Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats by gavage, at dose levels of 0, 60, 125 and 250 mg/kg bw/day, resulted in some effects in animals of either sex from all treatment groups, none of which were considered to be due to adverse systemic toxicity. The histopathological changes seen in the forestomach may be a result of local irritation of the test item rather than a true effect of systemic toxicity. The NOAEL for systemic toxicity was therefore determined to be 250 mg/kg bw/day. Additionally, no treatment related effects were detected in any of the reproductive parameters examined and therefore the NOEL for reproductive/developmental toxicity was greater than 250 mg/kg bw/day.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September 2011 and 02 May 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 331 to 368g, the females weighed 193 to 228g:
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet was used).
- Water (e.g. ad libitum): The animals were allowed free access to water (mains drinking water was supplied from polycarbonate bottles attached to the cage).
- Acclimation period: The animals were acclimatised for a total of twelve days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C.
- Humidity (%): The humidity was set to acieve target values of 55 ± 15% .
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: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 previously determined in a 14-day range finder study. Results from the previous study showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice monthly and stored at 4ºC in the dark.The test item was administered twice daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 8 ml/kg bw/day 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 regular intervals.
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 a nd 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 oestrus 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analysed for concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate. The concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate in the test item formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.The results indicate that the prepared formulations were within ± 5% of the nominal concentration.
Duration of treatment / exposure:
The test item was administered to rats for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
The test item was administered twice daily.
Details on study schedule:
Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated twice 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.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
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) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, (Day 5 post partum for seven females due to technician error) five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten males and ten females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were choosen based on the results of a 14-day range finder study.
Positive control:
No
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before the first dose, up to thirty minutes after each dosing occasion, and one and three hours after each dosing occasion, during the working week. Animals were observed immediately before the first dose, soon after each dosing occasion, and one hour after each dosing occasion at weekends (except for females during parturition where applicable). Observations were recorded.
FUNCTIONAL OBSERVATIONS: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity at least one hour after the second dose. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
BEHAVIOURAL ASSESSMENTS: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation FUNCTIONAL PERFORMANCE TESTS: Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
SENSORY REACTIVITY: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed:Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.
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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
FOOD CONSUMPTION: During the maturation period, weekly food consumption was recorded for each cage of adults. 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 on Days 1 and 4 post partum. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.
WATER CONSUMPTION: Water in take was measured daily throughout the study (with the exception of the mating phase and Day 20 of gestation for female Number 39 due to a technician error).
LABORATORY INVESTIGATIONS: Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained after the first dosing occasion and from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. HAEMATOLOGY: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)Erythrocyte count
(RBC)Haematocrit (Hct)Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leu cocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into
sodium citrate solution (0.11 mol/l).
BLOOD CHEMISTRY: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
UreaCalcium (Ca++)
GlucoseInorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
AlbuminAlanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)
PREGNANCY AND PARTURITION: Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours 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):
During mating a vaginal smear was prepared for each female and the stage of oestrus 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).
Sperm parameters (parental animals):
Parameters examined in all male parental generations: During histopathology, the male testes and epididymides were examined for spermatocoel granuloma formation.
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 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partumv)Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Physical Development: Live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
PATHOLOGY:Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.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 following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals; Prostate; Brain; Seminal vesicles; Epididymides; Spleen; Heart; Testes; Kidneys; Thymus; Liver; Thyroid (weighed post-fixation with parathyroid); Ovaries; Uterus (weighed with Cervix); Pituitary (post fixation)
HISTOPATHOLOGY: Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: Adrenals; Ovaries; Aorta (thoracic); Pancreas; Bone & bone marrow (femur including stifle joint); Bone & bone marrow (sternum); Pituitary; Prostate; Brain (including cerebrum, cerebellum and pons); Oesophagus; Caecum; Rectum; Coagulating gland; Salivary glands (submaxillary); Colon; Sciatic nerve; Duodenum; Seminal vesicles; Epididymides•; Skin (hind limb); Eyes*; Spinal cord (cervical, midthoracic and lumbar); Gross lesions; Heart; Spleen; Ileum (including peyer’s patches); Stomach; Jejunum; Thyroid/parathyroid; Kidneys; Trachea; Liver; Testes•; Lungs (with brochi); Thymus; Lymph nodes (cervical and mesenteric); Urinary bladder; Mammary gland; Uterus/CevixMuscle (skeletal); Vagina
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately fortyeight hours later
* = eyes fixed in Davidson’s fluid
All tissues were despatched for processing. The tissues from five selected control and 250 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 haematoxylin and eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 250 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.Since there were indications of treatment-related stomach changes, examination was subsequently extended to include similarly prepared sections of the stomach from five animals per sex from the low and intermediate groups.Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology examinations was also performed in this phase of
