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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
used for determination of limit dose
Principles of method if other than guideline:
For determination of limit dose the OECD 414 guideline was followed. The bottom dose level was selected based on information obtained from literature and was related to likely human exposure.
The maximum human intake has been estimated by MAFF (UK) 1986 to be 16 mg/day and this was calculated to be 25 mg/kg/day for a 60-70 kg human. A factor of 100 was then used to provide an appropriate margin of safety which thus gave a dose of 25 mg/kg/day in rats for the present study. The middle dose was spaced between these two doses using approximately a sixfold factor. The dose levels were then calculated as ppm in the diet (for a 300g rat eating 25g food per day). The rats were dosed on Days 1-22 inclusive of gestation, Day 1 being the day that mating was confirmed by a sperm-positive vaginal smear.
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
Molecular formula:
bis(2-ethylhexyl) adipate
Details on test material:
Purity: 99.2% w/w
Supplier: ICI France, Department Baleycourt.
Appearance: colourless liquid.
Batch: Y02259/003/003-4
Satisfactory chemical stability was proven at 300ppm and 12000 for 34 days, which is in excess of the maximum period of 21 days between fresh diet preparations.

Test animals

Details on test animals or test system and environmental conditions:
Test animals: Wistar rats of the Alpk: AP fSD strain (from the specific pathogen free (SPF) colony.
Initial weight: 218-278 g.

Animal Breeding Unit and Experimental Unit, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.
For the duration of the study, each rat was individually housed in rat
racks supplied by All Type Tools Ltd, Woolwich, London, UK . The cages
had solid stainless steel sides and the floor, back and front wer e
constructed of 14SWG stainless steel mesh . The internal measurements
were 34 .0 x 37 .5 x 20 .3cm3,with a floor area of 1275cm2 . The cages were
suspended over collecting trays lined with absorbent paper . On the front
of each cage was a card identifying the animal by individual number, dose
group and study . Tap water via an automatic watering system and food
were available ad libitum.
The temperature of the animal room was within the range of 19-24° C
(as recorded daily by a maximum and minimum thermometer) with a mean of
22°C . Relative humidity was within a recorded range of 44-70% (as assessed
by daily readings from a hygrometer) and mean of 54% . There were at least
12 air changes per hour . The artificial lighting was controlled by a
,time switch and provided alternate periods of 12 hours light and 12 hours
darkness throughout the study.

All diets were based on CT1 diet supplied by Special Diets Services Ltd,
Witham, Essex, UK. The experimental diets were prepared in 30kg batches from premixes and dispensed into glass feeding jars . Two batches of
diet were prepared at each level.

Diet sampling and analysis:
A sample was taken from each diet prepared . Samples were taken from the
diet feeding jars and analysed. Chemical stability of DEHA in CT1 diet was determined at 300 and 12000ppm .
Homogeneity of DEHA was also examined in a concurrent study (Tinston 1988) and found to be satisfactory .

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
From every prepared diet a sample was taken and analysed to verify the correct concentrations. Results were within 8% of the target concentration.

The amount of food consumed by each animal was measured daily by giving a weighed quantity of food contained in a glass jar on one day and calculating the amount consumed from the residue on the next day.
Details on mating procedure:
Wistar-derived, virgin female rats were paired overnight at the Breeding Unit with unrelated males of the same strain. On the following morning, vaginal smears from these females were examined for the presence of sperm. The day when spermatozoa were detected was designated Day 1 of gestation and on this same day, successfully mated females were delivered to the experimental unit at CTL . A total of 96 mated females was supplied over a two week period. On arrival, the rats were within the weight range 218-278g and were approximately 12 weeks of age. Twelve female rats were supplied on each of eight days.
Duration of treatment / exposure:
From Day 1 of gestation until termination on Day 22.
Frequency of treatment:
Each day
Duration of test:
The in life phase of the study was conducted from 15 September to 16 October 1987.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 300, 1800, 12000 ppm
nominal in diet
Doses / Concentrations:
0, 28, 170, 1080 mg/kg
nominal in diet
No. of animals per sex per dose:
Control animals:
yes, plain diet
Details on study design:
The study was divided into 24 replicates (randomised blocks) with each replicate containing one rat from each dosage group. Cages within the replicates were assigned to one of the four groups using computer-generated random number permutations. The individual animal numbers were then assigned sequentially within the relevant groups to give the rack plan. On arrival (Day 1 of gestation) each rat was allocated to a cage (and therefore a treatment group) randomly within the replicate and individually identified by ear punching with the number assigned to it from the experimental design. Replicates were filled sequentially with three replicates added to the study on each of the eight days on which rats were received .


Maternal examinations:
Clinical observations:
All animals were checked on arrival to ensure that they were physically normal externally . They were subsequently observed daily for any changes in behaviour or clinical condition and these were recorded.

Body weight:
The bodyweight of each animal was recorded daily on Days 1 to 22 inclusive of gestation .

Terminal Investigations:
On Day 22 of gestation all the animals were killed by over exposure to halothane BP (FLUOTHANE , ICI Pharmaceuticals, Macclesfield, Cheshire, UK) vapour . A post mortem was performed and all animals were examined macroscopically. The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination. The following data was recorded:
Number of corpora lutea in each ovary.
Number and position of implantations subdivided into:
(a) live foetuses.
(b) early intra-uterine deaths.
(c) late intra-uterine deaths.
Intra-uterine deaths were classified as follows:
Early intra-uterine deaths showed decidual or placental tissue only. Late intra-uterine deaths
showed embryonic or foetal tissue in addition to placental tissue. The implantations were assigned letters of the alphabet to identify their
position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.
Fetal examinations:
Each foetus was weighed and individually identified within the litter by means of a cardboard tag. After weighing, the foetuses were killed with an intra-cardiac injection of pentobarbitone,sodium solution, 200mg/ml, (EUTHATAL, May and Baker Ltd, Dagenham, Essex, UK).

