Bis(2-ethylhexyl) phthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study OECD 414

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Developmental toxicity in rats after inhalation exposure of di(2-ethylhexyl) phthalate (DEHP)

Author:

Merkle J, Klimisch HJ, Jäckh R

Year:

1988

Bibliographic source:

Toxicol. Lett. 42, 215-223

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phthalate

EC Number:

204-211-0

EC Name:

Bis(2-ethylhexyl) phthalate

Cas Number:

117-81-7

Molecular formula:

C24H38O4

IUPAC Name:

1,2-bis(2-ethylhexyl) benzene-1,2-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): DEHP
- Analytical purity: 99%
- Lot/batch No.: no data
- Stability under test conditions: stability of the aerosol verified throughout the testing period
- Source: BASF

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Virgin rats, SPF-Wistar /Chbb: Thom (Dr K. Thomae, Biberach, F.R.G.)
- Age at study initiation: 71-73 days old
- Weight at study initiation: 218+/-3.1 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum commercial food pellets (Kliba, Klingentalmühle, Kaiseraugst, F.R.G)
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 to 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: atomizer
- Method of holding animals in test chamber: slightly restrained in glass tubes fixed to a cylindrical inhalation chamber (50 L)
- System of generating particulates/aerosols: a polydisperse aerosol was generated with clean oil-free pressurized air. The ultrafine particles of the aerosol were separated by a glass cyclone and led to the inhalation system.
- Temperature : 22.1-22.5°C
- Humidity: 42.3-51.5%
- Air flow rate: 3.5m3/h
- Method of particle size determination: The particle size of the aerosol was determined with a cascade impactor (Stack Sampler Mark III, Andersen). The amounts impacted on seven different stages were determined by gas chromatography. From the equivalent aerodynamic diameter (MMAD) were calculated.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (see below)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the inhalation atmospheres were collected from the breathing zone of the animals wit ha sample velocity of 1.25m/s. The aerosol was impacted on quartz wool filled in a sample probe.. The DEHP vapor, passing through the quartz wool, was abserbed in two-glass freitted flasks, filled with xylene. The second glass-fritted flask separately to control the effectiveness of the sampling method. The quartz wool was eluted with xylene, mixed with the xylene solution of the first flask and analysed by gas chromatography (glass column 2m, 2mm diameter, 10 % UCW982 on 800/100 mesh Chromosorb W, AW-DMCS, furnace temperature 240°C, FID injection volume 1µl).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: until gestation or for a 2-week period
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily

Duration of test:

from GD 6 to GD 15

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.01, 0.05, 0.3 mg/l
Basis:
nominal conc.

No. of animals per sex per dose:

25 Males & 25 Females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The mated rats were sham-exposed in the inhalation apparatus to air without DEHP over 6 hours per day from D0 up to D6 post coitum.
At the end of gestation (D20 pc):
- 20/25 rats were subjected to caesarian section.
- 5/25 were allowed to litter and rear the pups until weaning (Day 21 after birth). During this period, physical development was assessed:
survival rate,
viability,
lactation indices,
righting test (D6 pp),
gripping reflex (D13+/-1 pp),
pupillar reflex (D20+/-1 pp),
hearing test (D21+/-1 pp)
eye/ear auricle,
incisivi,
fur development
body weight gain
skeletons were X-rayed and evaluated.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: days 3, 6, 9, 12, 15, 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Details: gross pathological investigations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
. number and distribution of dead and live fetuses,
. number and distribution of uterine scars (uterine scar: uterine implantation without implant)
. dead fetus: non live fetus with discernible digits.
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yesa
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- Body weight
- Sex
- External examinations: Yes: 1/3 per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 1/3 per litter

Statistics:

Williams test, Krauth test, Fisher test

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Conception rate was 90% in the control group and 95%, 85% and 80 % in groups treated at 0.01, 0.05 and 0.3 mg/ml, respectively.
No differences in body weight or body weight gain among dose groups and control group were recorded. During the whole experimental period the animals showed normal behaviour. Cage side observations and post-mortem macroscopic examination gave no indications for exposure-related effects. Only in the 0.3mg/l exposure group the dams showed a day 21 after littering a significantly lower body weight.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