the study.
Postmortem examinations (offspring):
PATHOLGY:Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Body weight and body weight change
Food and water consumptions during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Haematology, blood chemistry, adult absolute and body weight relative organ weights
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 of 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 for 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 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100 % 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 (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100
iii) Sex Ratio (% males) Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100
Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 250 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Although statistical analysis did not reveal any significant intergroup differences, a slight reduction in body weight gain was also evident in these males during Weeks 2 and 6; resulting in a reduction in overall body weight gain.No such effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 125 or 60 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 250 mg/kg bw/day showed a slight reduction in food consumption and food efficiency during the first two weeks of treatment. No adverse effect on food consumption or food efficiency was detected for females during the pre-mating, gestation or lactation phases of the study.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed an increase in water consumption during lactation. Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and can be associated with gastric irritancy rather than attributable to systemic toxicity. No such effects were detected in males throughout the treatment period or in females treated with 125 or 60 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the haematological parameters examined . Males from all treatment groups showed statistically significant reductions in prothrombin time. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance. Males treated with 250 mg/kg bw/day also showed a statistically significant reduction in platelet count and activated partial thromboplastin time. All individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were
considered not to be of toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined . Females treated with 250 and 125 mg/kg bw/day showed a statistically significant reduction in cholesterol and alkaline phosphatase. All individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 60 mg/kg bw/day showed a statistically significant increase in activity during the final 20% of the evaluation period. In the absence of a true dose related response or any support ing clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males from all treatment groups and females treated with 250 and 125 mg/kg bw/day showed statistically significant reductions in thyroid/parathyroid weight both absolute and relative to terminal body weight. Males treated with 250 mg/kg bw/day also showed a statistically significant increase in absolute and relative spleen weight. Females from all treatment groups showed statistically significant reductions in liver weight both absolute and relative to terminal body weight. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence
of true dose related responses or any histology correlates the intergroup differences were considered not to be of toxicological importance. Males treated with 60 mg/kg bw/day showed a statistically significant reduction in absolute and relative seminal vesicles weight. In the absence of a similar effect detected at 250 mg/kg bw/day or any histology correlates the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Seven males and three females treated with 250 mg/kg bw/day had sloughing on the non glandular region of the stomach. One male and one female treated with 125 mg/kg bw/day and one male treated with 60 mg/kg bw/day also had sloughing on the non-glandular region of the stomach and another female from the 125 mg/kg bw/day dose group had sloughing on the glandular and non glandular region of the stomach. No toxicologically significant effects were detected in females treated with 60 mg/kg bw/day. One male and one female treated with 250 mg/kg bw/day, one female treated with 125 mg/kg bw/day, two females treated with 60 mg/kg bw/day and one control male showed reddened lungs at necropsy. In the absence of treatment in the case of the control male or any toxic ologically significant histology correlates in treated groups the macroscopic findings were considered to be within the range of normal background alterations. One female treated with 250 mg/kg bw/day had increased pelvic space in the left kidney. In the absence of any histology correlates the intergroup difference wasconsidered of no toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Focal to multifocal squamous hyperplasia with hyperkeratosis, submucosal inflammation, and sometimes erosion in the forestomach were noted in a dose-related manner in treated animals of all dose groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on mating performance.No treatment-related effects on fertility were detected for treated animals when compared to controls.No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22½ to 24 days.In total eight females from control, nine females from the 60 mg/kg bw/day dose group and ten females from the 125 and 250 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 age.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic toxicity seen
Remarks on result:
other: not determinable due to absence of adverse systemic toxic effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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 specified
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
LITTER RESPONSE
In total eight females from control, nine females from the 60 mg/kg bw/day dose group and ten females from the 125 and 250 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
OFFSPRING LITTER SIZE AND VIABILITY No toxicologically significant effects were detected.No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
OFFSPRING GROWTH AND DEVELOPMENT No toxicologically significant effects were detected. There were no significant differences in litter, offspring weights or surface righting reflex. Statistical a nalysis of the data did not reveal any significant intergroup differences. No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, no milk in stomach, cold, weak, pale, no tail and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.
NECROPSY: Offspring:No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats by gavage, at dose levels of 0, 60, 125 and 250 mg/kg bw/day, resulted in some effects in animals of either sex from all treatment groups, none of which were considered to be due to adverse systemic toxicity. The histopathological changes seen in the forestomach may be a result of local irritation of the test item rather than a true effect of systemic toxicity. The NOAEL for systemic toxicity was therefore determined to be 250 mg/kg bw/day. Additionally, no treatment related effects were detected in any of the reproductive parameters examined and therefore the NOEL for reproductive/developmental toxicity was greater than 250 mg/kg bw/day.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and are compatible with the requirements of the OECD TG 422.

