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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
ETBE, batch no. S02-08-159-I3/1, >98% pure, colourless liquid, supplier: TOTAL France S.A., Paris-la-Défense, France (purified by SEPAREX, Champigneulles, France)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Animals: time-mated female Sprague Dawley rats (Charles River  Laboratories, L'Arbresle, France); 
Age at start of treatment: approx. 11 wk (bw 215-297 g)
Acclimation period: 5 d 
Housing: individually housed, stainless steel cages (wire mesh bottom) for duration of study
Diet: A04 C pelleted maintenance diet (UAR, Villemoisson, France), ad  libitum
Water: deionised water, ad libitum
Assignment to treatment groups: computer-generated weight randomisation

The animal room conditions are set as follows:
- temperature : 22 ± 2°C
- relative humidity : 50 ± 20%
- light/dark cycle : 12h/12h (7:00 - 19:00)
- ventilation : about 12 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
ETBE was administered by gavage (dosing needle) at doses of 0 (corn oil; 4 ml/kg bw), 250, 500 or 1000 mg/kg/day. Each animal was dosed once a day, at approximately the same time, from day 5 to day 19 post-coitum, inclusive.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing solutions were prepared weekly (based upon stability data  demonstrating no losses of ETBE over 9 d) and samples taken for analysis  (GC) to confirm received dose. Solutions displayed good homogeneity, and deviations from nominal  concentration were +/- 10%.
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c).
Duration of treatment / exposure:
gestation days 5-19
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500 or 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
24 mated female rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor on the basis of a previously conducted study (CIT/Study No. 24168 RSR), where ptyalism (excess salivation) and a slight, but statistically significant, decrease in body weight of pregnant females was noted at 1000 mg/kg/day. Consequently, dose-levels of 250, 500 and 1000 mg/kg/day were selected for this study.

Examinations

Maternal examinations:
GENERAL OBSERVATIONS
The dams were examined twice daily for mortality, and for the occurrence of clinical signs 3 times per day on  GD 5-14, and twice daily thereafter. Body weight and food consumption were recorded on 10 occasions between  GD 2-20.

Macroscopic post-mortem examination
All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of each placenta was also undertaken.
Ovaries and uterine content:
On day 20 post-coitum, all females were killed by asphyxiation using carbon dioxide. The weight of the gravid uterus was recorded at hysterectomy for each pregnant female with at least one live fetus.
The ovaries and uterus from all females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of implantation sites (or uterine scars).
Early resorptions refer to evidence of implantation without recognizable embryo or fetus. Late resorptions refer to dead embryo or fetus with external degenerative changes. In apparently non-pregnant females, the presence of implantation scars on the uterus were checked using an ammonium sulphide staining technique.
Fetal examinations:
The following examinations were performed on all litters from females with at least one live fetus. Whenever necessary, photographs were taken to document fetal findings and kept with the study archives.

Any abnormal fetal findings were recorded according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformation or variation:
- a malformation refers to a permanent structural change that is likely to adversely affect survival or health,
- a variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. It might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.

Body weight
The body weight of each live fetus was recorded.

External examination
Each fetus was subject to a detailed external examination, which included all visible structures, surfaces and orifices (including the oral cavity). Any dead fetuses were then discarded and the live fetuses sacrificed by subcutaneous injection of thiopental sodium.

Soft tissue examination (Wilson, J.G., 1965)
Approximately half of the live fetuses per litter were fixed in Harrisson’s fluid. A detailed soft tissue examination was performed using a free-hand serial sectioning technique (Wilson technique) which included observation of all the organs and structures of the head, neck, thorax and abdomen.

Skeletal examination (Peters P.W., 1977)
The remaining live fetuses per litter were fixed in ethyl alcohol and eviscerated. A detailed examination of the skeleton (bone + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included observation of all the bone structures and cartilage of the head, spine, rib cage, pelvis and limbs.

Sex of fetuses
The sex of each fetus was determined at the time of evisceration (after fixation in alcohol) or at the time of serial sectioning.
Statistics:
Data were expressed as group means (+/- SD) or as percentages. The unit  of comparison was the litter, with fetal body weight analysed by sex as  well as for both sexes combined. Means (normally distributed) were  compared by ANOVA and Dunnett's test, percentage values by Fisher's exact  probability test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PREGNANCY STATUS 
There were no significant or biologically relevant differences in  pregnancy status between the groups:
Dose-level (mg/kg/day)     0    250   500   1000
Mated females              24     24     24     24
Non-pregnant females       3     5      4      2
Pregnant, alive at GD20    21     19     20     22
Pregnancy rate             87%   79%    83%    92%
Litters                    21     19     20    22

There were no deaths in any group. Ptyalism (excess salivation) was noted in 5, 13 or 17 females from the 250, 500 and 1000 mg/kg bw/day dose groups at various times during the study, It occurred mainly during the middle part of the investigation and (with a single exception) was present immediately post-dosing and had resolved by one hour.
Comment: the report considers this a response to the unpalatable 'taste' of ETBE rather than an adverse clinical effect.

