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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Please refer to Section 13.2 for full read-across justification

Data source

Reference
Reference Type:
publication
Title:
Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat
Author:
Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DNR & Parks L
Year:
2000
Bibliographic source:
Toxicol Sci. 58(2):350-65

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Assessment of the sexual development of male rat offspring following perinatal exposure
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
Dioctylterephthalate (DOTP / DEHT)
IUPAC Name:
Dioctylterephthalate (DOTP / DEHT)
Constituent 2
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) terephthalate
EC Number:
229-176-9
EC Name:
Bis(2-ethylhexyl) terephthalate
Cas Number:
6422-86-2
Molecular formula:
C24H38O4
IUPAC Name:
bis(2-ethylhexyl) terephthalate
Constituent 3
Reference substance name:
Bis(2-ethylhexyl) benzene-1,4-dicarboxylate
IUPAC Name:
Bis(2-ethylhexyl) benzene-1,4-dicarboxylate
Details on test material:
98% purity; obtained from Aldrich

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River, NC, USA- Age at study initiation: 90 days- Weight at study initiation: Not stated- Fasting period before study: Not stated- Housing: Individual, in polycarbonate cages with pine shavings (maternal)- Diet: Purina Rat Chow (5001), ad libitum- Water: Filtered Tap Water, ad libitum- Acclimation period: Time-mated rats were received at GD 2ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24- Humidity (%): 40-50- Air changes (per hr): Not reported- Photoperiod (hrs dark / hrs light): 14/10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Time-mated female rats were gavaged with the phthalate esters di(2-ethylhexyl) phthalate (DEHP), benzyl butyl phthalate (BBP), diisononyl phthalate (DINP), diethyl phthalate (DEP), dimethyl phthalate (DMP) or dioctyl terephthalate (DOTP / DEHT) were administered by oral gavage to maternal female rats at a dose level of 750 mg/kg bw from Gestation Day 14 to Postnatal Day 3. 
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
90-day old nulliparous females were time-mated overnight (mating confirmed by sperm-positive smear)
Duration of treatment / exposure:
Gestation Day 14 to Postnatal Day 3.
Frequency of treatment:
Daily
Duration of test:
Male offspring were necropsied at 3-5 or 4-7 months of age
Doses / concentrations
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
8 (DOTP)19 (control)
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Signs of toxicity, bodyweights.
Ovaries and uterine content:
Not applicable - dams were allowed to litter normally
Fetal examinations:
Not applicable - dams were allowed to litter normally

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with DEHP and DNIP resulted in slight reductions in weight gain; mean maternal bodyweights were not significantly different to controls. Weight gain to PND3 was significantly reduced in DEHP-treated females.

Maternal developmental toxicity

Number of abortions:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yesDetails on maternal toxic effects:There were no treatment-related deaths or overt signs of toxicity. Treatment with DEHP and DNIP resulted in slight reductions in weight gain; mean maternal bodyweights were not significantly different to controls. Weight gain to PND3 was significantly reduced in DEHP-treated females. All dams were pregnant and delivered live offspring.

Results (fetuses)

Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The litter from one dam treated with BBP did not survive to PND 2; a further BBP-treated dam did not have any male pups at weaning.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Treatment with DINP, DEHP and BBP was shown to induce malformations in male offspring (84%, 92% and 7% incidence, respectively). Findings included retained nipple, cleft phallus, undescended testes, hypospadias and vaginal pouch; affected offspring often showed multiple malformations. Some males treated with DEHP or BBP showed complete agenesis of the prostate, seminal vesicles, bulbourethral and/or coagulating glands
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Anogenital distance in male pups was significantly reduced in BBP- and DEHP-treated groups. The reduction in AGD (~30%) was associated with reduced pup weight (~15%); anogenital index was not assessed. Male offspring from these groups also showed a significant (~35%) reduction in testis weight. At PND 8-9, males from the DEHP-treated group showed an increased incidence of haemorrhagic testes; a similar finding was seen in one male from the DINP-treated group. Histopathology of the testes from male offspring in the DEHP-treated group showed focal interstitial haemorrhage, granulomas (associated with focal haemorrhage) or infarcted areas (associated with more widespread haemorrhage and coagulative necrosis. Treatment with BBP, DEHP and DINP significantly induced areolae in male offspring.
Males from the DINP-, DEHP- and BBP-treated groups showed small testes, unilateral or bilateral testicular agenesis. Testes were flaccid, fluid-filled and devoid of sperm. Weights of the reproductive organs/tissues were significantly reduced in males from the DEHP- and BBP-treated groups.
Reduced mating behaviour was seen in DEHP-treated rats.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

The litter from one dam treated with BBP did not survive to PND 2; a further BBP-treated dam did not have any male pups at weaning.