This study was also designed to be compatible with the 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 twice daily by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 60, 125 and 250 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food 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 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post-partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post-partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality: There were no unscheduled deaths.

Clinical Observations.

Episodes of increased salivation were evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 125 mg/kg bw/day during the treatment period. An isolated incident of noisy respiration was also evident in one female treated with 250 mg/kg bw/day on Day 6. No such effects were detected in females treated with 125 mg/kg bw/day or animals of either sex treated with 60 mg/kg bw/day. Such effects on salivation are likely due to the palatability of the test substance which is a known skin irritant.

Behavioural Assessment.

There were no toxicologically significant changes in the behavioural parameters measured.

Functional Performance Tests.

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Body Weight.

Males treated with 250 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 2 and during the final week of treatment. No such effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 125 or 60 mg/kg bw/day.

Food Consumption.

Males treated with 250 mg/kg bw/day showed a slight reduction in overall food consumption when compared to controls. No such effects were detected in females treated with 250 mg/kg bw/day or in animals of either sex treated with 125 or 60 mg/kg bw/day.

Water Consumption.

No toxicologically significant effect on water consumption was detected.

Reproductive Performance:

Mating and Fertility: There were no treatment-related effects on mating or conception rates for treated animals.

Gestation Lengths.

There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability: Of the litters born, litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. There were no significant intergroup differences in sex ratio.

Offspring Growth and Development.

Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post-partum were comparable to controls.

Offspring Observations.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Laboratory Investigations:

Haematology: There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry: There were no toxicologically significant effects detected in the blood chemical parameters measured.

Pathology:

Necropsy: Sloughing on the non-glandular and/or glandular regions of the stomach were evident in a number of animals treated with 250 and 125 mg/kg bw/day and in one male treated with 60 mg/kg bw/day. No such effects were detected in females treated with 60 mg/kg bw/day.

Organ Weights.

No toxicologically significant effects were detected in the organ weights measured.

Histopathology.

Stomach: Focal to multifocal squamous hyperplasia with hyperkeratosis, submucosal inflammation, and sometimes erosion in the forestomach were noted in a dose-related manner in treated animals of all dose groups. The minimally increased incidence of mucosal glandular dilation in the glandular stomach of females from intermediate and high dose groups were considered secondary to the locally irritant effects in the forestomach.

