Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD Guideline 421 study (Reproduction / Developmental Toxicity Screening Test) the oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction.  At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day.  However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

An early and limited 3-generation study is available (Bornmann 1956). Eight female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted. In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to produce F3-Generation whose development was also observed. P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females. In F1, F2, F3 pups somatic development and maturity as well as fertility were not affected and comparable to the controls. Furthermore, 12 of 15 young female rats that had received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks were introduced to the Allen-Doisy-test which investigated the functionality of the hypophysis-ovar-axis by examination of vaginal smears for untypical cell morphology. Solvent controls are included in the test. Vaginal smears did not show any disturbance of oestrus cyclicity.

Additional, there is a subchronic study available (Leggett 2020). During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses ofb100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

These findings were confirmed by a subacute toxicity study available (Schladt 2013). During the 4-week treatment period male and female Wistar rats received 0, 100, 300 or 1000 mg/kg bw/day ‘Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate’ by gavage. The study was performed according to OECD TG 407 and GLP conditions. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 29 August 2017 Experimental Completion Date: date of final thyroid hormone report
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The purpose and integrity of the study is therefore unaffected.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The dose levels were chosen in collaboration with the Sponsor based on the results of previous toxicity work including an Oral (Gavage) PreNatal Development Toxicity Study in the Rat (Envigo Study number: 41404097). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Specific details on test material used for the study:
Identification: Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8)
Physical State/Appearance: Clear colorless liquid
Alternative Name: The test item is also known as Adimoll BO
Storage Conditions: Ambient temperature and humidity in darkness; used/formulated in light
Expiry Date: 28 November 2018
No correction for purity was made.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities. The Sprague-Dawley Crl:CD® (SD) IGS BR strain rat has been selected to match previous toxicity work with this test item and also because there is some historical control data available for the skeletal development of the Day 13 offspring.
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Kent, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 337 to 439g, and were approximately nine weeks old. The females weighed 225 to 294g, and were approximately eleven weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of another study (Envigo Study Number 41500056). Results showed the formulations to be stable for at least sixteen days. Formulations for this study were made and used within the known stability period; the bulk formulations were divided into daily aliquots and stored refrigerated (approximately 4°C in the dark) prior to use.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test item formulations were taken on two occasions and analyzed for concentration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given. The results indicate that the prepared formulations were within 101-108% of the nominal concentration.
Duration of treatment / exposure:
Approximately six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study. For the 14 days prior to pairing, pre-pairing vaginal smears were performed and assessed for females.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples. Where possible, litter size was standardized to ten offspring per litter. The excess offspring were retained in appropriate fixative and processed for possible skeletal evaluation.
vii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
viii. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. The remaining offspring were retained in appropriate fixative and processed for skeletal evaluation. All surviving offspring were killed and examined externally; the extent of any internal examination was dependent on the end point for which each offspring had been allocated.
ix. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Treatment group)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Low (Treatment group)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Intermediate (Treatment group)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High (Treatment group)
No. of animals per sex per dose:
12 males and 12 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor based on the results of previous toxicity work including an Oral (Gavage) PreNatal Development Toxicity Study in the Rat (Envigo Study number: 41404097). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded for all animals at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrous or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
Where possible, litters were culled to ten pups (five males and five females if possible) on Day 4 of age. Two culled offspring (same sex, if possible) were allocated for thyroid hormone analysis and the remaining culled offspring were allocated for possible skeletal examination.

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides, testes, seminal vesicles (with coagulating gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed after being placed in fixative (partial fixation).

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides♦ Prostate
Glans penis Seminal Vesicles (with coagulating gland)
Gross lesions Testes♦
LABC (levator ani-bulbocavernous) muscle Thyroid/Parathyroid
Mammary gland Uterus/Cervix (with oviducts)
Ovaries Vagina
Pituitary

Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered Formalin.
All tissues were dispatched to the histology processing Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
Necropsy
Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Offspring required for thyroid retention were killed by carbon dioxide asphyxiation with death confirmed by cervical dislocation. Remaining offspring were killed by intraperitoneal overdose of a suitable barbiturate agent, with death confirmed by the onset of rigor mortis.
Examination of offspring allocated to thyroid hormone assessments and skeletal evaluation was restricted to an external examination. Offspring allocated for retention of the thyroids had an external examination and limited internal examination. Any decedent offspring or offspring showing abnormalities had an internal examination where possible.

Skeletal Evaluation
Where possible for each litter, offspring at Day 4 and Day 13 post partum were retained in 70% Industrial Methylated Spirit (IMS), for post partum skeletal evaluation. Examination of the offspring was restricted to Day 13 post partum offspring.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (females where possible to maximise the number of males available for day 13 post partum visible nipple counts; if offspring were of the same sex, samples from the same litter were pooled). If ten or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
Serum and plasma samples were taken from all adult males and females at termination.
All serum samples were dispatched to the Test Site where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator. All plasma samples were retained at the Test Facility.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation,
Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Absolute Organ Weights, Body Weight-Relative Organ Weights, Skeletal Development Parameters (% incidence) and Thyroid Hormone (Thyroxine).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:
Post-implantation loss (%) = ((Number of implantation sites - Total number of offspring born)/Number of implantation sites) x 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
Viability Index 2 (%) = (Number of offspring alive on Day 13/Number of offspring alive on Day 4) x 100
Viability index 2 takes into consideration the offspring culled on Day 4 post partum.
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post partum, using the following formula:
(Number of males offspring/Total number of offspring) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 250, 500 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, increased post-dosing salivation was apparent for both sexes throughout the treatment period, although the incidence of this finding showed little consistency over time. Similar increased post-dosing salivation was apparent for both sexes at 500 mg/kg bw/day but at a much lower incidence. Such increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and probably indicates distaste or slight irritancy of the dosing formulations. Increased post-dosing salivation was also apparent for one female at 250 mg/kg bw/day but only for one day and this may reflect difficulty in dosing this particular animal on this isolated occasion.
Occasional instances of noisy respiration was apparent for a few animals (both sexes at all dosages including control) on the study. Whilst for females, the highest incidence of this clinical sign appeared at 1000 mg/kg bw/day, the incidence for females at lower dosages and for males at all dosages showed no dosage relationship. Additionally one male treated with 500 mg/kg bw/day and one male treated with 250 mg/kg bw/day showed pilo-erection and hunched posture for a short period of time. Overall, these findings were considered to reflect occasional difficulties of dosing particular animals on occasions during the study and not test item related.
One female treated with 250 mg/kg bw/day, one male and seven females treated with 500 mg/kg bw/day, and one male and three females treated with 1000 mg/kg bw/day showed generalized fur loss for a number of days. The higher incidence at 500 and 1000 mg/kg bw/day may be a consequence of the increased salivation at these dosages which may have prompted exaggerated grooming. This finding showed no dosage relationship, and was considered to be of no toxicological significance.
Occasional incidences of open wounds and/or scabbing were apparent for one, two and two males at 250, 500 or 1000 mg/kg bw/day respectively but not in any females in any dose group. This finding was considered to be incidental and unrelated to test item exposure.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one unscheduled death on the study. Control female 16 was killed for animal welfare considerations on Day 15 after showing hunched posture and a soft mass under the left forelimb. Necropsy revealed a tear in the esophagus and a brown fluid-filled mass beneath the left forelimb, indicating that the decline in clinical condition had been due to accidental trauma during the dosing procedure. The liver was also noted to be mottled in appearance.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on body weight or body weight gain for males throughout the study at 250, 500 or 1000 mg/kg bw/day.
There was no effect of treatment on body weight and body weight gain of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption of males throughout the pre-pairing and post-pairing phases of the study at 250, 500 or 1000 mg/kg bw/day.
There was no effect of treatment on food consumption of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption for either sex at 250, 500 or 1000 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No findings were apparent which could be related to the administration of the test item in the tissues examined.
There were no test item-related microscopic findings following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of follicles and corpora lutea in the ovaries.
Pairing of female 71 and male 59 (Group 3) did not result in a pregnancy. Male 59 had mild or minimal changes in the testes and epididymides which may have affected fertility.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day, with all treated females, except one female at 500 mg/kg bw/day, showing regular cycling.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of follicles and corpora lutea in the ovaries.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating
Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 250, 500 or 1000 mg/kg bw/day.

Fertility
There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 250, 500 or 1000 mg/kg bw/day.

Gestation Length
The intergroup distribution of gestation lengths observed during the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Description (incidence and severity):
There was no effect of treatment on post-natal survival of the offspring from birth to termination (Day 13 of age) at dose levels of 250 and 500 mg/kg bw/day.
At 1000 mg/kg bw/day, the high dose group, survival to Day 4 of age was slightly inferior to control, as indicated by a statistically significant decrease for viability index 1; between Days 1 and 4 (before culling) for control, 250, 500 and 1000 mg/kg bw/day groups viability index was 98.7% (±3.0%), 94.5% (±6.8%), 98.3% (±3.0%) and 88.0% (±12.8%) respectively. Subsequent viability index 2, survival between Day 4 after culling and Day 13 of age were comparable to controls; 99.1% (±3.0%), 96.5% (±7.0%), 99.1% (±3.0%) and 99.2% (±2.9%) for control, 250, 500 and 1000 mg/kg bw/day groups, respectively. The biological significance of the lower survival between Days 1 and 4 is therefore unclear.
Body weight and weight changes:
not specified
Description (incidence and severity):
At 1000 mg/kg bw/day offspring body weight gains for both sexes were statistically significantly lower than control during Days 4-7 and Days 7-13. However, differences from control for offspring body weight and cumulative body weight gain resulting from this lower gain only attained statistical significance on Day 13 of age. Lower litter weights were apparent at this dosage before culling on Day 4 post partum, compared to control, and no statistically significant effect was observed post culling on Day 4 or Days 7 and 13. Consequently, the lower litter weight is considered to reflect the slightly lower litter size before culling, compared to control.
There was no effect of maternal treatment on offspring body weight, body weight gains or litter weights on Days 1 to 13 at 250 or 500 mg/kg bw/day. Both sexes at 250 mg/kg bw/day and male offspring at 500 mg/kg bw/day showed statistically significantly lower body weight gains between Days 7-13 post partum, however, overall body weight gain at termination on Day 13 of age was only statistically significantly lower than control for males at 250 mg/kg bw/day. Given the lack of consistent dosage relationship for these findings there were considered to be incidental and to reflect normal biological variation. Supporting this, it was noted that one litter for each dose level appeared to have unusually low offspring body weight gains, and one control litter appeared to have unusually high values.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.
Histopathological findings:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index at dosages of 250, 500 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Evaluation of ano-genital distance for offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

The type, incidence and distribution of skeletal findings apparent during detailed skeletal examination of offspring at Day 13 of age did not indicate any effect of maternal treatment on development at 250, 500 or 1000 mg/kg bw/day.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
One control female was terminated early and one female treated at 500 mg/kg bw/day was non-pregnant. The following assessment is based on the 11, 12, 11 and 12 females that successfully maintained a litter to Day 13 of age at 0 (Control), 250, 500 or 1000 mg/kg bw/day respectively.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: neoplastic
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: neoplastic
Remarks on result:
other:
Remarks:
The No observed adverse Effect level for reproduction/developmental endpoints was reported to be the high dose level (1000mg/Kg body weight/day) on the basis of the effects seen on post- natal offspring survival and body weight gain at the highest dose level being limited, and the extent of effect were such that these findings were considered equivocal.
Critical effects observed:
no
Reproductive effects observed:
no

Summary of Reproductive Performance

 

Dose Group (mg/kg bw/day)

0 (Control)

250

500

1000

Males

 

 

 

 

Initial group size

12

12

12

12

Paired

11#

12

12

12

Induced pregnancy in female partner

11

12

11

12

Surviving to terminal necropsy

12

12

12

12

 

 

 

 

 

Females

 

 

 

 

Initial group size

12

12

12

12

Paired

11#

12

12

12

Non-Pregnant

0

0

1

0

Killedin extremis

1

0

0

0

Reared young to Day 13 of age

11

12

11

12

 # Female 16 killedin extremisprior to pairing, male 4 not paired with a female

Tabular Summary Report of Effects On Reproduction/Development

Observations

Dose Level (mg/kg bw/day)

0 (Control)

 250

500

1000

Paired animals

n

11M 11F

12M 12F

12M 12F

12M 12F

Females showing evidence of copulation

n

11

12

12

12

Pregnant females

n

11

12

12

12

Conception Days 1-4

n

11

12

11

12

Conception Day 14

n

0

0

1

0

Gestation = 22 Days

n

5

6

7

3

Gestation = 22 ½ Days

n

2

3

1

6

Gestation = 23 Days

n

4

2

3

3

Gestation = 23 ½ Days

n

0

1

0

0

Dams with live young born

n

11

12

12

12

Dams with live young at Day 13post partum

n

11

12

12

12

Implants/dam

χ

16.3

16.9

16.5

15.5

Live offspring/dam at Day 1post partum

χ

15.1

15.7

14.6

14.0

Live offspring/dam at Day 4 BCpost partum

χ

14.9

14.8

14.4

12.3

Live offspring/dam at Day 4 ACpost partum

χ

9.8

9.9

10.0

9.7

Live offspring/dam at Day 7post partum

χ

9.7

9.8

9.9

9.6

Live offspring/dam at Day 13post partum

χ

9.7

9.6

9.9

9.6

Sex ratio: % males at Day 1post partum

χ

53.3

47.7

52.8

52.3

Sex ratio: % males at Day 4 BCpost partum

χ

52.4

48.5

53.1

53.1

Sex ratio: % males at Day 4 ACpost partum

χ

48.2

50.5

50.0

50.7

Sex ratio: % males at Day 7post partum

χ

48.7

50.6

50.5

50.5

Sex ratio: % males at Day 13post partum

χ

48.7

50.6

50.5

50.5

Litter weight (g) at Day 1post partum

χ

97.49

96.68

92.78

86.28

Litter weight (g) at Day 4 BCpost partum

χ

132.27

122.09

126.67

105.35

Litter weight (g) at Day 4 ACpost partum

χ

87.24

82.42

88.60

83.84

Litter weight (g) at Day 7post partum

χ

140.70

130.11

142.95

127.61

Litter weight (g) at Day 13post partum

χ

287.00

261.38

279.17

254.88

Male offspring weight (g) at Day 1post partum

χ

6.68

6.41

6.54

6.50

Male offspring weight (g) at Day 4 BCpost partum

χ

9.18

8.54

8.99

8.87

Male offspring weight (g) at Day 4 ACpost partum

χ

9.14

8.51

8.95

8.84

Male offspring weight (g) at Day 7post partum

χ

14.85

13.61

14.59

13.61

Male offspring weight (g) at Day 13post partum

χ

30.12

27.75

28.48

27.03

Female offspring weight (g) at Day 1post partum

χ

6.22

6.03

6.19

6.00

Female offspring weight (g) at Day 4 BCpost partum

χ

8.57

8.10

8.72

8.54

Female offspring weight (g) at Day 4 ACpost partum

χ

8.60

8.09

8.77

8.56

Female offspring weight (g) at Day 7post partum

χ

14.04

12.98

14.23

13.08

Female offspring weight (g) at Day 13post partum

χ

28.88

26.59

27.87

26.18

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

3

1

3

4

1

n

5

7

1

6

2

n

1

4

5

0

3

n

2

0

1

1

4

n

0

0

1

0

6

n

0

0

0

1


TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT (continued)

