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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Justification for type of information:
Read across is based on the category approach. Please refer to attached category document.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989
Reference Type:
publication
Title:
Investigation of the Prenatal Toxicity of Orally Administered Diethylene Glycol in Rabbits
Author:
J. Hellwig,
H.J. Klimisch
R. Jackh
Year:
1995
Bibliographic source:
Fundam Appl Toxicol 28(1): 27-33

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethylene glycol
- Analytical purity: > 99.7% before the beginning of the study and 98.8% in the reanalysis after the end of the study.
- Stability: stability of the test substance over > 2 years was proven

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: the sexually mature, virgin Himalayan rabbits (Chbb: HM (outbred strain)) were supplied by Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 24 and 29 weeks old
- Weight at study initiation: mean body weight of pregnant animals only, ca. 2560 g (calculated from the means of the groups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 7 to day 19 p.i.) always at approx. the same time of day (in the morning). The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.). On day 29 p.i. all animals were sacrificed and examined macroscopically. The fetuses were dissected from the uterus, and further investigated with different methods. The test substance solutions were prepared only once for each study section because the stability of the test substance solution over a period of 21 days had been proven before treatment of the animals began. For the preparation of the solutions an appropriate amount of the test substance was weighed and subsequently dissolved in doubly distilled water. Due to technical reasons the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of 7 days elapsed before the next section.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance solutions over a period of 21 days could be demonstrated. The results of the analyses of the solutions of test substance confirmed the correctness of the prepared concentrations.
On the basis of the duration of use and the analytical findings the rabbit food used was found to be suitable. Federal Register, Vol. 44, No. 91, of May 9, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable contaminants.
On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation of May 22, 1986 served as a guideline for maximum tolerable contaminants.
Details on mating procedure:
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.
This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females.
The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).
Duration of treatment / exposure:
gestation day 7 - 19
Frequency of treatment:
daily
Duration of test:
30 days
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
Based on the results of a dose range-finding study, the following doses were selected for the prenatal toxicity study in rabbits:
- 100 mg/kg bw: should constitute the expected no observable effect level
- 400 mg/kg bw: with this dose marginal influences on dams and/or fetuses could not be ruled out
- 1000 mg/kg bw: with this dose signs of maternal toxicity (e.g. retarded body weight gain) may possibly have occurred. A higher dose level was not deemed to be necessary due to the recommendations in the test guidelines, which had to be taken into account.

