Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

GLP compliance:
not specified
Type of method:
in vivo

Test material

Reference
Name:
Unnamed

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
Primigravida pregnant ICR mice were purchased from Peking
University Health Science Center and were separated into two
groups (control and DBDPE treatments, seven to eight pregnant
mice per group). All pregnant mice were housed individually in an
environment maintaining the temperature of 25 ± 3 C and the
humidity of 50 ± 5% with 12hr/12hr light/dark cycle, and free access
to water and food during feeding.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
After five days of acclimatization,
the mice in the control group (corn oil, 5 mg/kg bw) and the DBDPE
treatment group (corn oil solution of DBDPE, final concentration of
DBDPE is 100 mg/kg bw) were gavaged every day from gestational
day 6 (GD6) to postnatal day 21 (PND21). On the PND2, the litters
were selected randomly to 8 pups. After weaning at PND21, male
offspring of both groups were selected because some previous
studies have demonstrated that male mice are more sensitive.
(Giulivo et al., 2016). Males in each group were fed
with low-fat diet (LFD: 10% calories from fat, Trophic Animal Feed
High-tech Co., Ltd, China) or high-fat diet (HFD: 60% calories from
fat, Trophic Animal Feed High-tech Co., Ltd, China) for 12 weeks.
Therefore, four groups were obtained: vehicle - LFD (V-L),
DBDPE - LFD (D-L), vehicle - HFD (V-H), and DBDPE-þ HFD
(D-H).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
15 days
Frequency of treatment:
Once daily
Duration of test:
99 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
0.1 mg/kg bw/day
No. of animals per sex per dose:
8 male pups per treatment group (VL, DL, VH, DH)
Control animals:
yes, concurrent vehicle
Statistics:
Resultswere presented as mean ± SEM.The graphswere drawn by GraphPad Prism Version 6.0. And SPSS 19.0 (IBM, USA) was used for
statistical analysis.Thenormality test of thedatawas performedusing the Shapiro-Wilk test and the homogeneity of the variance was
assessed using Levene's test. If therewas a normal distribution, T-test or ANOVA and Tukey's post hoc test were used to determine the
significance of variables between different groups. If there was a violation of normality, the data was subjected to nonparametric
analysis and adjusted for significance. P < 0.05 was considered significant in all tests. As for the metabolomics data, the SIMCA 13.0 software and the metaboanalyst 4.0 (http://www.metaboanalyst.ca) were used to analysis according to the previously mentioned method.

Results and discussion

Any other information on results incl. tables

Effects of DBDPE exposure on body weight, liver weight, and epididymis fat weight.

At 15 weeks, body weights of male mouse offspring in the DL group were significantly higher than those in the VL group (P < 0.0001), whereas there was no significant difference between the VL and DH group. Compared with LFD groups, the liver weight was increased in HFD groups significantly. However, no significant differences in liver weight were observed between DBDPE-exposed and vehicle-exposed mice fed either a LFD or HFD. The epididymis fat weight of mice in the DL group was significantly higher than those in the VL group, and mice in the HFD group had higher epididymis fat weight than LFD group.

 

Effect of DBDPE exposure on glucose homeostasis.

The effects of treatment with DBDPE or HFD on glucose homeostasis were determined by GTT and ITT. Mice exposed to the HFD (VH and DH groups) did not metabolize glucose as rapidly as control animals (VL group) during the GTT, and exposed to DBDPE caused further slowing in glucose metabolism. Similarly, glucose metabolism in VH and DH groups were significant retarded compared with VL group after being injected with insulin solution, without DBDPE effects.

 

Effect of DBDPE exposure on liver functions and injury.

We analyzed the levels of TG and TC in the liver and the results showed that the levels of TG in the two groups fed with HFD increased significantly, while the TC levels only increased significantly in group DL. In addition, the liver function impairment was roughly assessed by ALT and AST levels in serum. The results showed that the ALT level in the DL group was significantly lower, but the other groups did not change significantly. And the AST level were basically equivalent for each group.

 

Effect of DBDPE exposure on metabolomics.

A representative 600 MHz 1H NMR spectra of the liver aqueous extracts from mice was presented in Fig. S1. Chemical shift assignments for metabolites were labelled in Fig. S1 and summarized in Table S2 of the original publication. The 3D PCA score plot for all groups showed a trend of separation of samples as a whole. The OPLS-DA score plots showed that the groups can be clearly separated from each other. The R2X, R2Y, Q2 and CV-ANOVA p-values were shown in Table 1 of the original publication. The pairwise comparisons between four groups were used to determine the effect of DBDPE, HFD and their joint actions on the metabolic profile of mice (Wang et al., 2016). Based on the VIP values obtained from the OPLS-DA model, combined with the FDR values, we identified a total of 24 altered metabolites: bile acids, lipid, leucine, isoleucine, valine, lactate, alanine, butyric acid, lysine, acetate, glutamine, glutamate, glutathione, phosphocholine (PC), uridine, glycerophosphocholine (GPC), dimethylallylpyrophosphate

(DMAPP), alpha-glucose, glycogen, thiamine, nicotinurate, inosine and formate, which can be divided into amino acids, purines, lipids, vitamins and other small molecules involved in energy metabolism. The heatmap generated by the significantly changed metabolites is used to represent the clustering between

different individual samples. In fact, both HFD and DBDPE caused metabolomics changes, and combined exposure of HFD and DBDPE may enhance the drug-induced metabolomics changes.