Observations

Dose Level (mg/kg bw/day)

0 (Control)

 250

500

1000

Post natal (live births minus offspring alive on Day 13post partum) – Excluding offspring culled on Day 4 post partum

 

 

 

 

 

0

n

6

6

6

4

1

n

5

2

4

1

2

n

0

2

1

3

3

n

0

0

0

2

4

n

0

1

0

1

6

n

0

1

0

1

n= Number

χ = Mean

Conclusions:
The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction. At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day. However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for approximately six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Where possible for each litter, offspring at Day 4 and Day 13 post partum were retained in 70% Industrial Methylated Spirit (IMS), for post partum skeletal evaluation. Examination of the offspring was restricted to Day 13 post partum offspring. 

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results…….

Adult Responses

Mortality

There was one unscheduled death on the study. Control female 16 was killed for animal welfare considerations after showing hunched posture and a soft mass under the left forelimb. Necropsy revealed a tear in the esophagus and a brown fluid-filled mass beneath the left forelimb, indicating that the decline in clinical condition had been due to accidental trauma during the dosing procedure.

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 250, 500 or 1000 mg/kg bw/day.

Body Weight

There was no effect of treatment on body weight or body weight gain for males throughout the study at 250, 500 or 1000 mg/kg bw/day. 

There was no effect of treatment on body weight and body weight gain of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.

Food Consumption& Food Conversion Efficiency

There was no effect of treatment on food consumption of either sex throughout the study at 250, 500 or 1000 mg/kg bw/day.

There was no effect of treatment on food conversion efficiency of either sex during the pre-pairing phase of the study or for males during the post-pairing phase of the study at 250, 500 or 1000 mg/kg bw/day.

Water Consumption

There was no effect of treatment on water consumption for either sex at 250, 500 or 1000 mg/kg bw/day.


Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Mating

There was no effect of treatment on mating performance at dosages of 250, 500 or 1000 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at dosages of 250, 500 or 1000 mg/kg bw/day.

Gestation Length

There was no effect of treatment on gestation length at dosages of 250, 500 or 1000 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the number of implantations, post-implantation loss, live birth index or sex ratios at dosages of 250, 500 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day survival to Day 4 of age was slightly inferior to control, leading to non-statistically significant lower litter size compared to control. Subsequent survival to Day 13 of age and litter size was comparable to controls. There was no effect of treatment on post-natal survival of the offspring from birth to termination (Day 13 of age) at dose levels of 250 and 500 mg/kg bw/day.

Offspring Growth and Development

At 1000 mg/kg bw/day offspring body weight gains for both sexes were statistically significantly lower than control during Days 4-7 and Days 7-13, leading to statistically significant lower overall body weight gain, compared to control, at termination on Day 13 of age. However, differences from control for offspring body weight resulting from this lower gain only attained statistical significance on Day 13 of age. Lower litter weights were apparent at this dosage on Day 4 post partum, compared to control, but were considered to reflect the lower litter size, compared to control, apparent at this stage of the study.

There was no effect of maternal treatment on offspring body weight, body weight gains or litter weights on Days 1 to 13 at 250 or 500 mg/kg bw/day.

Evaluation of ano-genital distance for offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Offspring Observations

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment. 

Pathology

Necropsy

Offspring

Macroscopic necropsy findings did not indicate any effect of treatment for offspring at dosages of 250, 500 or 1000 mg/kg bw/day. 

Adults

Macroscopic necropsy findings did not indicate any effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day. 

Skeletal Examination

There was no effect of treatment on skeletal development apparent during detailed skeletal examination of offspring at Day 13 of age at 250, 500 or 1000 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.

Organ Weights

There was no effect of treatment on organ weights for male reproductive tissues, or thyroid weights for either sex at dosages of 250, 500 or 1000 mg/kg bw/day. 

Histopathology

No findings were apparent which could be related to the administration of the test item in the tissues examined.


Conclusion

The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction. At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day. However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested). 

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline mentioned, but information given is relevant for assessment.
Principles of method if other than guideline:
8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.
In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to produce F3-Generation whose development was also observed.
GLP compliance:
no
Species:
rat
Strain:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
no details reported
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.
In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to procuce F3-Generation whose development was also observed.
Details on mating procedure:
8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.
In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to procuce F3-Generation whose development was also observed.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
once daily
Details on study schedule:
8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.
In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to procuce F3-Generation whose development was also observed.
Remarks:
Doses / Concentrations:
1.0 mL/kg bw/day
Basis:

No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.
In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to procuce F3-Generation whose development was also observed.
Positive control:
no data
Parental animals: Observations and examinations:
Treatment of females did not cause any adverse effects.
Oestrous cyclicity (parental animals):
Females were observed for the first oestrus. There was no deviation from that observed in controls noted.
Sperm parameters (parental animals):
no data
Litter observations:
Number of liiters as well as mena number of pups per litter were comparable to the concurrent controls.
Postmortem examinations (parental animals):
not performed
Postmortem examinations (offspring):
not performed
Statistics:
no data
Reproductive indices:
no data,
P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.
Offspring viability indices:
no data,
F1, F2, F3 pups: Somatic development and maturity as well as fertility were not affected and comparable to the controls.
P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.
Dose descriptor:
other: overall NOAEL
Effect level:
1 other: ml/kg bw
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
male/female
Basis for effect level:
other: P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.
Dose descriptor:
dose level:
Effect level:
1 other: mL/kg bw/day
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
male/female
Basis for effect level:
other: P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.
Remarks on result:
other: Generation: up to F2
Dose descriptor:
dose level:
Effect level:
1 other: mL/kg bw(day
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
male/female
Basis for effect level:
other: F1, F2, F3 pups: Somatic development and maturity as well as fertility were not affected and comparable to the controls.
Remarks on result:
other: Generation: up to F3
Dose descriptor:
dose level:
Effect level:
1 other: mL/kg bw/day
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
male/female
Basis for effect level:
other: P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.
Remarks on result:
other: Generation: up to F2
F1, F2, F3 pups: Somatic development and maturity as well as fertility were not affected and comparable to the controls.
Dose descriptor:
dose level:
Generation:
other: F1, F2, F3
Effect level:
1 other: mL/kg bw/day
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
male/female
Basis for effect level:
other: F1, F2, F3 pups: Somatic development and maturity as well as fertility were not affected and comparable to the controls.
Remarks on result:
other: Generation: up to F3
Reproductive effects observed:
not specified
Executive summary:

8 female rats received daily 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in Oleum olivarum DAB 6 over a period of 6 weeks. Then they were mated with untreated males for 16 days to produce F1-Generation. Pregnant females were determined and the average number of pups per litter (F1-animals) was determined and the general development of the pups including beginning of the first oestrus was noted.

In another trial these F1-females were mated with F1-males to produce F2-Generation and the pregnancy of dams, mean number of pups per litter, general development and beginning of the first oestrus were noted. The same procedure was used to procuce F3-Generation whose development was also observed.

P, F1, F2: All females mated successfully and the mean number of pups per litter was comparable with those of the control females.

F1, F2, F3 pups: Somatic development and maturity as well as fertility were not affected and comparable to the controls.

Endpoint:
fertility, other
Remarks:
Allen-Doisy test daily during 6 weeks of treatment.
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Allen-Doisy test with relevant information for fertility assessment.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
15 young female rats received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks. 15 female control rats received oleum olivarum DAB 6 only. 12/15 female rats were used for Allen-Doisy test (vaginal smears were taken daily to examine functionality of hypophysis-ovar-axis).
GLP compliance:
no
Species:
rat
Strain:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
no details given
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
15 young female rats received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks. 15 female control rats received oleum olivarum DAB 6 only. 12 /15female rats were used for Allen-Doisy test (vaginal smears were taken daily to examine functionality of hypophysis-ovar-axis).
Details on mating procedure:
Allen-Doisy test: vaginal smears were taken daily to examine functionality of hypophysis-ovar-axis
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
daily
Details on study schedule:
15 young female rats received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks. 15 female control rats received oleum olivarum DAB 6 only. 12/15 female rats were used for Allen-Doisy test (vaginal smears were taken daily to examine functionality of hypophysis-ovar-axis).
Remarks:
Doses / Concentrations:
1.0 mL/kg bw
Basis:

No. of animals per sex per dose:
12/15 females for the Allen-Doisy test
Control animals:
yes, concurrent vehicle
Details on study design:
15 young female rats received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks. 15 female control rats received oleum olivarum DAB 6 only. 12/15 female rats were used for Allen-Doisy test (vaginal smears were taken daily to examine functionality of hypophysis-ovar-axis).
Positive control:
no data
Parental animals: Observations and examinations:
Animals were not affected by treatment.
Oestrous cyclicity (parental animals):
Allen-Doisy test:
Vaginal smears did not show any disturbance of oestrus cyclicity.
Sperm parameters (parental animals):
no data
Litter observations:
no data
Allen-Doisy test
Postmortem examinations (parental animals):
no data
For general toxicity see section 7.5.1 (Repeated dose toxicity: oral)
Postmortem examinations (offspring):
no data
Statistics:
no data
Reproductive indices:
Allen-Doisy test:
Vaginal smears did not show any disturbance of oestrus cyclicity.
Offspring viability indices:
no data
Allen-Doisy test:
Vaginal smears did not show any disturbance of oestrus cyclicity.
Dose descriptor:
other: NOAEL(fertility test)
Effect level:
1 other: mL/kg bw
Based on:
other: 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6
Sex:
female
Basis for effect level:
other: Vaginal smears did not show any disturbance of oestrus cyclicity.
Remarks on result:
other: Generation: Allen-Doisy test
no data
Reproductive effects observed:
not specified
Executive summary:

12 of 15 young female rats that had received daily per gavage 1.0 mL/kg bw of a 50 % solution of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in oleum olivarum DAB 6 over a period of 6 weeks were introduced to the Allen-Doisy test which investigated the functionality of the hypophysis-ovar-axis by examination of vaginal smears for untypical cell morphology. Solvent controls are included in the test. Vaginal smears did not show any disturbance of oestrus cyclicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is consistency across the available studies (screening test, subacute and subchronic toxicity study (Fulcher 2019, Schladt 2013, Leggett 2020) according to OECD TGs 421, 407 and 408 GLP and the 3-generation study and Allen-Doisy test (Bornmann 1956). They do not provide evidence that ‘Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate’ is a reproductive toxicant.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

According to column 1 of 8.7.3., Annex IX of the REACH Regulation an EOGRTS according to OECD TG 443 is appropriate if “available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD TGs 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.” There is no indication that 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is toxic to fertility.

In an OECD Guideline 421 study (Reproduction / Developmental Toxicity Screening Test) the oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction.  At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day.  However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

Additional, there is a subchronic study available (Leggett 2020). During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses ofb100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

These findings were confirmed by a subacute toxicity study available (Schladt 2013). During the 4-week treatment period male and female Wistar rats received 0, 100, 300 or 1000 mg/kg bw/day ‘Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate’ by gavage. The study was performed according to OECD TG 407 and GLP conditions. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

Overall, the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD TGs 421 or 422 screening studies) indicate no adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.” There is no indication that 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is toxic to fertility. An EOGRT study according to OECD TG 443 is therefore not required.

Effects on developmental toxicity

Description of key information

In an OECD Guideline 421 study (Reproduction / Developmental Toxicity Screening Test) the oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction.  At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day.  However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

An OECD TG 414 developmental toxicity study in rats was conducted and delayed ossification at multiple locations in the high dose group was reported (Rashid 2016). Oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested). There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested. The observed delayed ossification at multiple locations in the high dose tested were considered to represent a development effect of treatment with the test item and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity (delayed ossifications) was 500 mg/kg bw/day (intermediate dose level).

In a further OECD TG 414 developmental toxicity study in rabbits was conducted. The purpose of this study was to assess the influence of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905 -983-8; also known and presented hereafter as Adimoll BO), on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Days 6-28 after mating) in the New Zealand White Rabbit. Three groups of 24 females received Adimoll BO at doses of 100, 200 or 400 mg/kg bw/day by oral gavage administration. A similarly constituted Control group received the vehicle, 1% aqueous methylcellulose, at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

In the course of the study three animals at 400 mg/kg bw/day were killed for welfare reasons. The death of two animals was attributed to treatment and a relationship to treatment could not be discounted for the death of the third animal. One Control animal was humanly killed due to an adverse reaction to the dosing procedure and one animal receiving 100 mg/kg bw/day was humanly killed due to non-dose related abortion of the litter.

