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Toxicity to reproduction: other studies

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells
Author:
Daniel J. Spade, Cathy Yue Bai, Christy Lambright, Justin M. Conley, Kim Boekelheide, L. Earl Gray
Year:
2018
Bibliographic source:
Toxicology Letters 290 (2018) 55–61

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats. The anti-androgenic effects of phthalates have been more thoroughly quantified than testicular pathology due to the significant time requirement associated with manual counting of MNGs on histological sections. An automated counting method was developed in ImageJ to quantify MNGs in digital images of hematoxylin-stained rat fetal testis tissue sections. Timed pregnant Sprague Dawley rats were exposed by daily oral gavage from gestation day 17 to 21 with one of eight phthalate test compounds or corn oil vehicle.
GLP compliance:
not specified
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl phthalate
EC Number:
205-011-6
EC Name:
Dimethyl phthalate
Cas Number:
131-11-3
Molecular formula:
C10H10O4
IUPAC Name:
1,2-dimethyl benzene-1,2-dicarboxylate
Test material form:
liquid
Specific details on test material used for the study:
Gifted from the National Toxicology Program (Research Triangle Park, NC) and independently verified at 100% purity by Research Triangle Institute (Durham, NC) using gas chromatography with flame ionization detection.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Timed pregnant rats were exposed to one of the eight treatment compounds, or corn oil (Sigma-Aldrich; St. Louis, MO) vehicle in a 2.5 mL/kg body weight dosing solution by daily oral gavage from GD 17–21.
Duration of treatment / exposure:
GD 17-21
Frequency of treatment:
once daily
Doses / concentrations
Dose / conc.:
900 mg/kg bw/day (nominal)
No. of animals per sex per dose:
2 for DMP and 6 for vehicle

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (nominal)
Remarks on result:
not determinable due to absence of adverse toxic effects

Any other information on results incl. tables

Following phthalate exposure from GD 17–21, no significant differences in litter size were observed between any phthalate treatment group and control, and no fetal losses or resorptions were noted in any group. Maternal weight at termination of the study was not reduced in any group, but maternal weight gain was reduced by an average of 35.8 g in DPeP exposure. MNGs were clearly visible in H&E-stained sections of testes in the DBP, BBP, DPeP, and DEHP exposure groups. Samples treated with vehicle control, DMP, DEP, TBPH, and DOTP were largely devoid of MNGs. The induction of MNGs in DBP, BBP, DPeP, and DEHP-treated testes was statistically significant, regardless of whether the MNG rate was quantified as the number of MNGs per unit testis area, MNGs per unit seminiferous cord area, percent of cord cross sections with MNGs, or average number of MNGs per cord cross section. The background rate of MNGs in vehicle control samples and negative control phthalate samples (DMP, DEP, TBPH, and DOTP) was 1.91, 2.35, 3.35, 3.88, and 0.43 MNGs/mm2 testis, respectively, compared to 60.01, 64.13, 108.86, and 75.50 MNGs/mm2 testis for DBP, BBP, DPeP, and DEHP, respectively. There were no significant differences in MNG rate between vehicle controls and negative control  phthalates. There were no significant differences in the proportional seminiferous cord area in testis sections, and all metrics for MNG rate were strongly correlated.



Automated scoring of MNGs
The automated scoring procedure identified the same four phthalate treatment groups, BBP, DBP, DEHP, and DPeP, as significantly inducing
MNGs relative to vehicle-treated control. Manually counted MNGs correlated strongly with the counts derived from the automated image analysis (Pearson r=0.943).


Testosterone production
The four phthalate treatment groups that significantly induced MNGs (BBP, DBP, DPeP, and DEHP), also significantly decreased ex vivo testosterone production, by 69.2%, 75.4%, 74.9%, and 62.4%, respectively, relative to vehicle control. DOTP exposure also unexpectedly resulted in a statistically significant 30.5% reduction in ex vivo testosterone production. DMP did not decrease testosterone production.

Applicant's summary and conclusion

Executive summary:

In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats. The anti-androgenic effects of phthalates have been more thoroughly quantified than testicular pathology due to the significant time requirement associated with manual counting of MNGs on histological sections. An automated counting method was developed in ImageJ to quantify MNGs in digital images of hematoxylin-stained rat fetal testis tissue sections. Timed pregnant Sprague Dawley rats were exposed by daily oral gavage from gestation day 17 to 21 with one of eight phthalate test compounds or corn oil vehicle. Both the manual counting method and the automated image analysis method identified di-nbutyl phthalate, butyl benzyl phthalate, dipentyl phthalate, and di-(2-ethylhexyl) phthalate as positive for induction of MNGs. Dimethyl phthalate, diethyl phthalate, the brominated phthalate di-(2-ethylhexyl) tetrabromophthalate, and dioctyl terephthalate were negative. The correlation between automated and manual scoring metrics was high (r=0.923). Results of MNG analysis were consistent with these  compounds’ antiandrogenic activities, which were confirmed in an ex vivo testosterone production assay.