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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:
key study
Study period:
27 Sep 2005 - 07 Oct 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Experimental Toxicology and Ecology BASF Aktiengesellschaft
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-Phenoxyethanol
- Physical state: Liquid/colorless
- Analytical purity: >99.9 core peak-area %
- Lot/batch No.: 41183068E0
- Expiration date of the lot/batch: 23 Jul 2006
- Stability under test conditions: The stability of the test substance under storage
conditions over the test period was guaranteed by
the sponsor.
- Storage condition of test material: Room temperature, under N2 conditions, exclusion
of light

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH [former: Charles River Laboratories, Germany]
- Age at study initiation: 11 to 13 weeks
- Weight at study initiation: 144.9 – 185.3 g
- Housing: Rats were housed singly from day 0-20 p.c. in type DK III stainless steel wire mesh cages.
- Diet (ad libitum): Ground Kliba maintenance diet mouse/rat "GLP"
- Water (ad libitum): tap water
- Acclimation period: 6 days


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


IN-LIFE DATES: 1st cohort From: 27 Sep 2005 To: 17 Oct 2005; 2nd cohort From: 28 Sep 2005 To: 18 Oct 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water and a few drops Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance emulsions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification. For the preparation of the emulsions, appropriate amounts of the test substance were weighed into calibrated beakers and were topped up with tap water and a few drops Tween 80 (as emulsifier). Afterwards they were intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the emulsions homogeneous during the procedure of dosing the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in demineralized water for a period of at least 96 hours at room temperature were carried out before the study was initiated (in a similar batch). Samples of the test substance emulsions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the first concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (100 and 1,000 mg/kg body weight/day). Three samples (one from the top, middle and bottom in each case) were taken for each of these
concentrations from the beaker with a magnetic stirrer running.
Details on mating procedure:
The animals were paired by the breeder and supplied on day 0 post coitum (= detection of vaginal plug / sperm).
Duration of treatment / exposure:
gestation day 6-19 (post mating)
Frequency of treatment:
once daily
Duration of test:
28 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Animals were checked from day 0 to 20 p.c. at least once daily for clinical symptoms whereas check for mortality was conducted twice a day on working days, and once a day during weekend or public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 post mating (p.c.).


FOOD CONSUMPTION was recorded on days 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c..


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

The scope of examinations exceeded the respective test guideline requirements. It was enhanced by clinical pathology examinations (hematology and clinical chemistry). For this purpose, blood samples were collected from the retroorbital venus plexus at study termination.
Hematology
The following parameters were determined in blood:
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobin
• mean corpuscular hemoglobin concentration
• platelets
• differential blood count
• reticulocytes

• prothrombin time (Hepato Quick's test)


Clinical chemistry
An automatic analyzer was used to examine following clinical chemical parameters:
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatase
• γ-glutamyltransferase
• sodium
• potassium
• chloride
• inorganic phosphate
• calcium
• urea
• creatinine
• glucose
• total bilirubin
• total protein
• albumin
• globulins
• triglycerides
• cholesterol
• magnesium


Ovaries and uterine content:
At study termination and following blood samples collection, the animals were sacrificed. The sacrificed animals as well as the one animal of the 1,000mg/kg bw/day group that was prematurely killed because of moribund state were subjected to necropsy and gross pathology, and the ovaries and uterus were removed for following examination:

Gravid uterine weight (excepted for the prematurely sacrificed animal)
Number of corpora lutea
Number of implantations (implantation sites, live/dead implantations)
The conception rates, as well as the pre- and post-implantation losses, were calculated.
Fetal examinations:
Litter size, number of dead fetuses, fetal weight, sex ratio, external alterations. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded.

Following sacrifice, 50% of the fetuses were prepared for the purpose of skeletal examination.

The remaining half of the sacrificed fetuses was prepared for the purpose of visceral examination.
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Pairwise comparison of each dose group with the control group using FISHER'S EXACT-test (one-sided) for the hypothesis of equal proportions.
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Non-parametric one-way analysis using KRUSKAL-WALLIS-test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Test group 3 (1,000 mg/kg b.w./day):
• one high dose female was sacrificed in moribund condition, preterminally, lateral position, unsteady gait, vaginal hemorrhage, salivation and respiratory sounds were observed
• all dams at least once showed unsteady gait (from day 6 p.c. onwards) and transient salivation (from day 8 p.c. onwards), although not all animals exhibited these findings on any one day, those findings persisted for a short time (up to 3.5 hours) after the dosing
• individual dams were found in lateral position shortly after treatment (5/25), had vaginal hemorrhage (2/25) and/or urine smeared fur (2/25) on several occasions during dosing
• mean food consumption was statistically significantly reduced (at maximum 10 % below control) on days 6 - 13 p.c., if calculated for the entire treatment period (days 6 - 19 p.c.), the food intake was about 6 % below control
• mean body weights were slightly, but statistically significantly below control on days 8 – 20 p.c., on day 20 p.c. mean body weight was about 4 % below control
• statistically significantly lower body weight gain was noted after initiation of treatment (days 6 - 8 p.c., about 44 % below control), average weight gain during entire treatment period (days 6 - 19 p.c.) was statistically significantly below control (about 12 %)
• net maternal body weight was about 4 % below control, net maternal body weight gain was about 14 % below control