However, as there were no signs associated with dosing or clinical signs considered to be related to treatment and no effect on body weight gain, adjusted body weight and gravid uterine weight, or numbers of resorptions, pre- and post‑implantation losses, litter size, sex ratio, total litter, placenta and fetal weights and there were no macroscopic findings associated with treatment, the death of these animals was attributed to an all-or-nothing individual animal reaction to treatment. External, visceral and skeletal examination of the fetuses at 100, 200 or 400 mg/kg bw/day revealed no findings that were considered treatment related.

It is concluded that oral gavage administration of Adimoll BO during Days 6 to 28 of gestation at 100, 200 or 400 mg/kg bw/day was well-tolerated except for individual females at 400 mg/kg bw/day. There was no adverse effect of treatment on the outcome of pregnancy or embryo-fetal survival, development or growth up to the highest dose; therefore, the No‑Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 200 mg/kg bw/day and for embryo‑fetal toxicity was 400 mg/kg bw/day.

Delayed ossifications are not confirmed by the OECD 414 study in rabbits. Therefore the delayed ossification in rats is regarded as a toxicological non-relevant incidental finding. For the risk assessment the NOAEL of the OECD Guideline 421 study (Reproduction / Developmental Toxicity Screening Test) is used. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 29 August 2017 Experimental Completion Date: date of final thyroid hormone report
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).
Deviations:
yes
Remarks:
The purpose and integrity of the study is therefore unaffected.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Kent, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 337 to 439g, and were approximately nine weeks old. The females weighed 225 to 294g, and were approximately eleven weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of another study (Envigo Study Number 41500056). Results showed the formulations to be stable for at least sixteen days. Formulations for this study were made and used within the known stability period; the bulk formulations were divided into daily aliquots and stored refrigerated (approximately 4°C in the dark) prior to use.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test item formulations were taken on two occasions and analyzed for concentration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 101-108% of the nominal concentration.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrous or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
Approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Daily
Duration of test:
Approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Treatment group)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Low (Treatment group)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Intermediate (Treatment group)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High (Treatment group)
No. of animals per sex per dose:
12 males and 12 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor based on the results of previous toxicity work including an Oral (Gavage) PreNatal Development Toxicity Study in the Rat (Envigo Study number: 41404097). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study. For the 14 days prior to pairing, pre-pairing vaginal smears were performed and assessed for females.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples. Where possible, litter size was standardized to ten offspring per litter. The excess offspring were retained in appropriate fixative and processed for possible skeletal evaluation.
vii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
viii. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. The remaining offspring were retained in appropriate fixative and processed for skeletal evaluation. All surviving offspring were killed and examined externally; the extent of any internal examination was dependent on the end point for which each offspring had been allocated.
ix. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Maternal examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded for all animals at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrous or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides, testes, seminal vesicles (with coagulating gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed after being placed in fixative (partial fixation).

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides♦ Prostate
Glans penis Seminal Vesicles (with coagulating gland)
Gross lesions Testes♦
LABC (levator ani-bulbocavernous) muscle Thyroid/Parathyroid
Mammary gland Uterus/Cervix (with oviducts)
Ovaries Vagina
Pituitary
♦ preserved in Modified Davidsons fluid

Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered Formalin.
All tissues were dispatched to the histology processing Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented and represents the consensus view of both pathologists.
Ovaries and uterine content:
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
Where possible, litters were culled to ten pups (five males and five females if possible) on Day 4 of age. Two culled offspring (same sex, if possible) were allocated for thyroid hormone analysis and the remaining culled offspring were allocated for possible skeletal examination.

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Necropsy
Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Offspring required for thyroid retention were killed by carbon dioxide asphyxiation with death confirmed by cervical dislocation. Remaining offspring were killed by intraperitoneal overdose of a suitable barbiturate agent, with death confirmed by the onset of rigor mortis. Examination of offspring allocated to thyroid hormone assessments and skeletal evaluation was restricted to an external examination. Offspring allocated for retention of the thyroids had an external examination and limited internal examination. Any decedent offspring or offspring showing abnormalities had an internal examination where possible.

Skeletal Evaluation
Where possible for each litter, offspring at Day 4 and Day 13 post partum were retained in 70% Industrial Methylated Spirit (IMS), for post partum skeletal evaluation. Examination of the offspring was restricted to Day 13 post partum offspring.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (females where possible to maximise the number of males available for day 13 post partum visible nipple counts; if offspring were of the same sex, samples from the same litter were pooled). If ten or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
Serum and plasma samples were taken from all adult males and females at termination.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator. A complete Thyroid Hormone Analysis report is presented. All plasma samples were retained at the Test Facility.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation,
Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Absolute Organ Weights, Body Weight-Relative Organ Weights, Skeletal Development Parameters (% incidence) and Thyroid Hormone (Thyroxine).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Indices:
See "Any Other Information on Materials and Methods"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 250, 500 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, increased post-dosing salivation was apparent for both sexes throughout the treatment period, although the incidence of this finding showed little consistency over time. Similar increased post-dosing salivation was apparent for both sexes at 500 mg/kg bw/day but at a much lower incidence. Such increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and probably indicates distaste or slight irritancy of the dosing formulations. Increased post-dosing salivation was also apparent for one female at 250 mg/kg bw/day but only for one day and this may reflect difficulty in dosing this particular animal on this isolated occasion.
Occasional instances of noisy respiration was apparent for a few animals (both sexes at all dosages including control) on the study. Whilst for females, the highest incidence of this clinical sign appeared at 1000 mg/kg bw/day, the incidence for females at lower dosages and for males at all dosages showed no dosage relationship. Additionally one male treated with 500 mg/kg bw/day and one male treated with 250 mg/kg bw/day showed pilo-erection and hunched posture for a short period of time. Overall, these findings were considered to reflect occasional difficulties of dosing particular animals on occasions during the study and not test item related.
One female treated with 250 mg/kg bw/day, one male and seven females treated with 500 mg/kg bw/day, and one male and three females treated with 1000 mg/kg bw/day showed generalized fur loss for a number of days. The higher incidence at 500 and 1000 mg/kg bw/day may be a consequence of the increased salivation at these dosages which may have prompted exaggerated grooming. This finding showed no dosage relationship, and was considered to be of no toxicological significance.
Occasional incidences of open wounds and/or scabbing were apparent for one, two and two males at 250, 500 or 1000 mg/kg bw/day respectively but not in any females in any dose group. This finding was considered to be incidental and unrelated to test item exposure.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one unscheduled death on the study. Control female 16 was killed for animal welfare considerations on Day 15 after showing hunched posture and a soft mass under the left forelimb. Necropsy revealed a tear in the esophagus and a brown fluid-filled mass beneath the left forelimb, indicating that the decline in clinical condition had been due to accidental trauma during the dosing procedure. The liver was also noted to be mottled in appearance
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on body weight or body weight gain for males throughout the study at 250, 500 or 1000 mg/kg bw/day.
There was no effect of treatment on body weight and body weight gain of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption of males throughout the pre-pairing and post-pairing phases of the study at 250, 500 or 1000 mg/kg bw/day.
There was no effect of treatment on food consumption of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption for either sex at 250, 500 or 1000 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in organ weights for male reproductive tissues at dosages of 250, 500 or 1000 mg/kg bw/day.
There were no statistically significant differences in thyroid weights for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic necropsy findings did not indicate any effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.
An isolated incidence of increased pelvic space (hydronephrosis) in the right kidney was observed in a male treated with 250 mg/kg bw/day. Findings of this nature are consistent with normally expected low incidence findings in laboratory maintained rats within this laboratory.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings were apparent which could be related to the administration of the test item in the tissues examined.
There were no test item-related microscopic findings following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of follicles and corpora lutea in the ovaries.
Pairing of female 71 and male 59 (Group 3) did not result in a pregnancy. Male 59 had mild or minimal changes in the testes and epididymides which may have affected fertility.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index at dosages of 250, 500 or 1000 mg/kg bw/day.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index at dosages of 250, 500 or 1000 mg/kg bw/day.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The intergroup distribution of gestation lengths observed during the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 250, 500 or 1000 mg/kg bw/day.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day, with all treated females, except one female at 500 mg/kg bw/day, showing regular cycling.

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 250, 500 or 1000 mg/kg bw/day.

One control female was terminated early and one female treated at 500 mg/kg bw/day was non-pregnant. The following assessment is based on the 11, 12, 11 and 12 females that successfully maintained a litter to Day 13 of age at 0 (Control), 250, 500 or 1000 mg/kg bw/day respectively.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
effects on pregnancy duration
food consumption and compound intake
gross pathology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
necropsy findings
organ weights and organ / body weight ratios
pre and post implantation loss
water consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
not specified
Description (incidence and severity):
At 1000 mg/kg bw/day offspring body weight gains for both sexes were statistically significantly lower than control during Days 4-7 and Days 7-13. However, differences from control for offspring body weight and cumulative body weight gain resulting from this lower gain only attained statistical significance on Day 13 of age. Lower litter weights were apparent at this dosage before culling on Day 4 post partum, compared to control, and no statistically significant effect was observed post culling on Day 4 or Days 7 and 13. Consequently, the lower litter weight is considered to reflect the slightly lower litter size before culling, compared to control.
There was no effect of maternal treatment on offspring body weight, body weight gains or litter weights on Days 1 to 13 at 250 or 500 mg/kg bw/day. Both sexes at 250 mg/kg bw/day and male offspring at 500 mg/kg bw/day showed statistically significantly lower body weight gains between Days 7‑13 post partum, however, overall body weight gain at termination on Day 13 of age was only statistically significantly lower than control for males at 250 mg/kg bw/day. Given the lack of consistent dosage relationship for these findings there were considered to be incidental and to reflect normal biological variation. Supporting this, it was noted that one litter for each dose level appeared to have unusually low offspring body weight gains, and one control litter appeared to have unusually high values.
Reduction in number of live offspring:
not specified
Description (incidence and severity):
There was no effect of treatment on post-natal survival of the offspring from birth to termination (Day 13 of age) at dose levels of 250 and 500 mg/kg bw/day.
At 1000 mg/kg bw/day, the high dose group, survival to Day 4 of age was slightly inferior to control, as indicated by a statistically significant decrease for viability index 1; between Days 1 and 4 (before culling) for control, 250, 500 and 1000 mg/kg bw/day groups viability index was 98.7% (±3.0%), 94.5% (±6.8%), 98.3% (±3.0%) and 88.0% (±12.8%) respectively. Subsequent viability index 2, survival between Day 4 after culling and Day 13 of age were comparable to controls; 99.1% (±3.0%), 96.5% (±7.0%), 99.1% (±3.0%) and 99.2% (±2.9%) for control, 250, 500 and 1000 mg/kg bw/day groups, respectively. The biological significance of the lower survival between Days 1 and 4 is therefore unclear.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 or offspring body weight on Day 1 of age.
Changes in postnatal survival:
not specified
Description (incidence and severity):
There was no effect of treatment on post-natal survival of the offspring from birth to termination (Day 13 of age) at dose levels of 250 and 500 mg/kg bw/day.
At 1000 mg/kg bw/day, the high dose group, survival to Day 4 of age was slightly inferior to control, as indicated by a statistically significant decrease for viability index 1; between Days 1 and 4 (before culling) for control, 250, 500 and 1000 mg/kg bw/day groups viability index was 98.7% (±3.0%), 94.5% (±6.8%), 98.3% (±3.0%) and 88.0% (±12.8%) respectively. Subsequent viability index 2, survival between Day 4 after culling and Day 13 of age were comparable to controls; 99.1% (±3.0%), 96.5% (±7.0%), 99.1% (±3.0%) and 99.2% (±2.9%) for control, 250, 500 and 1000 mg/kg bw/day groups, respectively. The biological significance of the lower survival between Days 1 and 4 is therefore unclear.
External malformations:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The type, incidence and distribution of skeletal findings apparent during detailed skeletal examination of offspring at Day 13 of age did not indicate any effect of maternal treatment on development at 250, 500 or 1000 mg/kg bw/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
skeletal malformations
visceral malformations
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other:
Remarks:
The No observed adverse Effect level for reproduction/developmental endpoints was reported to be the high dose level (1000mg/Kg body weight/day) on the basis of the effects seen on post- natal offspring survival and body weight gain at the highest dose level being limited, and the extent of effect were such that these findings were considered equivocal.
Abnormalities:
no effects observed
Developmental effects observed:
no

Summary of Reproductive Performance

 

Dose Group (mg/kg bw/day)

0 (Control)

250

500

1000

Males

 

 

 

 

Initial group size

12

12

12

12

Paired

11#

12

12

12

Induced pregnancy in female partner

11

12

11

12

Surviving to terminal necropsy

12

12

12

12

 

 

 

 

 

Females

 

 

 

 

Initial group size

12

12

12

12

Paired

11#

12

12

12

Non-Pregnant

0

0

1

0

Killedin extremis

1

0

0

0

Reared young to Day 13 of age

11

12

11

12

 # Female 16 killed in extremis prior to pairing, male 4 not paired with a female

Tabular Summary Report of Effects On Reproduction/Development

Observations

Dose Level (mg/kg bw/day)

0 (Control)

