Bis(2-ethylhexyl) phthalate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1986 - May 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study realised according to the OECD guideline 412, with acceptable restrictions.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Di(2-ethylhexyl) phthalate (DEHP): A short-term repeated inhalation toxicity study including fertility assessment.

Author:

Klimisch HJ, Gamer AO, Hellwig J

Year:

1992

Bibliographic source:

Fd. Chem. Toxic. 30, 915-919

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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:

DEHP with a purity of 99.7%, determined by gas chromatography

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF Wistar/Chbb: Thom rats (Dr Thomae GmbH, Biberach, Germany)
- Age at study initiation: 9 wk old
- Weight at study initiation: 226 g (male) and 155 g (female)
- Fasting period before study: no
- Housing: maintained in individual Makrolon/wire cages in air-conditioned rooms
- Diet (e.g. ad libitum): ad libitum (Kliba 24-343-4 pellets)
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 1wk


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Head-nose exposure systems, each with a volume of 90 l and providing for the simultaneous exposure of 60 animals (INA 60, glass-steel construction, BASF AG) were used. The animals were restrained in glass tubes with their snouts projecting into the inhalation system. An aerosol was generated for each group with a two-component atomizer (Schlick 970, Schlick Coburg, Germany) supplied with the test substance by a metering pump (INFU 362, Indigel, Switzerland) and 1500 l/hr compressed air. After passing through a droplet separator, the aerosol was mixed with humidified supply air to form the final inhalation atmosphere that was fed into the inhalation system. The exhaust air system connected to the inhalation system was set to a slightly lower exhaust flow, compared with the supply flow, to ensure a positive pressure in the system and to avoid dilution of the aerosol in the animals' breathing zones. The air exchange rate was about 36-40 times per hr in the treated groups and 58 times per hr in the air control. The air-flows, relative humidity and temperature in the inhalation system were measured and recorded several times during each exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Hourly samples of the inhalation atmospheres were drawn from the breathing zone of the animals and absorbed in xylene through a glass tube filled with quartz wool and two fritted glass flasks. The samples were analyzed by gas chromatography .
Particle size measurement was performed with a cascade impactor (Anderson Stack Sampler Mark III) once during the study for each concentration. The deposits on the individual metal collecting disks were analysed by gas chromatography and the particle size distribution was calculated.

Duration of treatment / exposure:

6h

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1.0, 0.05 and 0.01 mg/l
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
230, 11 and 2.3 mg/kg for males and 360, 18 and 3.6 mg/kg
Basis:
other: estimated daily intake

No. of animals per sex per dose:

main groups (10 males and 10 females),
satellite group I (2 males and 2 females) for the investigations of peroxisome prolifération, fertility (males) and reversibility of effects.
satellite group II (15 males and 2-5 females) for evaluation of male fertility

Control animals:

yes, sham-exposed

Details on study design:

The animals of the main group and satellite group I were killed immediately after the exposure period, the former for haematology, clinical chemistry and pathology, the latter for a special examination of liver pathology.
Each male animal of satellite group II (15 per group) was mated overnight 2 and 6 wk after the end of exposure, with two non-treated mature female virgin rats of the same strain, until the vaginal smear was sperm positive; the maximum mating interval was 10 days. Two mating periods were chosen bearing in mind the duration of the spermatogenic cycle in the rat. 8 wk after the end of exposure all the animals of satellite group II (15 males and five females in groups 0 and 3, and 15 males and 2 females in groups 1 and 2) were killed and examined as detailed above to check for the reversibility or progression of any treatment effects.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before during and after exposure daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- When / How many animals: in all of the animals from the main groups, directly after the end of the exposure period and from five animals of each sex, in the control and high-dose groups of satellite group II, at the end of the 8-wk post-exposure period

CLINICAL CHEMISTRY: Yes / No / No data
- Animals fasted: No data
- When / How many animals: in all of the animals from the main groups, directly after the end of the exposure period and from five animals of each sex, in the control and high-dose groups of satellite group II, at the end of the 8-wk post-exposure period

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Routine macroscopic and microscopic pathological examinations of all animals of the main groups and some of the satellite animals was carried out. Body, liver, kidney, testes, adrenal, heart, lung and brain weights were determined. Light microscopic examinations of the lung, trachea, nasal cavity, liner, kidney, spleen, adrenals, heart, brain, spinal cord, sciatic nerve, gastrocnemius muscle, testes, epididymis, prostate, seminal vesicle and all organs with gross findings, were carried out, mainly on slides stained with haemotoxylin and eosin. on all animals of groups 0 and 3 in the main group. In the other groups only selected organs were investigated. Toluidine blueisafranin-stained semi-thin sections were prepared from Epon-embedded liver samples of perfusion-fixed animals (two male and two female animals per group) for detailed light microscopic examination and ultrathin sections were prepared for electron microscopy.