Examinations

Maternal examinations:
The consumption of food was determined daily during the entire study period. All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i.. The body weight change of the animals was calculated from these results. Furthermore, after terminal sacrifice the corrected body weight gain was calculated (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. Mortality: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).
Examinations of the dams at termination: On day 29 p.i. the dams were sacrificed by intravenous injection of a pentobarbital and the fetuses were dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded: - Weight of uterus before it was opened - Number of corpora lutea - Number and distribution of implantation sites classified as: --live fetuses --dead implantations: a) early resorptions (only decidual or placental tissues visible or from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy) b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible) c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
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
Fetal examinations:
Examination of the fetuses after dissection from the uterus: At necropsy each fetus was weighed and examined macroscopically for any external findings. 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. Soft tissue examination of the fetuses: After the fetuses had been sacrificed by C02, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed . The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the fetuses was determined by internal examination of the gonads. If heads of fetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these fetuses were severed from the trunk, fixed in BOUIN´s solution and later processed and assessed according to WIISON´s method. About 10 transverse sections were prepared per head. Skeletal examination of the fetuses: After the soft tissue examination all fetuses were placed in ethyl alcohol for staining of the skeletons. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed.
Statistics:
The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft (laboratory data processing, responsible:Dr. H.D. Hoffmann). Examinations of dams and fetuses: Dunnett's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation loss, resorptions and live fetuses. Fisher´s Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings. Significances resulting from these tests have been indicated in the tables (a for p < 0 .05, b for p < 0 .01).
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Food consumption of the substance-treated does was not influenced by the test substance administration. The observable differences between the control animals and the dams of test groups 1 - 3 (100, 400 and 1,000 mg/kg body weight/day), including the statistically significantly increased food consumption in the highest dose group on days 6 - 7 p.i. (pretreatment period), are without biological significance. There were no adverse effects on body weights or body weight changes which could be attributed to the test substance administration. All values are within the range of biological variation or of spontaneous nature, including the statistically significant decrease in mean body weight change of the intermediate dose females during days 0 - 29 p.i. (whole study period). The results of the corrected body weight gain (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened) do not clearly show any dose-related differences between the groups. The only statistically significant difference in comparison to the controls, the low value in test group 2 (400 mg/kg body weight/day), which is mainly caused by 2 does, is without biological relevance. Except one doe of test group 1 (100 mg/kg body weight/day), which showed a marked edema in the anogenital region during days 16 - 29 p.i. and one animal of test group 2 (400 mg/kg body weight/day), which had an accidental lesion of the left hindlimb (days 17 - 29 p.i.), there were no remarkable clinical observations in any doe of all groups. Both recorded clinical observations are spontaneous ones and are not related to the test substance administration. There were no mortalities.
There were no substantial differences concerning the uterus weights between the controls and the substance-treated groups. All values lie within the range of biological variation. Due to a technical error, the uterus weight of doe of test group 3 (1,000 mg/kg body weight/day) was not recorded.
The conception rate varied between 93 and 100%. Concerning all groups, there were no substance-related and/or statistically significant differences in the conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation loss, the number of resorptions or viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the one doe of test group 1 (100 mg/kg body weight/day) which did not become pregnant due to a hydrometra in the one and a blind ending uterine on the other side.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The sex distribution in test groups 1 - 3 (100 - 1,000 mg/kg body weight/day) was comparable with the control group. The observable differences are without any biological relevance. The mean placental weights in the substance-treated groups (100, 400 and 1,000 mg/kg body weight/day) were not substantially influenced. The differences observed in comparison to the control are without biological relevance and lie within the range of biological variation. There were no clearly dose-related or statistically significant differences between the groups concerning the mean fetal weights. All values are still within the range of biological variation; therefore the observable differences between the control and the substance-treated groups are assessed as to be of spontaneous nature. The external examination of the fetuses revealed no malformations in any group and only one kind of variation (pseudoankylosis) in 2 fetuses each of the control group, of test groups 2 (400 mg/kg body weight/day) and 3 (1,000 mg/kg body weight/day). There were no so-called unclassified observations (like placentae fused) in any fetus. The examination of the organs of the fetuses revealed two types of malformations. One high dose fetus showed a septal defect, a rather common finding in the rabbit strain used; this malformation is also present in the historical control data to about the same extent. Furthermore, two control fetuses and one fetus of the 400 mg/kg group had an agenesia of the gallbladder. Variations were detected in each group including the control. The very common finding (separated origin of carotids) in the rabbit strain used in this study occurred without a clear dose-response relationship, but was more frequently seen in the substance-treated groups, the differences in relation to the control being statistically significant ; however, if the relevant values of the substance-treated groups are compared with the corresponding historical control values, these values are within the range of biological variation. Therefore, it becomes obvious, that the number of control fetuses with this finding is unexpectedly low. Thestatistically significantly increased number of fetuses of test groups 1 - 3 (100, 400 and 1,000 mg/kg body weight/day) is therefore assessed as to be of incidental nature. The other variations (hypoplasia of gallbladder, dilated renal pelvis, ovary bipartite) occur without a dose-response relationship and/or are to be found in the same kind of magnitude in the historical control data. Moreover, several fetuses out of test groups 0 - 3 (0, 100, 400 or 1,000 mg/kg body weight/day) showed focal liver necrosis or blood coagula around the bladder (socalled unclassified observations). Various malformations of the ribs and/or the vertebral column were seen in 1 fetus of the control and the 400 mg/kg group and in 3 fetuses of the 1,000 mg/kg group. No other skeletal malformations were recorded for any group. The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal and frontal bones), the ribs (accessory ribs, flying ribs, or rudimentary cervical ribs), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape, fused or bipartite) and were found in all groups without a clear dose-response relationship and/or without any statistically significant differences between the groups. In all groups signs of retardations (incomplete or missing ossification of skull bones, sternebra(e), and/or talus) were found; they occurred without any differences of biological relevance between the groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
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:
In conclusion, the oral administration of diethylene glycol to pregnant Himalayan rabbits by stomach tube on day 7 through day 19 p.i. in dosages of 100, 400 and 1,000 mg/kg body weight/day led to no adverse effects which can be causally related to the test substance administration in both the does and in the fetuses. The observable differences between the control group and the substance-treated groups appeared either without a clear dose-response relationship and/or were assessed as being without biological relevance, because the relevant values/findings are to be found in a similar range within the historical control data.
Summarized, diethylene glycol caused, under the conditions of this study, up to and including a dose of 1,000 mg/kg body weight/day no signs of maternal toxicity and no signs of embryo-/fetotoxicity; especially no teratogenic effects could be detected. As to the OECD GUIDELINE for testing of chemicals (No. 414, adopted May 12, 1981) in the case of substances of low toxicity (i.e. "if a dose level of at least 1,000 mg/kg body weight/day produces no evidence of embryotoxicity or teratogenicity") further embryotoxicity studies at higher dose levels may not be considered necessary.