 250

500

1000

Paired animals

n

11M 11F

12M 12F

12M 12F

12M 12F

Females showing evidence of copulation

n

11

12

12

12

Pregnant females

n

11

12

12

12

Conception Days 1-4

n

11

12

11

12

Conception Day 14

n

0

0

1

0

Gestation = 22 Days

n

5

6

7

3

Gestation = 22 ½ Days

n

2

3

1

6

Gestation = 23 Days

n

4

2

3

3

Gestation = 23 ½ Days

n

0

1

0

0

Dams with live young born

n

11

12

12

12

Dams with live young at Day 13 post partum

n

11

12

12

12

Implants/dam

χ

16.3

16.9

16.5

15.5

Live offspring/dam at Day 1 post partum

χ

15.1

15.7

14.6

14.0

Live offspring/dam at Day 4 BC post partum

χ

14.9

14.8

14.4

12.3

Live offspring/dam at Day 4 AC post partum

χ

9.8

9.9

10.0

9.7

Live offspring/dam at Day 7 post partum

χ

9.7

9.8

9.9

9.6

Live offspring/dam at Day 13 post partum

χ

9.7

9.6

9.9

9.6

Sex ratio: % males at Day 1 post partum

χ

53.3

47.7

52.8

52.3

Sex ratio: % males at Day 4 BC post partum

χ

52.4

48.5

53.1

53.1

Sex ratio: % males at Day 4 AC post partum

χ

48.2

50.5

50.0

50.7

Sex ratio: % males at Day 7 post partum

χ

48.7

50.6

50.5

50.5

Sex ratio: % males at Day 13 post partum

χ

48.7

50.6

50.5

50.5

Litter weight (g) at Day 1 post partum

χ

97.49

96.68

92.78

86.28

Litter weight (g) at Day 4 BC post partum

χ

132.27

122.09

126.67

105.35

Litter weight (g) at Day 4 AC post partum

χ

87.24

82.42

88.60

83.84

Litter weight (g) at Day 7 post partum

χ

140.70

130.11

142.95

127.61

Litter weight (g) at Day 13 post partum

χ

287.00

261.38

279.17

254.88

Male offspring weight (g) at Day 1 post partum

χ

6.68

6.41

6.54

6.50

Male offspring weight (g) at Day 4 BC post partum

χ

9.18

8.54

8.99

8.87

Male offspring weight (g) at Day 4 AC post partum

χ

9.14

8.51

8.95

8.84

Male offspring weight (g) at Day 7 post partum

χ

14.85

13.61

14.59

13.61

Male offspring weight (g) at Day 13 post partum

χ

30.12

27.75

28.48

27.03

Female offspring weight (g) at Day 1 post partum

χ

6.22

6.03

6.19

6.00

Female offspring weight (g) at Day 4 BC post partum

χ

8.57

8.10

8.72

8.54

Female offspring weight (g) at Day 4 AC post partum

χ

8.60

8.09

8.77

8.56

Female offspring weight (g) at Day 7 post partum

χ

14.04

12.98

14.23

13.08

Female offspring weight (g) at Day 13 post partum

χ

28.88

26.59

27.87

26.18

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

3

1

3

4

1

n

5

7

1

6

2

n

1

4

5

0

3

n

2

0

1

1

4

n

0

0

1

0

6

n

0

0

0

1


TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT (continued)

Observations

Dose Level (mg/kg bw/day)

0 (Control)

 250

500

1000

Post natal (live births minus offspring alive on Day 13post partum) – Excluding offspring culled on Day 4 post partum

 

 

 

 

 

0

n

6

6

6

4

1

n

5

2

4

1

2

n

0

2

1

3

3

n

0

0

0

2

4

n

0

1

0

1

6

n

0

1

0

1

n= Number

χ = Mean

Conclusions:
The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction. At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day. However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for approximately six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Where possible for each litter, offspring at Day 4 and Day 13 post partum were retained in 70% Industrial Methylated Spirit (IMS), for post partum skeletal evaluation. Examination of the offspring was restricted to Day 13 post partum offspring. 

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results…….

Adult Responses

Mortality

There was one unscheduled death on the study. Control female 16 was killed for animal welfare considerations after showing hunched posture and a soft mass under the left forelimb. Necropsy revealed a tear in the esophagus and a brown fluid-filled mass beneath the left forelimb, indicating that the decline in clinical condition had been due to accidental trauma during the dosing procedure.

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 250, 500 or 1000 mg/kg bw/day.

Body Weight

There was no effect of treatment on body weight or body weight gain for males throughout the study at 250, 500 or 1000 mg/kg bw/day. 

There was no effect of treatment on body weight and body weight gain of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.

Food Consumption& Food Conversion Efficiency

There was no effect of treatment on food consumption of either sex throughout the study at 250, 500 or 1000 mg/kg bw/day.

There was no effect of treatment on food conversion efficiency of either sex during the pre-pairing phase of the study or for males during the post-pairing phase of the study at 250, 500 or 1000 mg/kg bw/day.

Water Consumption

There was no effect of treatment on water consumption for either sex at 250, 500 or 1000 mg/kg bw/day.


Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Mating

There was no effect of treatment on mating performance at dosages of 250, 500 or 1000 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at dosages of 250, 500 or 1000 mg/kg bw/day.

Gestation Length

There was no effect of treatment on gestation length at dosages of 250, 500 or 1000 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the number of implantations, post-implantation loss, live birth index or sex ratios at dosages of 250, 500 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day survival to Day 4 of age was slightly inferior to control, leading to non-statistically significant lower litter size compared to control. Subsequent survival to Day 13 of age and litter size was comparable to controls. There was no effect of treatment on post-natal survival of the offspring from birth to termination (Day 13 of age) at dose levels of 250 and 500 mg/kg bw/day.

Offspring Growth and Development

At 1000 mg/kg bw/day offspring body weight gains for both sexes were statistically significantly lower than control during Days 4-7 and Days 7-13, leading to statistically significant lower overall body weight gain, compared to control, at termination on Day 13 of age. However, differences from control for offspring body weight resulting from this lower gain only attained statistical significance on Day 13 of age. Lower litter weights were apparent at this dosage on Day 4 post partum, compared to control, but were considered to reflect the lower litter size, compared to control, apparent at this stage of the study.

There was no effect of maternal treatment on offspring body weight, body weight gains or litter weights on Days 1 to 13 at 250 or 500 mg/kg bw/day.

Evaluation of ano-genital distance for offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Offspring Observations

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment. 

Pathology

Necropsy

Offspring

Macroscopic necropsy findings did not indicate any effect of treatment for offspring at dosages of 250, 500 or 1000 mg/kg bw/day. 

Adults

Macroscopic necropsy findings did not indicate any effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day. 

Skeletal Examination

There was no effect of treatment on skeletal development apparent during detailed skeletal examination of offspring at Day 13 of age at 250, 500 or 1000 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.

Organ Weights

There was no effect of treatment on organ weights for male reproductive tissues, or thyroid weights for either sex at dosages of 250, 500 or 1000 mg/kg bw/day. 

Histopathology

No findings were apparent which could be related to the administration of the test item in the tissues examined.


Conclusion

The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated in adult animals, with no evidence of toxicity or effects on reproduction. At 1000 mg/kg bw/day, the biological significance of lower survival rate between Days 1 and 4, in the absence of any resulting statistically significant difference in litter size, and slightly lower offspring weight at termination was unclear and the NOEL for these observations is 500 mg/kg bw/day. However, based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested). 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
Principles of method if other than guideline:
The test item was administered by gavage to three groups each of twenty-four time-mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive, at dose levels 250, 500, or 1000 mg/kg bw/day. A further group of twenty-four time-mated females was exposed to the vehicle only (Arachis oil) to serve as a control.
Clinical signs, body weight change, food and water consumptions were monitored during the study.
All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter was examined for detailed skeletal development and the remaining half was subjected to detailed visceral examination.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD (SD) IGS BR
Details on test animals or test system and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches, two days apart. The first delivery consisted of two batches of thirty-two animals each at Day 1 and Day 0 of gestation, respectively, whilst the second delivery consisted of one batch of thirty-two animals at Day 1 of Gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation (sperm in the vaginal smear). At the start of treatment (Day 5 of gestation), the females weighed 200 to 335g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Test Item Preparation and Analysis
For the purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Arachis Oil. On each day of formulation preparation, for each concentration the required amount of Test Item was weighed out and added to the required volume of vehicle and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at 4°C in the dark until the day of use.
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of another study (Envigo Study Number 41500056), where formulations were shown to be stable for at least eight days when stored at 4°C in the dark. Formulations for this study were made and used within the known stability period.
Representative samples of dosing formulations were analyzed for concentration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No 905-983-8) at Envigo Research Limited Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 96-101% of nominal concentration confirming the accuracy of the preparation procedure.

Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Control 0 4 0 24 (1-24)
Low 250 4 62.5 24 (25-48)
Intermediate 500 4 125 24 (49-72)
High 1 000 4 250 24 (73-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of another study (Envigo Study Number 41500056), where formulations were shown to be stable for at least eight days when stored at 4°C in the dark. Formulations for this study were made and used within the known stability period.
Representative samples of dosing formulations were analyzed for concentration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No 905-983-8) at Envigo Research Limited Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 96-101% of nominal concentration confirming the accuracy of the preparation procedure.
Details on mating procedure:
The test item was administered by gavage to three groups each of twenty-four time-mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats.
Duration of treatment / exposure:
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Duration of test:
All females were terminated on Day 20 of gestation.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 female/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected on available toxicity data including a preliminary pre-natal development toxicity study (Envigo Study Number 41500056), where dose levels up to 1000 mg/kg bw/day were well tolerated. The oral route was selected as the most appropriate route of exposure for this type of study, and the results of the study are believed to be of value in predicting the potential toxicity of the test item to man.
Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 and on Day 5 (before the start of treatment) 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for obvious changes.
Ovaries and uterine content:
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

Implantation types were divided into:

Early Death:
No visible distinction between placental/decidual tissue and embryonic tissue

Late Death:
Separate embryonic/fetal and placental tissue visible

Dead Fetus:
A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for either skeletal alterations (skeletal examinations) or soft tissue alterations (visceral examinations).
Fetal examinations:
Visceral Examinations
At necropsy, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. These fetuses were subsequently transferred to distilled water prior to examination for visceral anomalies under a low power binocular microscope and following examination transferred to 10% Buffered Formalin for storage.
Visceral examinations commenced with an external assessment of general appearance, including the limbs, digits, genitals, anus, tail and umbilicus. Once completed the head, and subsequently the tongue was removed, for examination of the tongue, palate and surrounding tissue including the teeth, genioglossus and nasopharynx. Serial sections were made of the head and lower jaw to enable a detail examination of brain morphology and the lower jaw was also sectioned to allow examination of the incisors, multicuspid teeth and genioglossus muscle. The skin was opened up to allow further visceral examination and the internal sex of the fetuses was confirmed. The position of the umbilical artery and the liver, stomach spleen, pancreas and intestines assessed. These tissues were then removed to enable examination of the under lying abdominal tissues including the kidneys which were transversely cut to enable assessment of the cortex, medulla and papilla. Following these examinations the thoracic tissues were examined, commencing first with the diaphragm lungs, azygo us vein and thymus. The lungs and thymus were subsequently removed to allow assessment of the heart and cervicothoracic blood vessels. Examination of the heart included the external size and shape of the ventricles and atria, the entry and exit of the blood vessels around the heart and assessment of the foramen ovale, atrio-ventricular valves, semi-lunar valves, ventricular septum and general proportions of ventricular walls and cavities.

Skeletal Examinations
At necropsy, fetuses not allocated to visceral examinations were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and alterations and storage.
For assessment, fetuses were placed in a petri dish containing glycerol and examined using a microscope. Fetuses were examined whole but for evaluation and reporting purposes the skeleton was divided into the following regions: skull, vertebral column, ribs, sternebrae, pectoral girdle, pelvic girdle, fore limbs and hind limbs. A peer review (approximately 10% of the total number of litters examined) was performed as part of the overall skeletal examination procedures for the study.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea - number of implantations) : (number of corpora lutea)] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses) : (number of implantations)] x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = [(Total number of fetuses : Number of male fetuses)] x 100
Historical control data:
Historical control data are available for:
Normal Range Data for Pre-Natal Study Gestation Body Weights in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study Gestation Food Consumption in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Litter Data in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study External Fetal Observations in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Visceral Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Skeletal Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations up to a dose level of 1000 mg/kg bw/day that were considered to be related to the toxicity of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Throughout the dosing period, there was no effect of treatment with the test item at any dose level on body weight development.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Throughout the dosing period, there was no effect of treatment with the test item at any dose level on food consumption.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment at any dose level on food conversion efficiency.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any obvious intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of adult females on Day 20 of gestation did not identify any findings at 250, 500 or 1000 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no incidences of total litter loss by resorption and the following assessment is based on the 24 females each with live fetuses on Day 20 of gestation at 0 (control), 250, 500 and 1000 mg/kg bw/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no incidences of total litter loss by resorption and the following assessment is based on the 24 females each with live fetuses on Day 20 of gestation at 0 (control), 250, 500 and 1000 mg/kg bw/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females on the study were found to be pregnant at necropsy.
Other effects:
no effects observed
Details on maternal toxic effects:
No treatment-related effects were detected on litter data as assessed by the mean number of implantations, in utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, post-implantation loss or sex ratio at 250, 500 or 1000 mg/kg bw/day. At 500 mg/kg bw/day, the number of live male fetuses was statistically significantly lower than controls (control, low, intermediate and high dose: 7.0, 6.9, 5.3* and 6.4, respectively). There was no dose-related trend and with all individual values within the historical control data ranges (1-13), this observation was considered to be due to normal biological variation.