 

Specifically, compared with VL group, lactate, uridine, GPC, cyclic AMP, nicotinurate and inosine were increased in all other groups while thiamine, was lowered. In addition, bile acids, lipid, leucine,

isoleucine, valine, lysine, acetate and glutamine were reduced and glutathione, PC and DMAPP were raised in VH group compared with VL group. And, metabolites bile acids, lipid, butyric acid, glutamine, glutamate, glutathione, PC, DMAPP and formate were increased, glycogen was decreased after exposed to both DBDPE and HFD. In particular, after exposure to DBDPE, although there were fewer metabolites that were significantly changed in the LFD group, the trends were similar to those in the joint exposure group. More detailed results of metabolite changes between the groups are referenced in Table 2 of the original publication.

 

Effects of DBDPE exposure on the expression of some energy metabolism related genes.

In order to elucidate the mechanism of DBDPE-induced obesity, we determined the expressions of genes involved in lipid and glucose metabolism, which included fatty acid synthesis (SCD1, FASN, ACC, SPEBP-1c), fatty acid uptake and lipid storage (PPARg, CD36), b-oxidation (PPARa, Cpt1a), triglyceride synthesis

(DGAT1, DGAT2, Mogat1), triglyceride transport (Mttp), carbohydrate metabolism (G6pase, Gck) and bile secretion (Shp, Fxr). After DBDPE or HFD exposure, the expression of gene encoding the rate-limiting enzyme SCD1 for biosynthesis of unsaturated fatty acids was significantly reduced compared with VL group, and the DH group showed a more severe trend. And, the gene expression of FASN was significant increased only in the DH group compared with that in VL group. The gene expression of ACC was significant decreased in both VH and DH groups. There was no significant change in the gene expression of SPEBP-1c and Cpt1a. Compared with VL, the gene expression of PPARg in DL group and VH group were increased, and the increase caused by HFD is more significant. The mRNA levels of CD36

and PPARa were significant increase in VH group compared with VL group. As for DGAT1 and DGAT2, encoding the enzyme involved in de novo synthesis of triglyceride, showed no significant change in all groups. And, the expression of Mogat1, a key enzyme in triglyceride synthesis, had an increasing trend in the DL and VH groups and the DH group showed a more severe trend compared with VL group. The expression of microsomal triglyceride transfer protein Mttp had the similar changes to DGAT2. As glucose metabolism related genes, the expression of G6pase and Gck had different trends. The mRNA levels of G6pase was significant decreased in VH while the Gck was decreased in DL group. And Shp, compared with expression in the VL group, showed significant decreased in all other groups, while Fxr had significant increase in DL and VH groups.

Applicant's summary and conclusion

Conclusions:
In this study, the authors explored potential DBDPE toxic effects on male mouse offspring after perinatal exposure. During the perinatal period, pregnant ICR mice were exposed to DBDPE (0.1 mg/kg body weight) via oral gavage. After weaning, male offspring were fed on a low-fat diet and a high-fat diet, respectively. The authors measured and recorded body weight, liver weight, and epididymis fat mass, blood biochemical markers, metabolites changes in liver, and gene expression involved in lipid and glucose homeostasis. The authors report that perinatal exposure to DBDPE increased the risk of obesity in mouse offspring and affected triglyceride synthesis, bile secretion, purine synthesis, mitochondrial function and glucose metabolism; furthermore, the use of high-fat feeding may further exacerbate these effects. The authors then claim that their results show that early-life exposure to low doses of DBDPE may promote the development of metabolic dysfunction, which in turn can induce obesity. However the relevanc eof this protocol and the background variability of the parmeters remain unknown and the findings are of limited relevance for human health risk assessment.
Executive summary:

In this study, the authors explored potential DBDPE toxic effects on male mouse offspring after perinatal exposure. During the perinatal period, pregnant ICR mice were exposed to DBDPE (0.1 mg/kg body weight) via oral gavage. After weaning, male offspring were fed on a low-fat diet and a high-fat diet, respectively. The authors measured and recorded body weight, liver weight, and epididymis fat mass, blood biochemical markers, metabolites changes in liver, and gene expression involved in lipid and glucose homeostasis. The authors report that perinatal exposure to DBDPE increased the risk of obesity in mouse offspring and affected triglyceride synthesis, bile secretion, purine synthesis, mitochondrial function and glucose metabolism; furthermore, the use of high-fat feeding may further exacerbate these effects. The authors then claim that their results show that early-life exposure to low doses of DBDPE may promote the development of metabolic dysfunction, which in turn can induce obesity. However the relevanc eof this protocol and the background variability of the parmeters remain unknown and the findings are of limited relevance for human health risk assessment.