Registration Dossier

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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyl 2-ethylhexyl adipate
EC Number:
261-234-9
EC Name:
Benzyl 2-ethylhexyl adipate
Cas Number:
58394-64-2
Molecular formula:
C21H32O4
IUPAC Name:
benzyl 2-ethylhexyl adipate
Constituent 2
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
203-090-1
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
103-23-1
Molecular formula:
C22H42O4
IUPAC Name:
bis(2-ethylhexyl) adipate
Constituent 3
Chemical structure
Reference substance name:
Dibenzyl adipate
EC Number:
219-516-4
EC Name:
Dibenzyl adipate
Cas Number:
2451-84-5
Molecular formula:
C20H22O4
IUPAC Name:
dibenzyl adipate
Test material form:
liquid
Details on test material:
Batch No.: CH1611282

Test animals

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.

Administration / exposure

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).
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

open allclose all
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.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).