There was no effect of maternal treatment with the test item at any dose level on group mean fetal or litter weights. At 500 mg/kg bw/day, the total placental weight was statistically significantly lower than controls (control, low, intermediate and high dose: 8.689g, 8.093g, 7.452g*, 8.483g, respectively). There was no dose-relationship and all individual values were within the historical control data ranges (1.28g - 12.48g). Group mean placental weights were comparable with controls. The total placental weight observation at 500 mg/kg bw/day may be due to the slightly lower litter size at this dose level (control, low, intermediate and high dose: 13.9, 13.3, 12.3 and 13.9, respectively), which in itself was deemed to be an incidental observation.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected on litter data as assessed by the mean number of implantations, in utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, post-implantation loss or sex ratio at 250, 500 or 1000 mg/kg bw/day. At 500 mg/kg bw/day, the number of live male fetuses was statistically significantly lower than controls (control, low, intermediate and high dose: 7.0, 6.9, 5.3* and 6.4, respectively). There was no dose-related trend and with all individual values within the historical control data ranges, this observation was considered to be due to normal biological variation.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected on litter data as assessed by the mean number of implantations, in utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, post-implantation loss or sex ratio at 250, 500 or 1000 mg/kg bw/day. At 500 mg/kg bw/day, the number of live male fetuses was statistically significantly lower than controls (control, low, intermediate and high dose: 7.0, 6.9, 5.3* and 6.4, respectively). There was no dose-related trend and with all individual values within the historical control data ranges, this observation was considered to be due to normal biological variation.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected on litter data as assessed by the mean number of implantations, in utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, post-implantation loss or sex ratio at 250, 500 or 1000 mg/kg bw/day. At 500 mg/kg bw/day, the number of live male fetuses was statistically significantly lower than controls (control, low, intermediate and high dose: 7.0, 6.9, 5.3* and 6.4, respectively). There was no dose-related trend and with all individual values within the historical control data ranges, this observation was considered to be due to normal biological variation.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there was no consistent evidence of external or visceral abnormalities up to a dose level of 1000 mg/kg bw/day.

External evaluation of the fetuses/litters identified one small fetus from the 1000 mg/kg bw/day dose group (Litter 75) showing anal atresia, absent tail and malrotation of hindlimb with, visceral examination of this fetus also revealing malpositioned kidneys. In the absence of any supporting evidence from the remaining specimens, this cluster of abnormalities in one underdeveloped fetus was considered unlikely to be related to treatment with the test item.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal examination of the fetuses/litters revealed a number of findings, which achieved statistical significance in some instances. These finding were confined to delayed skeletal ossification and included the following:

At 1000 mg/kg bw/day, statistically significant intergroup differences included an increase in the number of fetuses/litters showing incomplete ossification of interparietal, occipital (supra-occipital) and femur regions. In addition, the number of fetuses/litters lacking ossification of the metacarpals was statistically significantly higher than controls whilst the number of fetuses/litters with ossified forepaw phalanges was statistically significantly lower than controls. A number of other notable differences in ossification parameters were also observed at this dose level; some of these differences showed dose-related trends and most individual mean values were outside the historical control data ranges. There was no evidence of an adverse impact of treatment with the test item up to a dose level of 1000 mg/kg bw/day on maternal or fetal weight. Taking into account the number and extent of skeletal findings, these observations were collectively considered to represent delayed ossification at multiple locations.

At 500 mg/kg bw/day, statistically significant differences were confined to an increase in the number of fetuses/litters showing ‘not ossified’ metacarpals and a lower number of fetuses/litters showing ossified forepaw phalanges. The effect on metacarpals extended to the 250 mg/kg bw/day dose group but this did not attain statistical significance. These changes were dose-related but the corresponding mean values remained within the historical control data ranges. It is also worth noting that the mean control value for metacarpals in the present study was relatively low in comparison with the corresponding control values in similar studies performed around the same timepoint at this Test Facility, whilst the mean control value for forepaw phalanges in the present study was relatively high in relation to the corresponding control values in these studies. The number of fetuses/litters with incomplete ossification of the nasal, humerus and femur regions from the 500 mg/kg bw/day dose group also showed slight increases in relation to controls, with the mean value for the latter parameter marginally exceeding the top end of the historical control data range. Statistical significance was not achieved for these findings and taking into account the overall results in this study and historical control data information, skeletal development observations at 500 or 250 mg/kg bw/day were considered to be within the normal biological variation and not adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination of the fetuses/litters from the 1000 mg/kg bw/day dose group showed a single fetus with enlarged cardiac atrium and thickened ventricular wall. Such observations are known to occur spontaneously in control rat populations of this strain and one fetus from the control group in this study also had an enlarged atrium. No other fetuses from the test item-treated dose group showed similar findings and, as such these observations were considered to have arisen spontaneously and are therefore unrelated to treatment with the test item. When compared with controls, other notable visceral findings included a statistically significant increase in the number of fetuses/litters from the 1000 mg/kg bw/day dose group showing dilated ureter (controls and high dose: 1.1 and 6.0*, respectively) and an increase in the number of fetuses/litters from all test item-treated dose groups exhibiting kinked ureter (control, low, intermediate and high dose: 2.7, 6.0, 6.2 and 8.9, respectively); the intergroup differences for the latter parameter did not attain statistical significance. A dose-relationship was only apparent for kinked ureter, but mean values, in particular from the control group, were below the historical control data ranges. Taking into account the lack of any associated findings and the minor nature of these variations, they were considered unlikely to represent an adverse effect of maternal treatment with the test item on fetal development.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).

There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested. The observed delayed ossification at multiple locations in the high dose tested were considered to represent a development effect of treatment with the test item and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity (developmental toxicity) was 500 mg/kg bw/day (intermediate dose level).
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
effects observed, treatment-related
Localisation:
other: delayed ossification at multiple locations
Description (incidence and severity):
Skeletal examination of the fetuses/litters revealed a number of findings, which achieved statistical significance in some instances. These finding were confined to delayed skeletal ossification and included the following:

At 1000 mg/kg bw/day, statistically significant intergroup differences included an increase in the number of fetuses/litters showing incomplete ossification of interparietal, occipital (supra-occipital) and femur regions. In addition, the number of fetuses/litters lacking ossification of the metacarpals was statistically significantly higher than controls whilst the number of fetuses/litters with ossified forepaw phalanges was statistically significantly lower than controls. A number of other notable differences in ossification parameters were also observed at this dose level; some of these differences showed dose-related trends and most individual mean values were outside the historical control data ranges. There was no evidence of an adverse impact of treatment with the test item up to a dose level of 1000 mg/kg bw/day on maternal or fetal weight. Taking into account the number and extent of skeletal findings, these observations were collectively considered to represent delayed ossification at multiple locations.

At 500 mg/kg bw/day, statistically significant differences were confined to an increase in the number of fetuses/litters showing ‘not ossified’ metacarpals and a lower number of fetuses/litters showing ossified forepaw phalanges. The effect on metacarpals extended to the 250 mg/kg bw/day dose group but this did not attain statistical significance. These changes were dose-related but the corresponding mean values remained within the historical control data ranges. It is also worth noting that the mean control value for metacarpals in the present study was relatively low in comparison with the corresponding control values in similar studies performed around the same timepoint at this Test Facility, whilst the mean control value for forepaw phalanges in the present study was relatively high in relation to the corresponding control values in these studies. The number of fetuses/litters with incomplete ossification of the nasal, humerus and femur regions from the 500 mg/kg bw/day dose group also showed slight increases in relation to controls, with the mean value for the latter parameter marginally exceeding the top end of the historical control data range. Statistical significance was not achieved for these findings and taking into account the overall results in this study and historical control data information, skeletal development observations at 500 or 250 mg/kg bw/day were considered to be within the normal biological variation and not adverse.
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Summary of Female Performance

            Number of Females at Dose Level (mg/kg bw/day)
 Category  0 (Control)  250  500  1000
Initial Group Size  24  24  24  24 
Deaths  0  0  0  0
 Pregnant  24  24  24  24

 With live Fetuses at Day 20

Gestation

 24  24  24  24

Summary Incidence of Daily Clinical Observations

 Dose Level (mg/kg bw/day) Number of Animals   Clinical Observations

Number Showing Effect

(Days post coitum affected)

0 (Control)   24  No abnormalities detected  -
 250  24  No abnormalities detected  -
 500  24

  No abnormalities detected

-

 1000  24

 Increased Post-Dose Salivation

1 (16)

Group Mean Body Weight Values

 

  Body Weight (g) at Day of Gestation                          

Dose Level

(mg/kg

bw/day)

 

 3

 5

 6

 7

 8

 11

 14

 17

 20

 0 (Control)

 mean

sd

n

246.4

28.9

24 

262.8

30.3

24 

264.2

30.7

24 

269.5

30.7

24 

273.4

30.8

24 

292.5

32.3

24 

310.6

32.7

24 

339.1

34.8

24 

385.6

38.3

24 

250 

mean

sd

n

246.1

21.5

24 

262.1

23.6

24 

262.5

22.9

24 

268.0

24.2

24 

273.7

24.8

24 

290.9

26.5

24 

309.8

27.8

24 

337.6

30.1

24 

381.5

35.7

24 

500 

 mean

sd

n

245.1

20.2

24 

261.3

20.4

24 

263.5

20.8

24 

268.1

22.0

24 

273.8

21.9

24 

289.4

23.2

24 

306.3

24.7

24 

333.7

26.0

24 

375.2

29.2

24 

1000 

 mean

sd

n

247.0

25.0

24 

264.8

27.3

24 

265.4

27.8

24 

270.8

27.8

24 

277.4

28.4

24 

295.2

30.6

24 

311.0

31.9

24 

339.8

33.9

24 

385.9

37.1

24 

Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

        Body Weight (g) on Days of Gestation

 Body Weight

Change (g)

during Days of

Gestation

Gravid

Uterus

Weight (g) 

Adjusted

Body

Weight (g)

Day 20 

Adjusted

Body

Weight

Change (g)

 Dose Level (mg/kg bw/day)

 5  20  5-20  

  Day 20

 5-20

 0 (Control)

mean

sd

n

262.8

30.3

24 

385.6

38.3

24 

122.8

14.2

24 

88.170

9.906

24 

297.5

32.8

24 

34.6

10.8

24 

 250

mean

sd

n

262.1

23.6

24 

 381.5

35.7

24

119.4

18.3

24 

83.622

15.563

24 

297.8

30.9

24 

35.8

11.5

24 

 500

mean

sd

261.3

20.4

24 

375.2

29.2

24 

113.8

14.9

24 

79.000

12.975

24 

296.2

27.0

24 

34.8

12.3

24 

 1000

 mean

sd

264.8

27.3

24 

385.9

37.1

24 

121.0

16.8

24 

87.686

14.208

24 

298.2

32.2

24 

33.4

9.9

24 

Summary Incidence of Necropsy Findings

            Dose Level (mg/kg bw/day)
   0 (Control)  250  500  1000
 Terminal death
Number of animals examined  24 24  24  24
 No abnormalities detected  24  24  23  24

Group Mean Litter Data Values

Dose Level

(mg/kg

bw/day) 

 

Number

of

Corpora

Lutea

 

 

Number

of Implants 

 

       Number of

Embryonic/Fetal

Deaths

Early Late Total

    Implantation

Loss

%

Pre Post

Number of Live

Implants       

Male Female Total

 %

Male

Fetuses

Mean

Male

Fetal

Weight

(g) 

Mean

Female

Fetal

Weight

(g) 

Mean

Fetal

Weight

(g) 

Mean

Placental

Weight

(g) 

 Litter

Weight

(g)

Total

Placental

Weight

(g) 

0 (Control) 

mean

sd

 14.4

1.9

24

14.1

1.8

24 

0.1

0.3

24 

0.1

0.3

24 

0.2

0.4

24 

 1.9

3.5

24

1.1

2.6

24 

7.0

2.4

24 

6.9

2.3

24 

13.9

1.8

24 

50.4

15.5

24 

4.121

0.244

24 

3.941

0.251

24 

4.039

0.223

24 

0.626

0.061

24 

56.074

6.693

24 

8.689

1.240

24 

250 

mean

sd

14.5

1.6

24 

13.8

2.3

24 

0.4

0.6

24 

0.1

0.3

24 

0.5

0.7

24 

4.8

8.7

24 

4.1

6.6

24 

6.9

2.2

24 

6.5

2.5

24 

13.3

2.5

24 

51.8

14.8

24 

4.115

0.266

24 

3.866

0.278

24 

3.996

0.256

24 

0.613

0.092

24 

53.453

11.093

24 

8.093

1.671

24 

500 

mean

sd

n

13.6

2.2

24 

13.1

2.1

24 

0.6

1.1

24 

0.2

0.4

24 

0.8

1.2

24 

3.8

5.4

24 

5.8

9.3

24 

5.3*

1.7

24 

 7.0

2.1

24

12.3

2.2

24 

43.5

13.3

24 

4.145

0.279

24 

3.980

0.212

24 

4.054

0.223

24 

0.604

0.065

24 

49.978

8.826

24 

7.452*

1.596

24 

1000 

mean

sd

14.8

2.7

24 

14.3

2.5

24 

0.3

0.6

24 

0.1

0.3

24 

0.4

0.8

24 

3.9

4.4

24 

2.5

5.3

24 

6.4

2.1

24 

7.5

2.3

24 

13.9

2.5

24 

46.2

13.8

24 

4.087

0.297

24 

3.858

0.234

24 

3.966

0.252

24 

0.613

0.067

24 

54.855

9.846

24 

8.483

1.737

24 

Summary Incidence of Fetal External Findings

                                    Dose level (mg/kg bw/day)
         0 (Control)        250        500        1000
                                    Number of fetuses (litters) examined
         334 (24)        320 (24)        296 (24)        333 (24)
 External findings  NF  NL  %†  NF  NL  %†  NF  NL  %†  NF  NL  %†

Total Number Affected

Hematoma - on head

Small

Malrotation of hind limbs

anal atresia

Tail absent

Subcutaneous hemorrhage - on neck

Hematoma - on right hind limb

Pale

3

0

2

0

0

0

0

0

1

3

0

2

0

0

0

0

0

1

0.9

0.0

0.6

0.0

0.0

0.0

0.0

0.0

0.3

4

2

1

0

0

0

0

1

0

3

2

1

0

0

0

0

1

0

1.4

0.7

0.3

0.0

0.0

0.0

0.0

0.4

0.0

2

0

1

0

0

0

0

0

1

2

0

1

0

0

0

0

0

1

0.6

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.3

3

0

2

1

1

1

1

0

0

2

0

2

1

1

1

1

0

0

0.8

0.0

0.6

0.3

0.0

0.0

0.3

0.0

0.0

Summary Incidence of Fetal Visceral Findings

 Visceral Findings                                   Dose Level (mg/kg bw/day)
         0 (Control)        250        500        1000
                                    Number of Fetuses (litters) Examined
         173 (24)        167 (24)        155 (24)        174 (24)

