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Reference
Endpoint:
toxicogenomics
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: For read-across justification see Section 13.
Principles of method if other than guideline:
To assess the potential to induce testicular mal-development (TMD) in the rat by studying effects on the expression of genes in pathways known to be involved in steroidogenesis and testes development that have previously been shown to be involved in the induction of TMD.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
developmental toxicity / teratogenicity
Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
24 female and 5 male Han Wistar rats (10-12 weeks old) were housed 3 per cage on sawdust in solid-bottom, polypropylene cages. The rats were acclimatised for at least 5 days before use. In the animal room, the environment was controlled to provide conditions suitable for the Wistar strain of rat. The temperature was maintained within a range of 19-23 deg C and relative humidity within a range of 40-70%. There were, nominally, 14-15 air changes per hour. Twelve-hour periods of light were cycled with twelve-hour periods of darkness.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Pregnant dams were treated daily with vehicle (corn oil) or L810TM by oral gavage on gestation days 12 to 19 inclusive. Di(2-ethylhexyl) phthalate (DEHP) was used as a positive control.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Gestation days 12 to 19 inclusively
Frequency of treatment:
Daily
Post exposure period:
Dams were killed on gestation day 19 and foetuses removed.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Testes from a minimum of 5 litters per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
Male rats were exposed in-utero by oral gavage of the dam and transcriptional profiling analysis of RNA extracted from neonatal testes was used to gain insight into the potential effects on pathways relevant to testicular mal-development (TMD). Male and female animals were randomly selected for mating. Owing to the propensity of rat vaginal plugs to drop out following mating, there was a need to mate animals one on one in wire bottom cages, so as to be able to confirm that mating had occurred and for the selection of time-mated females. Time-mated females were treated from gestation day (GD) 12-19 inclusive with either vehicle (corn oil) DEHP or L810TM (500 mg/Kg) by oral gavage all at a dose volume of 1 ml/Kg. On GD 19, dams were killed by inhalation of an increasing concentration of carbon dioxide. Foetuses were removed and killed by decapitation. Foetal gonads were dissected using a dissecting microscope. Testes from a minimum of 5 litters per treatment group were micro-dissected and snap frozen in liquid nitrogen. Pairs of snap frozen testes, two from each pup, were stored at -80°C prior to RNA isolation for microarray analysis and possible LCMS analysis.

Pools of foetal testes from a minimum of three pups per litter (6 foetal testes) were disrupted using a qiashredder column and purified using RNeasy mini columns according to the CXR method entitled `Method for Isolation of RNA from Foetal Rat Testes for use in Microarray Analysis¿ (Plummer et al, 2007). RNA integrity was checked using the Agilent Bioanalyser and the RNA nano or pico Lab chip kit according to the Agilent protocol entitled `RNA 6000 Nano/Pico Assay¿. Total RNA (100ng-1mg) was labelled prior to microarray hybridisation using the Agilent Quick Amp Labelling Kit One Colour (Agilent# 5190-0442). Agilent 4x44K Whole Rat Genome Oligo Microarray slides (G4131F) were hybridised, washed and then scanned on an Agilent Microarray Scanner.

Images from the scanner were processed using Agilent Feature Extraction Software v9.1. Rosetta ResolverTM 6 software was used to define a list of significantly altered genes (the ¿Signature List¿). Signature lists of significantly (p<0.01) altered genes were filtered to remove low intensity genes.

Bioinformatic analysis using Ingenuity Pathways AnalysisTM (IPA) software was used to identify genes in the signature lists that are associated with the TMD target pathways of testosterone synthesis and cryptorchidism. Lists of genes in these pathways relevant to TMD were constructed using from data derived from previous transcriptional profiling studies (Liu et al. 2005; Plummer et al. 2007).


Examinations:
Changes in gene expression in pathways relevant to TMD, (e.g steroidogenesis, cholesterol metabolism and transport and guvernacular ligament development), by transcription profiling analysis of RNA.
Positive control:
Di(2-ethylhexyl) phthalate (DEHP), at a dose of 500 mg/kg, was used as a positive control.
Details on results:
564 and 3406 gene expression changes (P<0.01), relative to control, were caused by DEHP (500mg/Kg) and L810TM, respectively.