No test substance-related adverse effects on dams in test group 1 (100 mg/kg bw/day) and test group 2 (300 mg/kg bw/day).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Gestational parameters:
At terminal sacrifice 22 - 25 females/group had implantation sites.
2-phenoxyethanol had no influence on gestational parameters up to and including the limit dose of 1,000 mg/kg bw/day.

Developmental toxicity:
2-phenoxyethanol induced no signs of developmental toxicity.

Fetal external, soft tissue and skeletal observations:
The external and skeletal malformations in one individual fetus from control and in 2 low dose group fetuses (0 and 100 mg/kg bw/day),


Fetal external, soft tissue and skeletal observations:
The external and skeletal malformations in one individual fetus from control and in 2 low dose group fetuses (0 and 100 mg/kg bw/day), which were related to the head/skull and sternebra, are considered to be spontaneous findings.
No soft tissue malformations were recorded at all. If all the different types of malformations are summarized, in total one out of 210 examined control fetuses [= 0.5 %] in one out of 25 litters [= 4.0 %], 2 out of 206 low dose fetuses [= 1.0 %] in 2 out of 25 litters [= 8.0 %], none of the 204 mid dose fetuses (from 23 litters) and none of the 180 high dose fetuses (from 22 litters) showed malformations.
The mean percentages of affected fetuses/litter with total malformations amounted to 0.4, 1.0, 0.0, and 0.0 % at 0, 100, 300 or 1,000 mg/kg bw/day respectively. These low, not dose-related incidences do not suggest any relationship to the test substance.
External variations did not occur in any of the fetuses in this study. Soft tissue variations, in the form of dilated renal pelvis or short innominate occurred in all test groups including the controls without any relation to dosing. The skeletal variations consisted primarily of small disturbances or transient delays of ossification. Most of the skeletal variations were equally distributed among the different test groups including controls, if normal biological variation is taken into account. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a relation to dosing, were not considered to be of any toxicological relevance and/or can be found at a comparable frequency in the historical control data. If all variations are summarized, in total 119 of the 210 examined control fetuses [= 57 %] in all 25 litters [= 100 %], 107 of the 206examined low dose fetuses [= 52 %] in all 25 litters [= 100 %], 110 out of 204 mid dose fetuses [= 54 %] in all 23 litters [= 100 %] and 104 out of 180 high dose fetuses [= 58 %] in all 22 litters [= 100 %] showed variations. The mean percentages of affected fetuses/litter with total variations amounted to 57.2, 52.3, 54.0, and 58.1 % at 0; 100; 300 or 1,000 mg/kg bw/day, respectively. These overall incidences do not suggest a treatment-relationship, but reflect the usual biological variation inherent in the strain of rats used for this experiment. A spontaneous origin is assumed for the unclassified external and the unclassified cartilage observation, that were recorded for very few fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 or 1,000 mg/kg bw/day). Distribution and type of these findings (i.e. blood coagulum around placenta, bipartite processus xiphoideus, notched cartilage between basisphenoid and basioccipital and notched manubrium) do not suggest any relation to treatment. Thus, the oral administration of phenoxyethanol to pregnant Wistar rats had no effect on development and morphology of offspring at any of the dose levels tested (100, 300 and 1,000 mg/kg bw/day).

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Hematology and serum enzyme examinations revealed no treatment-related changes. Clinical chemistry investigations showed decreased concentrations in total bilirubin, total protein, albumin and globulin as well as increased triglyceride values in the serum of the high dose animals (1,000 mg/kg bw/day). Similar effects on clinical chemistry parameters, with somewhat lower severity, were noted in the mid dose animals (300 mg/kg bw/day). All of these findings were rather related to adaptive metabolic changes, than indicative of particular organ toxicity. Although these effects were likely to be caused by the test material, they were not considered as adverse per se.

Applicant's summary and conclusion