0.3 mg/L air (nominal)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Early resorptions were seen throughout all groups, but without a dose-related response. One animals in the 0.05 mg/l group showed an early Salewsky resorption and produced no litter.
The number of live fetuses/dam was slightly, but significantly decreased in the 0.05 mg/l group and the percentage of resorptions/dam (mostly late resorptions) was elevated. Also, dead fetuses were observed in this group. These effects, however, are not regarded as exposure related, since there was no dose dependency. Placental weights showed no differences among the groups.
The percentage of anormal fetuses per litter showed no dose dependency; the percentage of litters with abnormal fetuses increased slightly with the exposure level.
At cesarean section, one fetuse in one litter of the 0.05 mg/l group (0.69% of the fetuses investigated per litter; 6.25% of litters investigated) showed abnormalities. No further anomalies, variations or retardations were seen at cesarean section in any experimental group.

Upon evisceration one fetus in the 0.05 mg/l group showed several anomalies. No further anomalies or variations were found at this stage of investigation. Some fetuses showed retardations (pelvis dilatation) without a dose-related trend.
The examination by microdissection revealed anomalies in the 0.3 mg/l (unilateral hydro-ureter) in three fetuses (4.69% of foetuses of one litter investigated per litter; 6.25% of litters investigated). This effect might be related to pelvis dilatation. No further anomalies or variations were detected.
Skeletal examination showed anomalies, variations, and retardation in all experimental groups and an increase at the highest exposure level. One fetus of the 0.05 mg/l group, which had lso exhibited several gross malformations, also has major skeletal anomalies. The other anomalies were mostly dislocated sternebrae ossification centres (ventral segments of the ribs asymmetrically fused with sternum). These anomalies occurred in a low incidence in all groups and are observed in similar and sometimes even higher incidences among the historical controls.
Number and kind of variations revealed no dose-dependency.
A slight increase in retardations in the fetuses of the highest dose group (0.3 mg/l) was recorded. These were mostly renal pelvis dilatations. This finding is not regarded as exposure-related since it is very common in this strain of rats and also observed in a very high incidence in historical controls.

In the post-exposure and lactation period, no differences in dam or offspring development were recorded. In the 0.3 mg/l group the body weight of the dams at day 21 pp was significantly decreased. Survival rats up to day 21 pp (100 % in the dose groups, 92 % in the controls), viability and lactations indices, development of auricles, incisive and eyes, righting and hearing test and gripping reflex were unaffected.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

0.3 mg/L air (nominal)

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 0.3 mg/l for maternal toxicity.
A NOAEL has been determined at 0.3 mg/l for foetal toxicity.

Executive summary:

The potential of DEHP to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to OECD Guideline N° 414 and in compliance with Good Laboratory Practices.

DEHP was administered by aerosol inhalation (nose only) to four groups of 25 bred female Wistar rats, 6 hours daily from gestation days 6 through 15. Dosage levels were 0, 0.01, 0.05 and 0.3 mg/l.

Animals were observed daily for mortality and morbidity. Clinical observations and body weights were recorded at appropriate intervals.

On gestation day 20, a laparohysterectomy was performed on 20/25 female. The uteri, placenta and ovaries were examined, and the number of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variations.

5/25 females were allowed to litter and rear the pups until weaning (Day 21 after birth). During this period, physical development was assessed: survival rate, viability, lactation indices, righting test (D6 pp), gripping reflex (D13+/-1 pp), pupillar reflex (D20+/-1 pp), hearing test (D21+/-1 pp) eye/ear auricle, incisivi, fur development body weight gain skeletons were X-rayed and evaluated.

All animals survived to the scheduled necropsy; No maternal toxicity has been observed. No clinical signs that could be attributed to DEHP were observed in the treated groups. Intrauterine growth and survival were unaffected by DEHP administration at all dose levels. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related.

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 0.3 mg/l for maternal toxicity. A NOAEL has been determined at 0.3 mg/l for foetal toxicity.