled on embryonic days 17.5 and 21.5 as well as testes and epididymides from adult males sampled on postnatal day 90. The reproductive phenotype of each adult male rat was also examined at necropsy, and blood was sampled from each adult male rat to quantify circulating plasma testosterone and LH levels. in utero exposure to DINP (125 and 750 mg/kg/day) caused no adverse effects on the assessed parameters both in the developing foetus and in the adult male. In contrast, DBP significantly affected the assessed parameters. Overall, these data indicate that in utero exposure to DINP (up to 750 mg/kg/day), a high molecular weight (HMW) phthalate, during the male MPW, had no adverse or anti-androgenic effect on the reproductive and endocrine systems in the developing male rat foetus and adult male rat, whereas in utero exposure to the low molecular weight (LMW) phthalate DBP did induce toxic effects.

An evaluation of scientific literature published before July 2015 that explores the effects of DINP (and DnHP and DCHP) exposure on developmental and fertility endpoints, was performed via a quantitative weight of evidence (QWoE) method, to determine whether the reported adverse effects fulfil the requirements for CLP classification of these chemicals as reproductive toxicants (Dekant and Bridges 2016). Papers were scored using the QWoE method based on quality/reliability and toxicological relevance/effects, in line with the WoE approaches included in the CLP Regulation (EC Regulation 1272/2008 part 3.7.2.3.1). For DINP, 13 papers that addressed developmental and fertility endpoints (including Boberg et al., 2011; Clewell et al., 2013a, 2013b; Exxon, 1996a, 1996b; Hellwig et al., 1997; Li et al., 2015 reported in this dossier) were scored. The overall weight of evidence indicated these papers were of good-to-high quality, and provided insufficient evidence (i.e no strong adverse findings) to support the allocation of a CLP reproductive classification category for DINP. This conclusion is consistent with the decision made by the European Chemicals Bureau (EU) Risk Assessment in 2003 as well as the decision made by ECHA RAC in 2018, that DINP does not require CLP classification for reproductive toxicity, based on evidence available in the scientific literature.

Justification for classification or non-classification

No classification for developmental or reproductive toxicity is indicated according to the general classification and labeling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling, and packaging (CLP) regulation (EC) No 1272/2008. DINP was evaluated by the Classification and Labelling Working Group with the conclusion that classification was not required for any endpoint. (European Commission Working Group, 2000a ; 2000b).