There was a transient reduction in maternal body weight gain of high dose  dams on GD 5-9 (-20%), with mean body weight gain decreased significantly  over the whole dosing period (-11%, p<0.05). Net body weight gain in the  1000 mg/kg/day group (corresponding to the maternal carcass weight at day  20 minus maternal bw on GD5) was also significantly lower (-17%, p<0.01)  when compared with the controls. 
Dose-level (mg/kg/day)        0     250   500   1000
Bw change GD 5-9               24    23     23     20 % 
diff. from control                 -4     -4    -20
Bw change GD 5-20            135   132   134   120*
% diff. from control                 -2    -1    -11
Net bw change GD 5-20    61.8  59.4  60.0  51.5**
% diff. from control                 -4    -3    -17
* = p<0.05; ** = p<0.01

The food consumption was unaffected by the treatment. No treatment-related macroscopic changes were present in the dams at  necropsy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
There were no differences in the number of corpora lutea and implantation  sites between the groups (hence no effects on pre-implantation losses).  There was no total litter resorption in any group. The number (21) and rate (1 per female) of early resorptions was slightly  greater in the 1000 mg/kg/day when compared to the control group (number  = 14, rate = 0.7 per female), hence the extent of post-implantation loss  was minimally increased in the high-dose females (7.5%) versus controls  (5.2%; non-significant). The report notes that this incidence was in the  range of historical control data for the laboratory (mean  post-implantation loss=3.4%, minimum=1.0%, maximum= 8.5%) and was  therefore considered unrelated to treatment.  The number of live fetuses were in a range 11.7 to 12.3 per female, with  no  treatment-related or statistically significant differences present  between the groups. No dead fetuses were found in any group. The sex ratio in the low dose group (53.5% male, 46.5% female) was  significantly different from the control value. This difference appeared  principally related to an unusually low proportion of males (40.8%) and a  correspondingly higher number of females (59.2%) in the control group.  The sex ratio in the other treated groups (47.3-48.8% male, 51.2-52.7%  female) was within the normal range (no dose-response relationship  present). The report considered this observation in the low dose litters  a chance event, unrelated to treatment with ETBE. Fetal weight was unaffected by the treatment.

FETAL EXAMINATION
A single occurrence of umbilical hernia (malformation) and a bent tail  (variation) were present in the 250 mg/kg/day group (no other findings in  any group). These were considered unrelated to treatment due by the study  report due to isolated nature of the findings and absence of any  dose-response. 

SOFT TISSUE EXAMINATION - MALFORMATIONS
There were no malformations in the control or the low or intermediate  treatment groups. At 1000 mg/kg/day an absence of kidneys, ureters and  adrenals was observed in single fetus. These were considered unrelated to  treatment by the study report due to isolated nature of the findings and  absence of any dose-response.

- VARIATIONS
The following findings were noted among the groups:
- dilated ureters in one fetus from the control, 250 m/kg/day and 1000  mg/kg/day groups;
- short uterine horns in one fetus from the 500 mg/kg/day;
- dilated renal pelvis in one fetus from the 250 and 1000 mg/kg/day.
These few findings, which were not dose-related and randomly distributed  across the groups, were considered by the report to be unrelated to the  treatment.

SKELETAL EXAMINATION - MALFORMATIONS
The following malformations were recorded:
- misaligned sternebra in one control fetus;
- at 250 mg/kg/day, fused rib and misshapen sacral vertebra in one fetus;  split sternebra and fused ribs in one other fetus;
- at 500 mg/kg/day, bilateral misshapen ilium noted in one fetus;
- at 1000 mg/kg/day, absence of one pair of ribs observed in two fetuses, one of which also had an absence of thoracic vertebra.
Because of their low incidence and the absence of any dose-relationship,  these malformations were not considered by the report to be related to  the treatment with ETBE.

- VARIATIONS
There was a significant increase in the incidence of unossified 4th  metacarpal at the highest dose: 43/136 fetuses (31.6%, p<0.05) were  affected versus 27/135 (20%) in the control group. This was not  statistically significant when expressed on a fetus/litter basis.  Cartilage was generally present suggesting that this was due to slightly  delayed ossification rather than to a persistent alteration. As the  findings were isolated, not statistically significant or dose-related at  the level of the litter and not associated with any significant delay in  ossification of other bones, and since the total incidence of skeletal  variations in treated animals did not increase, the report concludes  these are of no toxicological significance. There were no other findings.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

In the developmental toxicity study ptyalism was observed at all dose levels (i.e., from 250 mg/kg bw/day onwards). The study authors considered this effect related to unpalatable nature of the dosing solutions / to the unpalatable 'taste' of ETBE. In the EU RAR this effect is not taken into account as an adverse effect, therefore, this effect a not considered a significant health effect.

Applicant's summary and conclusion