Conclusion

The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats by gavage, at dose levels of 0, 60, 125 and 250 mg/kg bw/day, resulted in some effects in animals of either sex from all treatment groups, none of which were considered to be due to adverse systemic toxicity. The histopathological changes seen in the forestomach may be a result of local irritation of the test item rather than a true effect of systemic toxicity. The NOAEL for systemic toxicity was therefore determined to be 250 mg/kg bw/day. Additionally, no treatment related effects were detected in any of the reproductive parameters examined and therefore the NOEL for reproductive/developmental toxicity was greater than 250 mg/kg bw/day.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric Acid, linear and branched Octyl Esters [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
For the Source substance, there is evidence from a screening for reproduction and developmental toxicity study (OECD 422). The study had NO(A)EL of 125 (125 or 250 for males/females), and 250 mg/kg bw/day (highest dose tested) for reproduction and development. In the Source study, 250 mg/kg bw/day was the highest dose tested; the NOEL at 125 mg/kg bw/day was due to local irritancy (stomach) and was most probably not due to systemic toxicity. Such effects in the rat may be considered as not relevant to man. The NOAEL can therefore be considered to be 250 mg/kg bw/day. Reproduction (mating and fertility) and in utero development were unaffected.
The data suggest that this type of substance may be predicted to have no appreciable effects on reproduction and development and to be of low to moderate systemic toxicity at relatively high doses. It may be predicted that such a study would allow read-across to the Target substance which shares several common characteristics with the Source substance.
4. DATA MATRIX
Please refer to attached justification.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic toxicity seen
Remarks on result:
other: not determinable due to absence of adverse systemic toxic effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse toxicity seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September 2011 and 02 May 2012.
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity S tudy with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 331 to 368g, the females weighed 193 to 228g:
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet was used).
- Water (e.g. ad libitum): The animals were allowed free access to water (mains drinking water was supplied from polycarbonate bottles attached to the cage).
- Acclimation period: The animals were acclimatised for a total of twelve days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C.
- Humidity (%): The humidity was set to acieve target values of 55 ± 15% .
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 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 previously determined in a 14-day range finder study. Results from the previous study showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice monthly and stored at 4ºC in the dark.The test item was administered twice daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 8 ml/kg bw/day 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 regular intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analysed for concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate. The concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate in the test item formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.The results indicate that the prepared formulations were within ± 5% 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 oestrus 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:
The test item was administered to rats for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
The test item was administered twice daily.
Duration of test:
Up to eight weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten males and ten females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated twice 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.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
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) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, (Day 5 post partum for seven females due to technician error) five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically
Maternal examinations:
CLINICAL OBSERVATIONS: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before the first dose, up to thirty minutes after each dosing occasion, and one and three hours after each dosing occasion, during the working week. Animals were observed immediately before the first dose, soon after each dosing occasion, and one hour after each dosing occasion at weekends (except for females during parturition where applicable). Observations were recorded.
FUNCTIONAL OBSERVATIONS: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity at least one hour after the second dose. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
BEHAVIOURAL ASSESSMENTS: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation
FUNCTIONAL PERFORMANCE TESTS: Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
SENSORY REACTIVITY: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.
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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
FOOD CONSUMPTION:During the maturation period, weekly food consumption was recorded foreach cage of adults. 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 on Days 1 and 4 post partum. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.
WATER CONSUMPTION: Water intake was measured daily throughout the study (with the exception of the mating phase and Day 20 of gestation for female Number 39 due to a technician error).