 

 NF  NL  %†  NF  NL  %†  NF  NL  %†  NF  NL  %†

External/General

Mass

Anal atresia

Tail - absent

Hindlimb - malrotated

digit - fused

Digit - extra

1

0

0

0

0

1

0

0

0

0

0.6

0.0

0.0

0.0

0.0

0.0

0

0

0

0

0

0

0

0

0

0

0

0.0

0.0

0.0

0.0

0.0

0.0

0

0

0

0

1

1

0

0

0

0

1

1

0.0

0.0

0.0

0.0

0.6

0.6

0

1

1

1

0

0

0

1

1

1

0

0.0

0.5

0.5

0.5

0.0

0.0 

Head

Tongue- short

Rugae - non-uniform patterning

Nasal cavity - enlarged

Brain - olfactory ventricle - enlarged

Brain - lateral ventricle - enlarged

Brain - third ventricle - enlarged

Brain - cerebral aqueduct - enlarged

1

16

0

1

1

1

1

1

10

0

1

1

1

1

0.6

8.9

0.0

0.5

0.5

0.5

0.6

0

15

0

0

0

0

0

0

8

0

0

0

0

0

 

0.0

8.6

0.0

0.0

0.0

0.0

0.0

0

14

1

0

0

0

0

0

11

1

0

0

0

0

0.0

8.6

0.7

0.0

0.0

0.0

0.0

0

7

0

0

0

0

0

0

6

0

0

0

0

0

0.0

4.5

0.0

0.0

0.0

0.0

0.0

Abdomen

Liver - additional lobe between right and left median

Umbilical artery - left-sided

Testis - undescended

Testis - partially undescended

Ureter - kinked

Ureter - dilated

Kidney - malpositioned

Renal pelvis cavitation - increased

Renal papilla - absent

1

1

0

1

5

2

0

9

1

1

1

0

1

5

2

0

7

1

0.5

0.5

0.0

0.5

2.7

1.1

0.0

5.1

0.6

0

1

0

4

11

6

0

9

0

0

1

0

2

8

5

0

9

0

0.0

0.6

0.0

2.1

6.0

3.3

0.0

5.3

0.0

0

0

0

1

11

2

0

13

4

0

0

0

1

6

1

0

8

4

0.0

0.0

0.0

0.6

6.2

1.0

0.0

8.2

2.4

0

1

1

5

16

11

1

11

1

0

1

1

5

13

9

1

9

1

0.0

0.6

0.5

2.8

8.9

6.0*

0.5

6.6

0.5

Thorax

Thymus - lobe partially undescended

Right subclavian artery - retro-oesophageal

Atrium - enlarged

Ventricular wall - thickened

Lungs - irregular surface throughout

 

3

0

1

0

1

  

3

0

1

0

1

  

2.0

0.0

0.5

0.0

0.5

 

7

1

0

0

0

  

5

1

0

0

0

  

4.5

0.6

0.0

0.0

0.0

 

8

0

0

0

0

 

5

0

0

0

0

  

4.7

0.0

0.0

0.0

0.0

  

5

0

1

1

0

 

4

0

1

1

0

2.5

0.0

0.5

0.5

0.5

 Total

 32

 17

 18.4

 38

 20

 21.8

 37

 18

 22.6

 34

19 

 19.1

NOTE: a fetus may appear in more than one category

Summary Incidence of Fetal Skeletal Findings

 Skeletal Findings                                   Dose Level (mg/kg bw/day)
         0 (Control)        250        500        1000
                                    Number of Fetuses (litters) Examined
         148 (22)        155 (23)        145 (23)        151 (24)

 

 NF  NL  %†  NF  NL  %†  NF  NL  %†  NF  NL  %†

Skull

Fontanelle (anterior) - large

Nasal - incomplete ossification

Frontal - incomplete ossification

Frontal - unossified area

Intraorbital process of frontal - incomplete ossification

Parietal - incomplete ossification

Parietal - unossified area(s)

Interparietal - incomplete ossification

Intraparietal - unossified area(s)

Occipital (Supra-occipital) - incomplete ossification

Occipital (Supra-occipital) - unossified area(s)

Squamosal - incomplete ossification

Squamosal - unossified area(s)

Jugal - incomplete ossification

Zygomatic process of maxilla - incomplete ossification

Zygomatic process of maxilla - fused to jugal 

Zygomatic process of squamosal - incomplete ossification

Premaxilla - incomplete ossification

Hyoid - incomplete ossification

Hyoid - not ossified

Mandible - incomplete ossification

Presphenoid - incomplete ossification

Presphenoid - not ossified

Basisphenoid - incomplete ossification

1

8

5

2

0

10

1

19

1

11

1

16

2

9

16

0

3

3

5

4

1

6

0

0

1

6

5

2

0

5

1

10

1

5

1

9

2

4

11

0

3

3

5

4

1

3

0

0

0.6

4.9

3.1

1.2

0.0

5.9

0.6

11.7

0.7

6.6

0.7

9.9

1.2

5.6

9.7

0.0

2.0

1.9

3.0

2.5

0.8

3.5

0.0

0.0

0

9

3

1

0

6

0

15

0

10

2

12

0

3

10

0

2

0

9

5

0

0

0

0

0

5

3

1

0

6

0

10

0

6

2

10

0

2

7

0

2

0

7

4

0

0

0

0

0.0

6.2

2.0

0.7

0.0

3.9

0.0

9.5

0.0

6.3

1.4

7.4

0.0

1.9

6.6

0.0

1.2

0.0

5.8

3.6

0.0

0.0

0.0

0.0

0

16

8

0

0

11

1

22

0

9

1

18

1

4

12

1

1

2

11

3

0

0

0

1

0

9

4

0

0

6

1

10

0

6

1

8

1

2

7

1

1

2

7

3

0

0

0

1

0.0

10.0

4.9

0.0

0.0

7.0

1.4

14.5

0.0

5.8

1.0

11.5

0.7

2.5

8.1

0.6

0.6

1.3

6.9

2.0

0.0

0.0

0.0

0.6

1

27

22

9

3

31

0

48

1

29

3

44

1

11

30

0

5

5

13

5

0

5

1

0

1

12

10

5

1

12

0

20

1

13

3

14

1

7

12

0

5

4

11

4

0

3

1

0

0.5

17.0

14.1

5.5

2.5

20.2

0.0

31.1**

0.5

18.4*

2.2

29.1

1.0

7.7

19.1

0.0

3.0

3.3

8.1

2.8

0.0

3.4

0.6

0.0

Vertebral Column

Ventral arch of vertebra 1 - ossification present

Cervical (neural) arch - incomplete ossification

Thoracic centrum - incomplete ossification

Thoriac centrum - not ossified

Thoracic centrum - bipartite ossification

Thoracic centrum - dumb-bell-shaped

Thoracic centrum - asymmetrically ossified

Sacral (neural) arch - incomplete ossification

Sacral (neural) arch - not ossified

Caudal vertebrae - less than 4 ossified

35

6

5

0

2

7

0

22

2

32

16

4

4

0

2

7

0

11

2

15

22.1

3.6

3.1

0.0

1.2

4.7

0.0

13.9

1.2

19.0

31

4

5

0

2

9

0

18

1

38

17

4

4

0

2

8

0

9

1

14

19.6

2.4

3.5

0.0

1.3

6.4

0.0

12.0

0.6

25.5

31

2

2

1

0

10

0

22

2

35

14

2

2

1

0

8

0

9

2

13

21.9

1.3

1.3

0.7

0.0

8.0

0.0

15.1

1.3

24.5

13

5

4

0

1

12

1

41

13

42

10

4

4

0

1

9

1

17

6

18

8.1

3.0

3.0

0.0

0.8

7.9

0.8

25.6

8.1

26.4

Ribs

Ossification centre - associated with 7th cervical vertebra

Ossification centre - associated with 1st lumbar vertebra

One or more ribs - wavy

Oneor more rips - thickened

Rib - short

Rib - rudimentary

Rib - incomplete ossification

Costal cartilage - misaligned

Costal cartilage - not fused to sternebra

0

2

0

2

2

2

1

4

7

0

2

0

2

2

2

1

3

7

0.0

1.2

0.0

1.5

1.2

1.3

0.6

2.7

4.3

1

3

1

1

4

5

0

3

8

1

3

1

1

3

3

0

2

5

0.6

2.0

0.7

0.7

2.3

2.6

0.0

2.1

7.8

1

2

1

1

0

3

1

5

15

1

2

1

1

0

3

1

4

12

0.6

1.2

0.6

0.6

0.0

2.1

0.6

3.3

11.6

1

6

0

0

0

2

0

2

9

1

5

0

0

0

2

0

2

6

0.6

4.3

0.0

0.0

0.0

1.2

0.0

1.2

5.7

Sternebrae

Sternebra - incomplete ossification

Sternebra - not ossified

Sternebra - bipartite ossification

Sternebra - missaligned

Xiphoid cartilage - partially split

3

1

2

5

14

 

2

1

2

4

10

1.8

0.6

1.2

3.0

9.5

5

0

1

3

10

4

0

1

2

6

3.5

0.0

0.7

2.2

6.3 

0

2

1

9

10

0

2

1

9

8

0.0

1.3

0.7

6.3

7.6

1

0

0

2

8

1

0

0

2

7

0.6

0.0

0.0

1.3

4.3

Pectoral Girdle

Scapula - misshapen

4

3

2.6

3

2

1.6

1

1

0.7

1

1

0.5

Pelvic Girdle

Ischium - incomplete ossification

Pubis - not ossified

Pubis - incomplete ossification

3

1

8

2

1

5

1.8

0.6

5.0

4

0

6

3

0

4

2.6

0.0

4.0

0

1

5

0

1

5

0.0

0.6

3.3

2

10

20

2

5

11

1.2

5.5

12.7

Forelimbs

Metacarpal - not ossified

Metacarpal - incomplete ossification

Forepaw phalanges - 1 or more – ossified

Humerus - incomplete ossification

Humerus - hole

23

3

41

2

1

8

2

18

2

1

13.9

1.8

24.9

1.4

0.5

35

3

34

1

1

14

2

13

1

1

24.2

2.1

21.3

0.5

0.6

37

8

21

5

0

15

5

10

3

0

25.8*

5.0

14.4*

3.4

0.0

89

4

2

7

0

23

3

2

4

0

54.5***

2.4

1.2***

4.8

0.0

Hindlimbs

Metatarsal - 1st - ossified

Metatarsal - incomplete ossification

Femur - incomplete ossification

5

0

10

3

0

6

3.3

0.0

6.7

0

0

10

0

0

6

0.0

0.0

8.1

2

1

18

2

1

9

1.4

0.6

12.8

0

4

35

0

3

15

0.0

2.3

22.6**

 Total  128  24 80.0  125  24  82.5  112  24  78.8  129  24  80.7

NOTE: a fetus may appear in more than one category

Conclusions:
The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).

There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested. The observed delayed ossification at multiple locations in the high dose tested were considered to represent a development effect of treatment with the test item and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity (delayed ossifications) was 500 mg/kg bw/day (intermediate dose level).
Executive summary:

Methods

The test item was administered by gavage to three groups each of twenty-four time-mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive, at dose levels 250, 500, or 1000 mg/kg bw/day. A further group of twenty-four time-mated females was exposed to the vehicle only (Arachis oil) to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

No clinical signs of test item toxicity were detected for any of the animals throughout the study.

Body Weight

There was no effect of treatment with the test item at any dose level on body weight development.

Food Consumption

Dietary intake across all groups of females receiving the test item remained similar to controls throughout the treatment period.

Water Consumption

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Post Mortem Studies

There were no macroscopic findings for any of the females receiving the test item at dose levels of 250, 500 or 1000 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development.

Fetal Examination

There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested.

Skeletal evaluation of the fetuses/litters from the 1000 mg/kg bw/day dose group identified a number of findings relating to delayed ossification in various regions. In the absence of any maternal toxicity or an effect of treatment on fetal growth, these observations were collectively considered to represent an adverse effect of the test item on fetal development. Delayed ossifications were also noted in the lower dose groups; however, taking into account the overall findings in this study and historical control data information, these were considered to be within normal biological variation and not adverse.

Conclusion

The oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested). There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested. The observed delayed ossification at multiple locations in the high dose tested were considered to represent a development effect of treatment with the test item and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity (delayed ossifications) was 500 mg/kg bw/day (intermediate dose level).

Endpoint:
developmental toxicity
Remarks:
This study was requested by the European Chemicals Agency (ECHA; Decision number: CCH‑D‑2114448639-34-01/F). 
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (arrival of pre-mated animals) - 09 December 2019 Experimental completion date (fetal pathology) 05 February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Strain/Species New Zealand White rabbit.
Supplier Envigo RMS UK.
Number of animals ordered 96 time-mated females.
Day of delivery Gestation Day 1.
Age of the animals at the start of the study (Day 1 of gestation) 16 to 22 weeks old.
Weight range of the animals at the start of the study (Day 1 of gestation) 2.59 to 4.60 kg.

Allocation and Identification
Allocation On arrival.
Method Randomly to group and cage position. Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
Identification of animals Each animal was assigned a number and identified uniquely using a microchip (located subcutaneously).
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.

Animal Care and Husbandry
Environmental Control
Multispecies facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated, at least 15 air changes per hour.
Temperature and relative humidity Monitored and maintained within the range of 15-21°C and 45-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 14 hours light: 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Plastic suspended cages fitted with perforated floor panels (4200 cm2 x 45 cm or 651 inches2 x 18 inches) with built-in resting platforms (25cm or 10 inches from floor and 55 x 22 cm or 30 x 12 inches) mounted in batteries. Undertrays lined with absorbent paper that was changed at least three times a week.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries, each containing six cages.
Number of animals per cage One.