Litter size was comparable in all groups. Pup weights were significantly lower in the DEHP- and BBP-treated groups.

Anogenital distance in male pups was significantly reduced in BBP- and DEHP-treated groups. The reduction in AGD (~30%) was associated with reduced pup weight (~15%); anogenital index was not assessed. Male offspring from these groups also showed a significant (~35%) reduction in testis weight. At PND 8-9, males from the DEHP-treated group showed an increased incidence of haemorrhagic testes; a similar finding was seen in one male from the DINP-treated group. Histopathology of the testes from male offspring in the DEHP-treated group showed focal interstitial haemorrhage, granulomas (associated with focal haemorrhage) or infarcted areas (associated with more widespread haemorrhage and coagulative necrosis. Treatment with BBP, DEHP and DINP significantly induced areolae in male offspring. Mean age at puberty (preputial separation) was not affected by treatment in any group, with the exception of extremely malformed males in the BBP- and DEHP-treated groups. Treatment with DINP, DEHP and BBP was shown to induce malformations in male offspring (84%, 92% and 7% incidence, respectively). Findings included retained nipple, cleft phallus, undescended testes, hypospadias and vaginal pouch; affected offspring often showed multiple malformations. Some males treated with DEHP or BBP showed complete agenesis of the prostate, seminal vesicles, bulbourethral and/or coagulating glands. Males from the DINP-, DEHP- and BBP-treated groups showed small testes, unilateral or bilateral testicular agenesis. Testes were flaccid, fluid-filled and devoid of sperm. Weights of the reproductive organs/tissues were significantly reduced in males from the DEHP- and BBP-treated groups; bodyweights and non-reproductive organ weights were not affected by treatment. Reduced mating behaviour was seen in DEHP-treated rats. 

Applicant's summary and conclusion

Conclusions:
As dioctyl terephthalate (DOTP / DEHT) is shown to be rapidly hydrolysed to terephthalic acid, it can also be concluded that terephthalic acid will similarly not affect the sexual differentiation of male rats.
Executive summary:

In this study, the authors examined a number of phthalate esters to determine any effects on the sexual differentiation of male rat offspring due to anti-androgenic properties. The substances di(2-ethylhexyl) phthalate (DEHP), benzyl butyl phthalate (BBP), diisononyl phthalate (DINP), diethyl phthalate (DEP), dimethyl phthalate (DMP) or dioctyl terephthalate (DOTP / DEHT) were administered by oral gavage to maternal female rats at a dose level of 750 mg/kg bw from Gestation Day 14 to Postnatal Day 3. None of the substances tested caused overt maternal toxicity or reduced litter size. Dams administered DEHP showed reduced maternal weight gain over the dosing period by about 15 g; DEHP and DINP reduced gestation weight gain by 24 g and 14 g, respectively. Treatment with DEHP and BBP treatments reduced pup weight at birth (15%). Male pups from dams treated with DEHP and BBP showed reduced (~30%) anogenital distance and reduced (~35%) testis weight. A high proportion of male offspring of dams administered DEHP, BBP and DINP displayed feminine areolas/nipples (87%, 70% and 22%, respectively). A significant incidence of reproductive malformations was also apparent in male offspring of these groups (82%, 84% and 7.7%, respectively. Perinatal maternal exposure to the phthalate esters DEHP, BBP and DINP was therefore shown to alter the sexual differentiation of male offspring, suggesting an anti-androgenic mode of action. Similar effects were not seen with the phthalate esters DOTP, DEP and DMP. DEHP and BBP were shown to be of approximately equivalent potency; DINP exhibited potency an order of magnitude lower.

As dioctyl terephthalate (DOTP / DEHT) is shown to be rapidly hydrolysed to terephthalic acid, it can also be concluded that terephthalic acid will similarly not affect the sexual differentiation of male rats.