Other examinations:

Fertility
The interval from the beginning of the mating period to the first sperm-positive vaginal smear was recorded. Female animals were killed 14 days after either the detection of sperm or the last day of the mating period. The corpora lutea were counted and the uterine contents were examined for live or dead implantations and resorptions. The male and female fertility indices as well as pre- and post-implantation losses were calculated.

Statistics:

Body weights, and haematological and clinicochemical parameters were evaluated by analysis of variance (ANOVA) followed by Dunnett's test. For post-mortem body- and organ weight analysis a Williams t-test was used. The fertility assessment data were analysed wither by Fisher's exact test or by the Krauth test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities or clinical symptoms were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight changes (growth) were similar in all groups.

HAEMATOLOGY
No significant changes could be detected in the following parameters: a number of red and white blood tells, platelets and reticulocytes, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and differential blood count. The thromboplastin time was not affected.

CLINICAL CHEMISTRY
No elevation in plasma concentrations of the enzymes, alanine aminotransferase, aspartate aminci-transférase or alkaline phosphatase, occurred. There were no significant changes in the plasma concentrations of sodium, potassium, calcium, chloride, urea, creatinine. glucose, total bilirubin, triglycerides, cholesterol, total protein and globuline. An increase in plasma albumin concentration in the animals of the highest concentration group was observed, and this was reversed in male but not female animals during the post-exposure period. Additionally, the inorganic phosphate was increased in the male rats at this concentration.

ORGAN WEIGHTS
The absolute liver weight of female animals and the relative liver weights of male and female animals of the highest concentration group were elevated at the end of the exposure period. Furthermore, the relative lung weight of male animals in this group was significantly increased. These findings were reversed during the post-exposure period (satellite groups).

GROSS PATHOLOGY
No substance-related gross lesions could be detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
A few sporadic changes that were not attributable to the treatment were seen in the trachea, urinary tract, male reproductive organs and spleen. Semiquantitative grading of foam-cell content and alveolar septal thickening in the lungs revealed a slight increase in these findings in the main group at the highest concentration, which correlated well with the increased organ weight and was reversed within the post-exposure period. No light-microscopic changes (in routine histology and semi-thin sections), corresponding to the increased liver weights at the highest concentration were observed. No testicular toxicity was detected.
No substructural changes attributable to the treatment were seen by electron microscopy; in particular, no prolifération of peroxisomes occurred.

OTHER FINDINGS: FERTILITY:
There were no indications of substance-related effects on male reproductive function.

Effect levels

Dose descriptor:

NOAEC

Effect level:

50 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The analytical concentrations matched well with the target concentrations. The mass median aerodynamic diameter of 1.2µm or below insured a readily inhaled aerosol.

Applicant's summary and conclusion

Executive summary:

In a repeated dose toxicity study realised according to the OECD guideline 412, Wistar rats (10 males and females per group in the main dose group; 2 males and females per group in satellite group I, 15 males and 2-5 females per group in satellite group II; an equal number of control rats in each group; 9 weeks old at the beginning of exposure) were exposed in head-nose inhalation systems to DEHP (99.7% pure) aerosols of respirable particle size (mass median aerodynamic diameter 1.2 ± 2.9-9.5 μm) or air (control). Exposure duration was 6 hours per day, 5 days per week for 4 weeks at 0, 0.01, 0.05, or 1.0 mg/litre (0, 10, 50, or 1,000 mg/m3). The animals of the main dose group were sacrificed at the end of the exposure period. Before sacrifice, male rats from satellite group II had a recovery period of 2 or 6 weeks after termination of exposure. Livers of animals from satellite groups I and II were examined by light and electron microscopy. No animals died during the study. Clinical examination and blood chemistry parameters did not reveal treatment-related effects. Body weights of treated rats and controls were similar. In the highest dose group, a significant increase in relative lung weights was seen in male rats. This was accompanied by foam cell proliferation and thickening of the alveolar septi. Absolute liver weights (females) and relative liver weights (both sexes) were slightly but significantly increased but there were no corresponding histological findings. All these effects were reversible within the post-exposure observation period. No testicular toxicity was detected histologically. Electron microscopical examination of liver samples from all three concentration groups and controls at the end of exposure and after the post-exposure period did not reveal clear ultrastructural changes in hepatocytes that could be attributed to the exposure or to peroxisome proliferation.

The NOAEL in this study is 50 mg/m3.