2-(2-ethoxyethoxy)ethanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2001 to June 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted under a guideline equivalent protocol

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ICH S5(R2) Detection of toxicity to reproduction for medicinal products & toxicity to male fertility

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethanol

EC Number:

203-919-7

EC Name:

2-(2-ethoxyethoxy)ethanol

Cas Number:

111-90-0

Molecular formula:

C6H14O3

IUPAC Name:

2-(2-ethoxyethoxy)ethan-1-ol

Details on test material:

- Name of test material (as cited in study report):Transcutol HP; Diethylene glycol mono ethyl ether
- Physical state:Clear colourless liquid
- Analytical purity:99.98%
- Lot/batch No.:D 4089
- Storage condition of test material:at approximately +4C, protected from light and under nitrogen.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Iffa Credo, Bp 0109, 69592 l'Arbresle Cedex, France
- Age at study initiation:10-13 weeks old
- Weight at study initiation:199.4-264.5g
- Fasting period before study:none
- Housing:individually hosed in plastic cages with autoclaved sawdust as bedding
- Diet (e.g. ad libitum):ad libitum, pelleted commercial complete rodent diet (Diet reference A04 C-10)
- Water (e.g. ad libitum):ad libitum, mains
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- Humidity (%):55+/-15
- Air changes (per hr):minimum 15
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: October 8, 2001 To: October 25, 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test article was prepared daily as a solution in the vehicle at the concentrations of 150 and 500mg/ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 5 ml samples were taken from the formulations (including control) on the first day of treatment and on one day during the last week of treatment. One sample of each formulation was immediately analysed by gas chromatography and the second samples were kept frozen in the event of re-analysis.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant. Animals were received at the testing facility on day 0 of gestation.

Duration of treatment / exposure:

From day 6 to day 17 of gestation inclusive

Frequency of treatment:

Once daily

Duration of test:

Animals were killed on day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were based on the results of another study (number 935/107)
- Rationale for animal assignment (if not random):random
- Other: Volume of administration: 4ml/kg/day (control and high dose groups) or 2 mg/kg/day (low and intermediate dose groups).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily at the beginning and at the end of each working day (including weekends) to detect any which were dead or moribund.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily. During the treatment period, the animals were observed once before and at least once after the treatment to detect any abnormalities in appearance, behaviour or other signs of reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were individualy weighed on days 0, 6, 11, 15, 18 and 20 of gestation.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Yes. Animals were dissected and examined for macroscopic pathological changes in maternal organs. Any abnormalities observed were recorded and preserved in 10% neutral formalin for possible histopathological examinations. No further examinations were performed.

OTHER:

Ovaries and uterine content:

For each female, the ovaries and uterus were removed and examined, including examination of the placentae. Uterus and ovaries were not weighed.
The following data were recorded:

pregnancy status
number of corpora lutea
number and distribution of live fetuses
number and distribution of embryonic/fetal deaths
individual fetal weights
fetal sex

The embryonic/fetal deaths were classified as:
early: only placenta visible at termination
late: both placenta and embryonic tissue visible at termination

The uterus of all females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter. Following maceration of the soft tissues with aqueuous potassium hydroxide, staining of the skeleton with Alizarin red then passage into glycerol
- Head examinations: Yes
The remaining fetuses were preserved in Harrisson's fluid for fixed visceral examination by the modified Wilson-Barrow technique. Fixed-fetal examinations were performed under low power magnification.

Statistics:

Data was checked for homogeneity of variance across groups using Bartlett's test. Homogenous data were then analysed by parametric methods, i.e. ANOVA followed by Dunnett's test if ANOVA was significant. Non-homogenous data, were analysed by non-parametric methods, i.e. Kruskal-Wallis test followed by Dunn's test if the Kruskal-Wallis was significant. The numbers of resorptions and all litter-based percentages were analysed using the above non-parametric methods.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher's two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Indices:

Pre-implantation loss (in %) = [(Number of corpora lutea - Number of implantations)/Number of corpora lutea]x100
Post-implantation loss (in %) = [(Number of implantations - Number of viable fetuses)/Number of implantations]x100

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no unscheduled deaths throughout the study. There were no treatment-related clinical signs in any group during the study. The body weight gain and mean food consumption during the first five days of treatment (days 6 to 11 of gestation) was statistically significantly lower in comparison with the control group. There were no adverse effect of treatment on mean body weight or mean food consumption for the other treated groups. No treatment-related lesions were found at necropsy examination of the dams.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were at least 24 pregnant females at term in all groups. One dam given 1000mg/kg/day had a single early resorption and no live fetuses. The mean number of uterine implantation as slightly lower in the 300mg/kg/day group due to an incidental increase in pre-implantation loss. Pre-implantation data were comparable in the other treated and control groups. There were no adverse influences of treatment on embryo-fetal survival. Mean fetal weights and sex ratio were comparable in all groups. There were no fetal malformations in any group. There was, however, a dose-related increase in the incidence of fetuses with reduced ossification, principally of the cranial bones, in the 1000 and 2000mg/kg/day groups. None of these reached statistical significance however until the 2000mg/kg dose group.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Percentage fetal incidence of selected skeletal abnormalities of potential note.

	Historical data	Control	300mg/kg	1000mg/kg	2000mg/kg
Incomplete ossification of:
Cranium – interperietal	11.6	5.5	10.7	19.3	36.4**
Cranium – supraoccipital	11.8	7.5	17.2	17.2	25.3*
Cranium – parietal	3.7	0.7	0.8	4.1	11.7*
Facial – squamosal	1.3	1.4	1.6	3.4	7.1
Mandibular: hyoid	0.6	0.7	2.5	3.4	15.6**
Vertebrae: thoracic 9-13^th	38.0(1)	22.6	25.4	31.7	43.5
Vertebrae: thoracic 1-4^th	38.0(1)	5.5	20.5	9.0	27.9
No ossification of:
Mandibular: hyoid	2.5	0.0	1.6	0.7	9.1**
Other findings
Sternebrae: asymmetric	0.4	0.7	4.1	2.1	6.5*
Number of ribs = 13/14	0.7	602	6.6	11.7	13.6
Number of ribs = 14/14	0.3	5.5	4.1	11.7	11.7
Vertebrae, cervical: advanced ossification of centrum	15.7	61.0	36.9	27.6	11.7*

(1) recorded for the thoracic vertebrae as one unit

* P<0.05

** P<0.01

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study a NOAEL was determined at 1000mg/kg/day for maternal toxicity and 300mg/kg/day for embryotoxicity based on dose related observations but 1000mg/kg based on statistical evaluation.

Executive summary:

In this developmental toxicity study in rats conducted under ICH guidelines, 2 -(2 -ethoxyethoxy)ethanol was administered by gavage from day 6 to day 17 of gestation inclusive at dose levels of 0, 300, 1000, or 2000mg/kg/day. Animals were killed on day 20 of gestation for examination of their uterine contents including examination of the placentae. The high dose level of 2 -(2 -ethoxyethoxy)ethanol resulted in slight maternal toxicity characterised by reductions in body weight gain and food consumption. Evidence of embryo-fetal toxicity was restricted to minor skeletal findings which principally included a dose-related increase in the incidence of reduced ossification of cranial bones in the 1000 and 2000mg/kg/day groups, which were not considered to be indicative of a teratogenic potential of 2 -(2 -ethoxyethoxy)ethanol. Therefore, under the conditions of this study a NOAEL could be determined at 1000mg/kg/day for maternal toxicity and 300mg/kg/day for embryotoxicity based on dose related observations but 1000mg/kg based on statistical evaluation.