From a toxicological point of view, it is more relevant to analyze the effects of compounds in the context of functional pathways rather than looking at individual genes. Hence, analysis of all the signature gene expression changes (>1.5 fold) in the IPA database against gene/pathway information. Representation analysis of bias towards effects on particular pathways in the gene lists relative to the IPA database, identified pathways (1-7) that were significantly over-represented in the DEHP and TOTM gene lists. DEHP treatment caused bias towards effects in the pathways of steroidogenesis and steroid metabolism. Genes in this pathway, namely 3-hydroxy 3-methylglutaryl-Coenzyme A reductase (HMGCR), cytochrome P450 short chain cleavage (CYP11A), cytochrome P450 17 alpha-hydroxylase (CYP17A), steroidogenic acute regulatory protein (STAR), delta(5)-3-beta, hydroxysteroid dehydrogenase-1 (HSD3B1) and sterol isomerase (EBP), were down-regulated.

By contrast, the L810TM signature gene list showed a bias towards effects on the pathways of hepatic stellate cell activation, glucocorticoid signalling, integrin signalling, androgen signalling and MAPK signalling. None of these effects were considered to be relevant to TMD.

To compare the effects of the different treatments on genes responsible for the testosterone hormone synthesis, the data from the gene lists were superimposed on a TMD pathway derived from previous mechanistic studies on certain phthalates (CXR0206, CXR0409), (Plummer et al. 2007). In the present study, the results showed that DEHP caused a repression of genes involved in testes development and cholesterol and testosterone biosynthesis.

L810TM had no significant repressive effect on genes in the TMD pathway,
Conclusions:
MEHP and DEHP treatment caused repression in a number of genes previously identified as being involved in the induction of TMD by certain phthalate esters. L810TM did not repress TMD target genes. It is concluded that it is highly unlikely that L810TM would cause testicular dysgenesis in rats under these treatment conditions.
Executive summary:

A study on effects on gene expression associated with developmental toxicity in male rat foetal testes by transcriptional profiling indicate that the substance does not cause repression of genes in the testicular mal-development pathway indicating that the substance is unlikely to cause testicular dysgenesis in rats.

MEHP and DEHP treatment caused repression in a number of genes previously identified as being involved in the induction of TMD by certain phthalate esters. L810TM did not repress TMD target genes. It is concluded that it is highly unlikely that L810TM would cause testicular dysgenesis in rats under these treatment conditions.

Description of key information

tential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed in utero to L810TM or the positive controls.

The potential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed in utero to L810TM or the positive controls.The potential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed in utero to L810TM or the positive controls. The potential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed in utero to L810TM or the positive controls.

The potential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed i

ated substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and testes development previously shown to be involved in the induction of TMD by certain phthalate esters were studied using transcriptional profiling techniques on RNA extracted from the testes of rats exposed in utero to L810TM or the positive controls. The potential of a structurally related substance, octyl-decyl trimellitate (L810TM) to induce testicular mal-development(TMD) has been studied in the rat and compared with the positive controls, di- (2 -ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2 -ethylhexyl) phthalate (MEHP). The effects of L810TM on the expression of genes in pathways involved in steroidogenesis and t

Additional information

Rats were exposed to L810TM in utero by the administration of daily oral gavage doses to pregnant dams between gestational day 12 and 19. The foetal testes were obtained by micro-dissection and prepared to facilitate the isolation of RNA, which was subsequently analysed using whole rat genome microarrays. The studies focussed on assessment of effects in pathways relevant to rat testicular mal-development (TMD). The effects of the two substances were compared with those of diethylhexyl phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP), an active metabolite of DEHP, both of which were used as a positive controls.

MEHP & DEHP (500 mg/Kg) caused a repression of genes in TMD pathways involved in cholesterol synthesis and transport (HMGCS, HMGCR, STAR, SCARB1, FDFT1, FDPS), Steroidogenesis (Cyp11a, HSD3B1, SC4MOL) and testes development (INSL3, INHA). L810TM did not cause significant repression of genes in TMD pathway.

In conclusion, it is highly unlikely that the substance will cause testicular mal-development in the rat under these treatment conditions.