Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 1987 to 16 October 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A GLP study conducted to sound scientific principles, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. As the study was conducted with the structural analogue, bis(2-ethylhexyl) adipate, it has been assigned a reliability score of 2.

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)
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 six-fold 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
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Bis(2-ehtylhexyl) Succinate
- Appearance: colourless liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS:
- Strain: Wistar rats of the Alpk: AP fSD strain
- Age at study initiation: aproximately 12 weeks
- Weight at study initiation: 218 - 278 g
- Housing: individually housed in rat racks supplied by All Type Tools Ltd, Woolwich, London, UK
- Water: tap water ad libitum via an automatic dispensary system

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24 °C
- Humidity (%): 44 - 70 %
- Air changes (per hr): 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 15 September 1987 to 16 October 1987

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
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
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 . 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:
Daily
Duration of test:
All females were sacrificed on Day 22 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 300, 1800, 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 28, 170, 1080 mg/kg
Basis:
nominal in diet
No. of animals per sex per dose:
24 females per dose group
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, 200 mg/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 test material resulted in slight maternal toxicity (small maternal reduction in body weight gain and reduced food
consumption). At 1800 ppm test material, there was no evidence of maternal toxicity.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
170 mg/kg bw/day
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:
Administration of 1800 ppm test material resulted in minimal foetotoxicity (reduced ossification and increase in the incidence of visceral variants). A dietary level of 300 ppm test material was a clear no-effect level for embryonic development.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
28 mg/kg bw/day
Based on:
test mat.
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 test material in diet was satisfactory. Concentrations of test material were within acceptable limits.

 

Maternal clinical observations: All rats survived to scheduled termination.

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

 

Maternal body weight: Administration of 12000 ppm test material 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 test material groups.

 

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

Foetal examinations: 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 test material, 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 test material.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the maternal toxicity No Observed Adverse Effect Level was determined to be 170 mg/kg bw/day while the developmental toxicity No Observed Effect Level was determined to be 28 mg/kg bw/day.
Executive summary:

The developmental toxicity of the test material was determined in a GLP study which was conducted to a methodology similar to that which is outlined in the standardised guideline OECD 414. During the study 24 females were mated with unrelated males and received test material in the diet, at dose levels of 0, 300, 1800, 12000 ppm, from day 1 to day 22 of gestation. During treatment females were observed daily for clinical signs and individual body weights were recorded. On day 22 of gestation all females were sacrificed and examined macroscopically.

 

The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed. The ovaries and uterine content were examined after termination and the number of corpora lutea and number and position of implantations were recorded. After weighing, each foetus was sacrificed and 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 examined for macroscopic abnormalities. The 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.

 

All maternal females survived until scheduled sacrifice and no adverse clinical signs of toxicity were observed. Maternal food consumption was statistically significantly reduced in the 12000 ppm group from Days 2-18 inclusive of pregnancy. There were no adverse effects on food consumption in the 300 or 1800 ppm test material group. In line with the reduction in food consumption in the high-dose females, administration of 12000 ppm test material 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 macroscopic findings (post mortem) were considered not to be related to test material 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.

 

Examination of the feotuses revealed that 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 test material, 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 test material.

 

Overall, under the conditions of the study, the NOAEL (maternal toxicity), based on reduction in body weight and food intake at 12000 ppm (1080 mg/kg), was determined to be 170 mg/kg bw/day while the NOEL (fetotoxicity), based on reduced ossification and increase in the incidence of visceral variants observed at the highest doses, was determined to be 28 mg/kg bw/day.