Environmental Enrichment
Aspen chew block A soft white untreated Aspen wood block provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring Attached to the cage.
Paper ball A paper ball (cage paper) was provided to each cage from Day 20 after mating to allow expression of nesting behavior and was replaced as necessary.

Diet Supply
Diet Envigo Teklad 2930, pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Restricted to 200g/animal/day.
If an individual showed a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with up to 50 mL of water) was offered, the consumption was recorded.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided where necessary.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are provided by the water supplier.
Certificates of analysis were also received from the suppliers of the Aspen chew blocks. Certificates of analysis are stored in the archive.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose.
Details on exposure:
Method of preparation
The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. The formulation was transferred to the final containers, via syringe whilst magnetically stirring.

A series of formulations at the required concentrations were prepared in ascending order.

Frequency of preparation Weekly.
Storage of formulation Refrigerated (2-8°C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. There were no discrepancies.

Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study number SP63HG.
• Ambient temperature (15 to 25°C) for three days.
• Refrigerated (2-8°C) for 18 days.
Achieved concentration Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item.

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at Constant doses in mg/kg bw/day.

Volume dose 5 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as treated groups.

Frequency Females were treated from Day 6 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.

Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M065/19).

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 50 μg/mL.

Concentration of Dose Formulations
The formulations for the first and last preparation were sampled. Group 2, 3 and 4 were sampled 4 × 1 mL (accurately weighed) from the middle of the formulation by Pharmacy personnel. For Group 1, 2 × 3 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel.

Two samples from Groups 2, 3, and 4 and duplicate aliquots from one Group 1 sample were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.
Details on mating procedure:
Method
Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related. After mating each female injected intravenously with 25 i.u. luteinising hormone.

Day 0 of gestation On the day of mating.

Delivery to Covance Day 1 after mating; supplied in four deliveries for logistical reasons
Duration of treatment / exposure:
Females were treated from Day 6 to Day 28 (inclusive) after mating,
Frequency of treatment:
Once per day
Duration of test:
Day 0-6: Mating
Day 6-28: Treatment
Day 29: Necropsy
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle only
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 animlas per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies (ECHA; Decision number: CCH-D-2114448639-34-01/F). The New Zealand White strain was used because of the historical control data available in this laboratory.

Route of Administration
The oral (gavage) route of administration was selected as it was requested by ECHA (Decision number: CCH-D-2114448639-34-01/F).

Rationale for Dose Level Selection
Dose levels were selected for this study, based on the results from a preliminary study (Covance Study Number: DH32LS) treated at 100, 500, 750, 1000 mg/kg bw/day. In that study, treatment at 750 or 1000 mg/kg bw/day was not tolerated and some animals were killed for welfare reasons and the groups were terminated early. One female treated at 500 mg/kg bw/day showed total inappetence, marked body weight loss and abortion on Day 20. In the remaining females at 500 mg/kg bw/day, overall body weight gain was low and gravid uterine weight was marginally low and food consumption was unaffected.

Reproductive performance, measured as the number of implantations, resorptions, implantation losses (%) and the number of live young, was unaffected. There was no effect of treatment on any parameter at 100 mg/kg bw/day. Therefore, doses of 0, 100, 200 and 400 mg/kg bw/day were selected for administration in this follow-on main study.
Maternal examinations:
Serial Observations

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.
A complete necropsy was performed in all cases.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
• Pre-dose observation.
• One to two hours after completion of dosing.
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 1 (Day 1 after mating) and Days 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on arrival (Day 1 after mating) and on Days 3 and 6 to 29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating up to Day 28.
Ovaries and uterine content:
Reproductive Assessment

For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn Number of: Corpora lutea., Intrauterine deaths (classified as early or late resorptions), Resorption sites (classified as early or late), Fetuses (live and dead).

Apparently non pregnant animals and for apparently empty uterine horns The absence or number of uterine implantation sites was confirmed using Salewski stain.

Females exhibiting pregnancy loss Expelled uterine contents were identified and examined, as appropriate.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities, using modified Staples Technique and the sex of each fetus was also recorded.

Fixation Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.
Remaining whole fetuses and the torsos from the decapitated fetuses were eviscerated and fixed in Industrial Methylated Spirit.

Processing Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities, using Modified Wilson Technique.
Alizarin Red stained fetuses and torsos Assessed for skeletal development and abnormalities, using Dawsons Technique.
Statistics:
Please refer to "Any other comments on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) / Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = ((Number of implantations - Number of live fetuses) / number of implantations) x 100

All group values and SD were calculated from the individual litter values.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs associated with dosing or clinical signs considered to be related to treatment at 100, 200 or 400 mg/kg bw/day.

Repetitive swaying was evident in one animal receiving 100 mg/kg bw/day (No. 35) and in one animal receiving 400 mg/kg bw/day (No. 80) on Days 26-27 of gestation. Observations of swaying in rabbits can be normal behaviour; therefore, these incidences were considered to be unrelated to treatment.

Two animals receiving 100 mg/kg bw/day (No’s 43 and 45) had few fecal pellets on Day 29. Both animals showed low or no food intake the previous day and No. 43 was not pregnant. In the absence of this finding in any other animal at 100 mg/kg bw/day, or in any animal at 200 or 400 mg/kg bw/day, it was considered not to be treatment related.

On Day 1 of gestation (receipt from the supplier), four animals were considered thin and persisted in one animal (No. 62) receiving 200 mg/kg bw/day to Day 12 and in another animal receiving 400 mg/kg bw/day (No. 90) to Day 23. As this sign was evident before treatment commenced, it was considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
One Control animal (No. 9) showed persistent body weight loss from Day 1 (receipt from the supplier) to Day 18 of gestation (600 g) and food consumption was low from Day 6; however, the animal remained in good clinical condition. The animal was euthanised for welfare reasons on Day 18 of gestation, due to the extent of the observed weight loss. The animal was macroscopically normal and the uterus contained four live fetuses and six early resorbed fetuses, from 10 implantations. The death of this animal was considered due to an adverse individual animal reaction to the dosing procedure (daily insertion of oral catheter into the stomach and associated restraint procedures).

One animal receiving 100 mg/kg bw/day (No. 42) had red ‘bloody’ discharge from the vagina on Days 22-27 and aborted the pregnancy on Day 27 of gestation. The animal was macroscopically normal and the pregnancy consisted of 12 late resorbed fetuses. As there were no similar deaths at 100, 200 or 400 mg/kg bw/day, it is considered this death may have been an individual animal response to the handling/dosing procedures during late gestation and was therefore not attributable to treatment with Adimoll BO.

One animal receiving 400 mg/kg bw/day (No. 92) showed persistent body weight losses from Day 8 of gestation to Day 20 (690 g) and food consumption was noticeably lower than other animals at this dose from Day 12. From Day 17, the animal had low fecal output and on Day 20 had consumed little hay or water and had low urine output. The animal was euthanised for welfare reasons on Day 20 of gestation. Macroscopic examination revealed that the stomach contained bedding material (probably that supplied for the animal during transit to the necropsy area) and the uterus contained nine live fetuses. A relationship to treatment with Adimoll BO for the death of this animal cannot be ruled out.

One animal receiving 400 mg/kg bw/day (No. 93) showed persistent body weight loss from Day 6 (commencement of treatment) to Day 16 of gestation (640 g), low food intake from Day 6 and total inappetence from Day 12, but remained in good clinical condition. The animal was euthanised for welfare reasons on Day 16. The animal was macroscopically normal and was pregnant with nine live fetuses. A relationship to treatment with Adimoll BO for the death of this animal cannot be ruled out.

One animal receiving 400 mg/kg bw/day (No. 96) showed persistent body weight losses from Day 1 (receipt from the supplier) to Day 12 of gestation (720 g) and total inappetence from Day 2 to Day 12. The animal consumed little hay and was thin on Day 12. The animal was euthanised for welfare reasons on Day 12 of gestation. The animal was macroscopically normal and was pregnant with 11 live fetuses. As the inappetence and body weight loss commenced before treatment commenced, the death of this animal may be attributed to an adverse reaction to transit from the supplier; however treatment with Adimoll BO may also have been a contributory factor for the death of this animal.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain was unaffected by treatment at 100, 200 or 400 mg/kg bw/day.

Gravid uterine weight and adjusted body weight change were unaffected by treatment at 100, 200 or 400 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption at 200 or 400 mg/kg bw/day was low on the majority of days during Day 6 (commencement of treatment) to Day 12 of gestation (88% or 75% of Control, respectively), but was similar to Control thereafter.

Overall food consumption was unaffected by treatment at 100 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related macroscopic findings at 100, 200 or 400 mg/kg bw/day.

One female treated at 200 mg/kg bw/day (No. 60) had two small areas (both 2-9 mm) of firm, pale tissue (masses) in the liver and two females treated at 100 mg/kg bw/day had several bilateral clear cysts in the kidney. In the absence of similar findings in females at 400 mg/kg/day, these macroscopic abnormalities were considered unrelated to administration with Adimoll BO.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One animal receiving 100 mg/kg bw/day (No. 42) had red ‘bloody’ discharge from the vagina on Days 22-27 and aborted the pregnancy on Day 27 of gestation. The animal was macroscopically normal and the pregnancy consisted of 12 late resorbed fetuses. As there were no similar deaths at 100, 200 or 400 mg/kg bw/day, it is considered this death may have been an individual animal response to the handling/dosing procedures during late gestation and was therefore not attributable to treatment with Adimoll BO.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
one animal treated at 100 mg/kg bw/day showed total resorption of the litter
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of resorptions, pre- and post-implantation losses, litter size and the ratio of male to female fetuses were unaffected by treatment at 100, 200 or 400 mg/kg bw/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One Control animal, three animals that received 100 mg/kg bw/day, one animal that received 200 mg/kg bw/day and two animals that received 400 mg/kg bw/day were not pregnant
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Total litter, placenta and fetal weights were considered to be unaffected by treatment at 100, 200 or 400 mg/kg bw/day.

Total litter weight was marginally low at 400 mg/kg bw/day (91% of Control), but fetal weight was unaffected. This was therefore attributed to the marginally lower number of fetuses at this dose, when compared with Control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Total litter, placenta and fetal weights were considered to be unaffected by treatment at 100, 200 or 400 mg/kg bw/day.
Total litter weight was marginally low at 400 mg/kg bw/day (91% of Control), but fetal weight was unaffected. This was therefore attributed to the marginally lower number of fetuses at this dose, when compared with Control.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Following maternal treatment at 100, 200 or 400 bw mg/kg bw/day, there was a single fetus at each dose exhibiting similar major head and limb abnormalities (Acephalostomia/Cranioschisis with short long bones/limbs). As this was evident at very low incidence and there was no relationship to dose, an association with treatment was not inferred.

PLEASE REFER TO ATTACHED - TABLE 7
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, there was an increase in the incidence of delayed/incomplete ossification/unossifed metacarpals and phalanges, when compared to Control; however, as there was no relationship to dose and the incidence was within the Historical Control Data (HCD) range, an association with treatment was not inferred.

PLEASE REFER TO ATTACHED - TABLE 8
Visceral malformations:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: There was no adverse effect of treatment on the outcome of pregnancy or embryo-fetal survival, development or growth up to the highest dose
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
external: limb
external: paw
Description (incidence and severity):
Following maternal treatment at 100, 200 or 400 bw mg/kg bw/day, there was a single fetus at each dose exhibiting similar major head and limb abnormalities (Acephalostomia/Cranioschisis with short long bones/limbs).
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
no
Conclusions:
It is concluded that oral gavage administration of Adimoll BO during Days 6 to 28 of gestation at 100, 200 or 400 mg/kg bw/day was well-tolerated except for individual females at 400 mg/kg bw/day. There was no adverse effect of treatment on the outcome of pregnancy or embryo-fetal survival, development or growth up to the highest dose; therefore, the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 200 mg/kg bw/day and for embryo fetal toxicity was 400 mg/kg bw/day.
Executive summary:

The purpose of this study was to assess the influence of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905 -983-8; also known and presented hereafter as Adimoll BO),on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Days 6-28 after mating) in the New Zealand White Rabbit. This study was requested by the European Chemicals Agency (ECHA; Decision number: CCH‑D‑2114448639-34-01/F). Three groups of 24 females received Adimoll BO at doses of 100, 200 or 400 mg/kg bw/day by oral gavage administration. A similarly constituted Control group received the vehicle, 1% aqueous methylcellulose, at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

Three animals at 400 mg/kg bw/day were killed for welfare reasons. The death of two animals was attributed to treatment and a relationship to treatment could not be discounted for the death of the third animal.

One Control animal was humanly killed due to anadverse reaction to the dosing procedure and one animal receiving 100 mg/kg bw/daywas humanly killed due to non-dose related abortion of the litter.

There were no signs associated with dosing or clinical signs, considered to be related to treatment, in the remaining animals at100, 200 or 400 mg/kg bw/day.

Overall body weight gain, adjusted body weight and gravid uterine weight on Day 20 of gestation were unaffected by treatment at100, 200 or 400 mg/kg bw/day.

Food consumption at 200 or 400 mg/kg bw/day was transiently low during Days 6 to 12 of gestation; however this was considered non-adverse and not biologically relevant.

There were no treatment related macroscopic findings in the adult at100, 200 or 400 mg/kg bw/day.

Reproductive parameters, namely, the numbers of resorptions, pre- and post-implantation losses, litter size and the ratio of male to female fetuses, total litter, placenta and fetal weights were unaffected by treatment at100, 200 or 400 mg/kg bw/day.

There were no treatment related findings effects on the development or growth of the fetus at100, 200 or 400 mg/kg bw/day.