LABORATORY INVESTIGATIONS: Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained after the first dosing occasion and from the lateral tail vein . Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. HAEMATOLOGY: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH) - mean corpuscular volume (MCV) - mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut) - lymphocytes (Lymph) - monocytes (Mono) - eosinophils (Eos) -basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).
BLOOD CHEMISTRY: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea; Calcium (Ca++); glucose; Inorganic phosphorus (P); Total protein (Tot.Prot.); Aspartate aminotransferase (ASAT); Albumin; Alanine aminotransferase (ALAT); Albumin/Globulin (A/G) ratio (by calculation); Alkaline phosphatase (AP); Sodium (Na+); Creatinine (Creat); Potassium (K+); Total cholesterol (Chol); Chloride (Cl-); Total bilirubin (Bili); Bile acids (Bile)
REPRODUCTIVE SCREENING:
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 oestrus 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 approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours 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
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 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)Physical Development:Live offspring were assessed for surface righting reflex on Day 1 post partum.
PATHOLOGY: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous
overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum. 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 and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.ORGAN WEIGHTS:The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Ad
renals; Prostate; Brain; Seminal vesicles; Epididymides; Spleen; Heart; Testes; Kidneys; Thymus; Liver; Thyroid (weighed post-fixation with Parathyroid); Ovaries; Uterus (weighed with Cervix); Pituitary (post fixation)
HISTOPATHOLOGY: Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: Adrenals; Ovaries; Aorta; (thoracic); Pancreas; Bone & bone marrow (femur including stifle joint); Bone & bone marrow (sternum); Pituitary; Prostate; Brain (including cerebrum, cerebellum and pons); Oesophagus; Caecum; Rectum; Coagulating gland; Salivary glands (submaxillary); Colon; Sciatic nerve; Duodenum; Seminal vesicles; Epididymides•; Skin (hind limb); Eyes*; Spinal cord (cervical, mid-thoracic and lumbar); Gross lesions; Heart; Spleen; Ileum (including peyer’s patches); Stomach; Jejunum; Thyroid/parathyroid; Kidneys; Trachea; Liver; Testes•; Lungs (with brochi); Thymus; Lymph nodes (cervical and mesenteric); Urinary bladder; Mammary gland; Uterus/Cevix; Muscle (skeletal);Vagina
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
* = eyes fixed in Davidson’s fluid
All tissues were despatched for processing. The tissues from five selected control and 250 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 haematoxylin and eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 250 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.Since there were indications of treatment-related stomach changes, examination was subsequently extended to include similarly prepared sections of the stomach from five animals per sex from the low and intermediate groups.Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology examinations was also performed in this phase of the study.
Ovaries and uterine content:
For all females, the uterus was examined for signs of implantation and the number of uterine implant ations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).The number of corpora lutea was recorded.
Fetal examinations:
Not performed.
Statistics:
The following parameters were subjected to statistical analysis:Quantitative functional performance dataBody weight and body weight changeFood and water consumptions during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Haematology, blood chemistry, adult absolute and body weight relative organ weights
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 5 of age).
i) Implantation Losses (%) Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100 %
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 (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 )
Historical control data:
Normal range data for the different parameters examined were used in comparison against the test
data.
Details on maternal toxic effects:
Maternal toxic effects:yes
Details on maternal toxic effects:
ADULT RESPONSES:
MORTALITY: There were no unscheduled deaths.
CLINICAL OBSERVATIONS: Episodes of increased salivation were evident in animals of either sex treated with 250 mg/kg bw/day from Day 7 (females) and Day 12 (males) onwards. An isolated incident of noisy respiration was evident in one female treated with 250 mg/kg bw/day on Day 6.At 125 mg/kg/day, increased salivation was also evident in males between Days 12 and 41.No such effects were detected in females treated with 125 mg/kg bw/day or animals of either sex treated with 60 mg/kg bw/day.
BEHAVIOURAL ASSESSMENTS:Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.
FUNCTIONAL PERFORMANCE TESTS: There were no toxicologically significant changes in functional performance.Males treated with 60 mg/kg bw/day showed a statistically significant increase in activity during the final 20% of the evaluation period. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.
SENSORY REACTIVITY ASSESSMENTS: There were no treatment-related changes in sensory reactivity.
BODY WEIGHT: Males treated with 250 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Although statistical analysis did not reveal any significant intergroup differences, a slight reduction in body weight gain was also evident in these males during Weeks 2 and 6; resulting in a reduction in overall body weight gain.No such effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 125 or 60 mg/kg bw/day.
