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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, near-guideline study, fully adequate for assessment, read-across.
Justification for type of information:
Bis (2-ethylhexyl) adipate (DEHA) is appropriate for read-across because it is considered as a similar substance, based on similar structure, toxicokinetic and toxicological profile useful for this interpolation (please see also read-across justification).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
used for determination of limit dose
Deviations:
no
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:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 99.2% w/w
Supplier: ICI France, Department Baleycourt.
Appearance: colourless liquid.
Batch: Y02259/003/003-4

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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.

Housing:
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.

Diet:
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
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 .
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.
No. of animals per sex per dose:
Group 1: female animals 1-24
Group 2: female animals 25-48
Group 3: female animal 49-72
Group 4: female animal 73-96
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 .

Examinations

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.
Statistics:
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

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

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
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

Details on embryotoxic / teratogenic effects:
Administration of 1800 ppm DEHA resulted in minimal foetotoxicity (reduced ossification and increase in the incidence of visceral variants). A dietary level of 300 ppm DEHA was a clear no-effect level for embryonic development.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
28 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

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

abnormalities.

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 increased in the 1800 and 12000 ppm groups and

slightly dilated ureter being increased in the 12000 ppm group. 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 and increase in

the incidence of visceral variants 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