Conclusion

It is concluded that oral gavage administration of Adimoll BO during Days 6 to 28 of gestation at 100, 200 or 400 mg/kg bw/day was well-tolerated except for individual females at 400 mg/kg bw/day. There was no adverse effect of treatment on the outcome of pregnancy or embryo-fetal survival, development or growth up to the highest dose; therefore, the No‑Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 200 mg/kg bw/day and for embryo‑fetal toxicity was 400 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline studies.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

An OECD TG 414 developmental toxicity study in rats was conducted and delayed ossification at multiple locations in the high dose group was reported (Rashid 2016). Oral administration of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905-983-8) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested). There were no major structural abnormalities or observations that could be considered functional deficiencies observed in fetuses at any dose level tested. The observed delayed ossification at multiple locations in the high dose tested were considered to represent a development effect of treatment with the test item and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity (delayed ossifications) was 500 mg/kg bw/day (intermediate dose level).

In a further OECD TG 414 developmental toxicity study in rabbits was conducted. The purpose of this study was to assess the influence of Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No. 905 -983-8; also known and presented hereafter as Adimoll BO), on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Days 6-28 after mating) in the New Zealand White Rabbit. Three groups of 24 females received Adimoll BO at doses of 100, 200 or 400 mg/kg bw/day by oral gavage administration. A similarly constituted Control group received the vehicle, 1% aqueous methylcellulose, at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

External, visceral and skeletal examination of the fetuses at 100, 200 or 400 mg/kg bw/day revealed no findings that were considered treatment related.

It is concluded that oral gavage administration of Adimoll BO during Days 6 to 28 of gestation at 100, 200 or 400 mg/kg bw/day was well-tolerated except for individual females at 400 mg/kg bw/day. There was no adverse effect of treatment on the outcome of pregnancy or embryo-fetal survival, development or growth up to the highest dose; therefore, the No‑Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 200 mg/kg bw/day and for embryo‑fetal toxicity was 400 mg/kg bw/day.

Delayed ossifications are not confirmed by the OECD 414 study in rabbits. Therefore the delayed ossification in rats is regarded as a toxicological non-relevant incidental finding. For the risk assessment the NOAEL of the OECD Guideline 421 study (Reproduction / Developmental Toxicity Screening Test) is used. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

Overall, based on the available developmental studies it is assumed that the delayed ossifications reported in the developmental toxicity study in rats will not lead to any structural or permanent effects.

Toxicity to reproduction: other studies

Description of key information

Additional, there is a subchronic study available (Leggett 2020). During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

These findings were confirmed by a subacute toxicity study available (Schladt 2013). During the 4-week treatment period male and female Wistar rats received 0, 100, 300 or 1000 mg/kg bw/day ‘Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate’ by gavage. The study was performed according to OECD TG 407 and GLP conditions. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

Referring to the evaluation of reproductive organs in males and females, treatment with 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate'was tolerated without impairment. Thus, the NOAEL (reproductive organs) is 1000 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Remarks:
OECD Guideline 408 study: Reproductive organs of all rats were weighed and the reproductive organs were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
In the OECD 408 study the reproductive organs of all rats were weighed and the reproductive organs were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain/Species Crl:CD® (SD) IGS BR rat.
Supplier Charles River (UK) Ltd.
Number of animals 45 males and 45 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization 12 days before commencement of treatment.
Age of the animals at start of treatment 41 to 47 days.
Weight range of the animals at the start of treatment Males: 201 to 254 g
Females: 151 to 195 g
Allocation and Identification
Allocation Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment commenced
Body weight range extreme One female

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated; 15 air changes /hour.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Alarm systems Activated on ventilation failure and when temperature/ humidity limits exceeded.

Animal Accommodation
Cages Polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage Three or four of the same sex.
Bedding Softwood based bark-free fiber bedding, sterilized by autoclaving, which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material A soft white untreated wood block; provided to each cage throughout the study (except during overnight for urine collection) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C pelleted Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology, blood chemistry and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during the period of urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The oral route of administration was chosen as requested by ECHA (Decision number CCH D-2114448639-34-01/F).

Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg bw/day.
Volume dose 4 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.


Formulation
Correction factor None.
Vehicle Arachis oil BP.

Method of preparation The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The remaining concentrations were then formulated in ascending order of concentration.

Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8°C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. There were no discrepancies.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 250 mg/mL (Covance Study Number SP63HG) and at 3.70 to 251 mg/mL (Envigo Study Number 41500056) were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations confirmed the following:

24 hours stability at ambient temperature (15 to 25°C) at 1 to 250 mg/mL.

16 days stability at refrigerated temperature (2 to 8°C) at 3.70 to 251 mg/mL.

Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 6 and 12 of treatment were analyzed for achieved concentration of the test item.

Analysis
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Duration of test:
90 days
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males/ 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
The doses used in this study (0, 100, 300 and 1000 mg/kg bw/day) were selected in conjunction with the Sponsor.

A dose of 1000 mg/kg bw/day was considered a suitable high dose for use in this study based on the results of a four-week toxicity study in rats (Bayer Pharma AG Study Number T100005-6), at doses of 100, 300 and 1000 mg/kg bw/day. In that study, changes were seen in males in the liver at 300 mg/kg bw/day and in the kidneys at 100 mg/kg bw/day. The changes in the liver were considered an adaptive response to treatment with a xenobiotic and therefore was considered non-adverse. The changes in the kidneys were regarded to be adverse in the male (alpha-2u-globulin-mediated nephropathy). Therefore the no observed adverse effect level (NOAEL) for repeated oral administration of Adimoll BO in the four week study was 1000 mg/kg bw/day for female Wistar rats, but a NOAEL could not be established in male Wistar rats. To assist in determining doses for the present study, an OECD 421 study (Envigo study Number: JD44CD) in Sprague-Dawley rats, treated at 250, 500 and 1000 mg/kg bw/day resulted in a NO(A)EL of 1000 mg/kg bw/day. It was accepted that in that screening study, no hematology or blood chemistry investigations were required, and only limited histopathology evaluations were performed.
Statistics:
Please refer to "Any other information on materials and methods incl. tables" (see IUCLID section 7.5.1 - Repeated dose toxicity: oral_Adimoll BO_HB57JT).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina). No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.
Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).
No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.
Conclusions:
During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.
Executive summary:

A subchronic study is available (Leggett 2020). During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

According to column 1 of 8.7.3., Annex IX of the REACH Regulation an EOGRTS according to OECD TG 443 is appropriate if “available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD TGs 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.”  

There is no indication that 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is toxic to fertility. An EOGRT study according to OECD TG 443 is therefore not required.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A subacute toxicity study according to OECD TG 406 and GLP in which reproductive organs aree also examined.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD TG 407
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 Horst, The Netherlands
- Age at study initiation: 6 weeks
- Mean Weight at study initiation: male 180.1 g, female 136.1 g
- Housing: in groups with 2 or 3 animals
- Diet ad libitum
- Water ad libitum
- Acclimation period: approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Ethanol/ Kolliphor HS 15/ tap water (10 %/40 %/50 %, v/v/v).
Details on exposure:
'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is an industrial chemical, which consists of benzyl 2-ethylhexyl adipate, bis(2-ethylhexyl) adipate and dibenzyl adipate. 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was administered orally by gavage to 5 male and 5 female Wistar (HsdRCCHan:Wist) rats per dose group using ethanol/ Kolliphor HS15®/ tap water (10%/40%/50%, v/v/v) as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw for a period of 4 weeks. This GLP study was performed according to OECD guideline 407.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and stability of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' in the administration vehicle were checked prior to study start. This analytical investigation showed 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' to be stable for 4 days in the concentration range used.
Analyses during the study verified that the 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' content agreed with the target concentrations.
Duration of treatment / exposure:
30 days
Frequency of treatment:
once daily
Duration of test:
30 days
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is an industrial chemical, which consists of benzyl 2-ethylhexyl adipate, bis(2-ethylhexyl) adipate and dibenzyl adipate.
'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was administered orally by gavage to 5 male and 5 female Wistar (HsdRCCHan:Wist) rats per dose group using ethanol/ Kolliphor HS15®/ tap water (10%/40%/50%, v/v/v) as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw for a period of 4 weeks. This GLP study was performed according to OECD guideline 407.

The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Necropsy: week 5

Organ weight:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries/oviducts, prostate, seminal vesicles with coagulation glands, spleen, testes, thymus, uterus with cervix

Histopathological evaluation:
adrenal gland bone, sternum bone marrow, sternum brain (three regions), cecum cervix coagulating gland, colon, duodenum, epididymis, eye, femur/knee joint (with bone marrow), heart, ileum, jejunum, kidney, liver, lung, lymph node iliac, lymph node mesenteric, ovary/oviduct, parathyroid gland, Peyer’s patch, prostate gland, rectum, sciatic nerve, seminal vesicle, skeletal muscle, spinal cord (three regions), spleen, stomach (nonglandular and glandular), testis, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina, macroscopic abnormalities
Statistics:
Dunnett-test, Dunnett exact homogenous test, adjusted Mann Whitney-U-test, Bonferroni/Mann Whitney-U-test
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
female
Basis for effect level:
other: 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was tolerated without impairment
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Based on hyaline droplet formation in the kidneys, a typical male rat specific effect which is without toxicological relevance for humans.
Dose descriptor:
other: NOAEL (reproductive organs)
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Reproductive organs were not affected by treatment with 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate'.
For general toxicity:
Survival was not affected by the treatment with 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate'. Body weight development as well as food and water intake in treated groups was not relevantly affected by the treatment.
Neither hematology nor clinical chemistry gave evidence for treatment-related adverse effects up to 1000 mg/kg bw/day.

Liver:
In the liver, a minimal centrilobular hepatocellular hypertrophy was observed in males starting at 300 mg/kg bw/day. At 1000 mg/kg bw/day, the change corroborated the minimally higher mean liver weight recorded, and a macroscopically swollen liver noted in one animal. This minor liver change was considered to indicate a metabolic adaptation of the liver due to induction of hepatic microsomal enzymes and not as an adverse effect. The increase in concentration of bile acids at 1000 mg/kg bw/day might also be a result of this adaptive effect on liver metabolism. However, taken into account the known variability for this parameter, the relatively slight difference to control and the absence of statistical significance a toxicological relevant is not assumed

Kidneys:
In the kidney of males multifocal minimally dilated cortical and medullary tubules were seen at histopathology starting at 300 mg/kg bw/day. They were associated, in a proportion of the animals concerned and mainly at 1000 mg/kg bw/day, with multifocal minimal or mild cortical tubular degeneration. In addition, there was an increased incidence and severity of renal hyaline droplets in corticotubular cells in males, considered to represent α2-microglobulin and which were observed starting at 100 mg/kg b.w./day. The occurrence of α2-microglobulin is commonly recognized to be a male rat-specific event without toxicological relevance for humans.

Reproductive organs:
Determination of organ weights revealed at 1000 mg/kg bw/day decreased absolute and relative weights of prostate and ovaries and increased weights of thymus in females. However, as the differences to control were slight and/or statistical significance was absent and no treatment related histopathological findings were observed. A toxicological relevant effect was not assumed from these data.
Executive summary:

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is an industrial chemical, which consists of benzyl 2-ethylhexyl adipate, bis(2-ethylhexyl) adipate and dibenzyl adipate.

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using ethanol/Kolliphor HS15®/ tap water (10%/40%/50%, v/v/v) as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw for a period of 4 weeks.

This GLP study was performed according to OECD guideline 407.

The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Effects on liver of males starting at 300 mg/kg bw/day and on kidneys of males starting at 100 mg/kg bw/day are reported. The effects on liver are considered as adaptive response on the treatment with a xenobiotic and not as adverse. The effects on the kidney are regarded to be adverse for the male rat and are without toxicological relevance for the human situation.

Under the conditions described the no observed adverse effect level (NOAEL) for repeated oral administration of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate'was 1000 mg/kg bw/day for female Wistar rats. A NOAEL for the male rat could not be established.

Referring to the evaluation of reproductive organs in males and females, treatment with 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was tolerated without impairment. Thus, the NOAEL (reproductive organs) of 1000 mg/kg bw/day is established.

Additional information

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' is an industrial chemical, which consists of benzyl 2-ethylhexyl adipate, bis(2-ethylhexyl) adipate and dibenzyl adipate.

'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using ethanol/Kolliphor HS15®/ tap water (10%/40%/50%, v/v/v) as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw for a period of 4 weeks.

This GLP study was performed according to OECD TG 407.

The animals were regularly observed and weighed and food and water intakes were determined.In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Effects on liver of males starting at 300 mg/kg bw/day and on kidneys of males starting at 100 mg/kg bw/day are reported. The effects on liver are considered as adaptive response on the treatment with a xenobiotic and not as adverse. The effects on the kidney are regarded to be adverse for the male rat and are without toxicological relevance for the human situation.

Under the conditions described the no observed adverse effect level (NOAEL) for repeated oral administration of 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate'was 1000 mg/kg bw/day for female Wistar rats. A NOAEL for the male rat could not be established.

Referring to the evaluation of reproductive organs in males and females, treatment with 'Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate' was tolerated without impairment. Thus, the NOAEL (reproductive organs) of 1000 mg/kg bw/day.

Additional, there is a subchronic study available (Leggett 2020). During the 90 day treatment period three groups, each comprising ten males and ten females, received Adimoll BO at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 4 mL/kg bw. A similarly constituted control group received the vehicle (Arachis oil BP) at the same dose volume. The study was performed according to OECD TG 408 and GLP conditions. Reproductive organs of all rats were weighed (epididymides, seminal vesicles and coagulating gland, testes, prostate, uterus and cervix) and the reproductive organs of the treated and of the control animals were examined macropathological and histopathological (epididymides; prostate; testes; mammary; ovaries; oviducts; uterine cervix; uterus; vagina).

No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available studies, the No Observed Adverse Effect Level (NOAEL) for parental animals, reproductive and developmental endpoints was considered to be 1000 mg/kg bw/day (the highest dosage tested).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.

Additional information