Assessment of Teratogenicity:
Each foetus was examined for external abnormalities and for cleft palate. All foetuses were then examined internally for visceral abnormalities under magnification, sexed, eviscerated and fixed in methanol. The head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities. (The brains of one litter, female 72, 1800ppm, inadvertently were not examined.) The carcasses were then returned to methanol for subsequent processing and staining with Alizarin Red S. The stained foetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed and the result converted to a four point scale. Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external, visceral or skeletal examination) defects. Variations were also recorded and classified as minor defects or variants depending on the historical frequency of occurrence in rats of this strain.
The following data were considered by analysis of variance :
(i) Maternal bodyweight gain.
(ii) Maternal food consumption.
(iii) The numbers of implantations and live foetuses per female.
(iv) Percentage pre-implantation loss and percentage post-implantation loss (calculated on an individual litter basis). The percentage pre-implantation loss and post-implantation loss were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The analyses of variances were weighted by the denominator in the proportion.
(v) The percentage of implantations which were early intra-uterine deaths (calculated on an individual litter basis).
(vi) Gravid uterus weight, litter weight and mean foetal weight (calculated on an individual litter basis).
(vii) Mean manus and pes score per foetus (calculated on an individual litter basis).
(viii) The percentage of foetuses with minor external/visceral defects only, external/visceral variants and minor skeletal defects only (calculated on an individual litter basis).

The analyses of variance allowed for the replicate structure of the study design and were carried out using the GLM procedure in SAS (1985). Unbiased estimates of the treatment group means were provided by the least square means (LSMEANS option in SAS). Individual treatment group means were compared with the control group mean using Student's t-test based on the error mean square in the analysis.

Further analysis:
Fisher's Exact Test, comparing each treated group with the control group.

All statistical tests were one-sided with the following exceptions which were two-sided: maternal bodyweight gain, maternal food consumption and the proportion of male foetuses.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: reduced body weight gain and food consumption

Details on maternal toxic effects:
Administration of 12000 ppm DEHA resulted in slight maternal toxicity (small but significant maternal reduction in body weight gain (-13%) and food consumption). At 1800 ppm DEHA, there was no evidence of maternal toxicity.

No treatment related clinical signs, mortality or macroscopic anomalies were observed.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
ca. 170 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: reduced ossification and increase in the incidence of visceral variants, not considered adverse

Details on embryotoxic / teratogenic effects:
Administration of 1800 and 12000ppm DEHA resulted in minimal foetotoxicity (reduced ossification and increase in the incidence of visceral variants, when compared to control groups), which were not considered adverse. Incidences of slightly dilated ureter were significantly increased in the high dose group compared to concurrent control values, but were well within historical control data. Though an increase in kinked ureters was proposed by the authors, the increase was slight, not significant, and within historical control data.

A dietary level of 300 ppm DEHA was a clear no-effect level for embryonic development. There was no effect on foetal weight, litter weight, gravid uterus weight, number of intra-uterine deaths, or number of external abnormalities. At the highest dose, there was a minimal and not significant decrease in litter size (10.7 vs. 11.8), but the change was too small to be of toxicological significance. No historical control data on litter size was given. Additionally, there was no effect on number of corpora lutea, implantations, and post-implantation loss.

Effect levels (fetuses)

open allclose all
Dose descriptor:
Effect level:
ca. 1 080 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
Effect level:
ca. 1 080 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Dose descriptor:
Effect level:
28 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

no effects observed

Overall developmental toxicity

Developmental effects observed:

Any other information on results incl. tables

Diet analysis: chemical stability of DEHA in diet was satisfactory.

Concentrations of DEHA were within acceptable limits.

Clinical observations: All rats survived to scheduled termination.

The clinical findings observed were considered not to be related to DEHA administration.

Maternal body weight: Administration of 12000 ppm DEHA was associated with a small but statistically significant reduction in bodyweight gain compared with the control group which was most marked at the start of the feeding period.

Maternal food consumption: Maternal food consumption was statistically significantly reduced in the 12000ppm group from Days 2-18 inclusive of pregnancy. There were no adverse effects on food consumption in the 300 or 1800 ppm DEHA groups.

Maternal macroscopic findings (post mortem): macroscopic changes were considered not to be related to DEHA treatment.

There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external


The incidence of minor external and visceral defects was unaffected by treatment although two visceral variants were increased at the top two dose

levels; kinked ureter being slightly and not significantly increased in the 1800 and 12000 ppm groups and slightly dilated ureter being increased in the 12000 ppm group. Incidences for both effects were within historical control data for this laboratory. Overall, minor skeletal defects were increased in a dose-related manner at 1800 and 12000 ppm DEHA, while skeletal variants and pes score were increased at the top dose only. These findings indicate slightly poorer ossification at the 1800 and 12000 ppm dose levels. The reduced ossification are considered to be the result of slight foetotoxicity. There was no treatment-related effect on skeletal or visceral variants at 300 ppm DEHA.

Applicant's summary and conclusion