FOOD CONSUMTION AND FOOD EFFICIENCY: Males treated with 250 mg/kg bw/day showed a slight reduction in food consumption and food efficiency during the first two weeks of treatment. No adverse effect on food consumption or food efficiency was detected for females during the pre-mating, gestation or lactation phases of the study. Statistical analysis of gestation and lactation data revealed no significant differences.WATER CONSUMPTION:Females treated with 250 mg/kg bw/day showed an increase in water consumption during lactation. Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and can be associated with gastric irritancy rather than attributable to systemic toxicity. No such effects were detected in males throughout the treatment period or in females treated with 125 or 60 mg/kg bw/day.
REPRODUCTIVE PERFORMANCE:
Mating: There were no treatment-related effects on mating performance. Fertility:No treatment-related effects on fertility were detected for treated a nimals when compared to controls.Two control females and one female treated with 60 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in either the male or female reproductive organs however, the male partner treated with 60 mg/kg bw/day revealed a moderate multifocal squamous hyperplasia and hyperkeratosis with slight multifocal submucosal inflammation in forestomach. These findings are considered to possibly be the cause of unsuccessful mating. In the absence of any similar infertility effects detected at 250 mg/kg bw/day the intergroup differences were considered not to be related to test item toxicity.
Gestation Length: No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22½ to 24 days.
LABORATORY INVESTIGATIONS:
HAEMATOLOGY:There were no toxicologically significant effects detected in the haematological parameters examined.Males from all treatment groups showed statistically significant reductions in prothrombin time. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance. Males treated with 250 mg/kg bw/day also showed a statistically significant reduction in platelet count and activated partial thromboplastin time. All individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance.
BLOOD CHEMISTRY: There were no toxicologically significant effects detected in the blood chemical parameters examined.Females treated with 250 and 125 mg/kg bw/day showed a statistically significant
reduction in cholesterol and alkaline phosphatase. All individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histology correlates the intergroup
differences were considered not to be of toxicological importance.
PATHOLOGY:
ORGAN WEIGHTS: There were no toxicologically significant effects detected in the organ weights measured. Males from all treatment groups and females treated with 250 and 125 mg/kg bw/day showed statistically significant reductions in thyroid/parathyroid weight both absolute and relative to terminal body weight. Males treated with 250 mg/kg bw/day also showed a statistically significant increase in absolute and relative spleen weight. Females from all treatment groups showed statistically significant reductions in liver weight both absolute and relative to terminal body weight. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of true dose related responses or any histology correlates the intergroup differences were considered not to be of toxicological importance. Males treated with 60 mg/kg bw/day showed a statistically significant reduction in absolute and relative seminal vesicles weight. In the absence of a similar effect detected at 250 mg/kg bw/day or any histology correlates the intergroup differences were considered not to be of toxicological significance. NECROPSY:Adults:Seven males and three females treated with 250 mg/kg bw/day had sloughing on the non glandular region of the stomach. One male and one female treated with 125 mg/kg bw/day and one male treated with 60 mg/kg bw/day also had sloughing on the non glandular region of the stomach and another female from the 125 mg/kg bw/day dose group had sloughing on the glandular and non glandular region of the stomach.No toxicologically significant effects were detected in females treated with 60 mg/kg bw/day. One male and one female treated with 250 mg/kg bw/day, one female treated with 125 mg/kg bw/day, two females treated with 60 mg/kg bw/day and one control male showed reddened lungs at necropsy. In the absence of treatment in the case of the control male or any toxicologically significant histology correlates in treated groups the macroscopic findings were considered to be within the range of normal background alterations. One female treated with 250 mg/kg bw/day had increased pelvic space in the left kidney. In the absence of any histology correlates the intergroup difference was considered of no toxicological importance.
HISTOPATHOLOGY:The following treatment related microscopic findings were detected:Stomach: Focal to multifocal squamous hyperplasia with hyperkeratosis, submucosal inflammation, and sometimes erosion in the forestomach were noted in a dose-related manner in treated animals of all dose groups. The minimally increased incidence of mucosal glandular dilation in the glandular stomach of females from intermediate and high dose groups were considered secondary to the locally irritant effects in the forestomach.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse toxicity seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse toxicity seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

LITTER RESPONSE

In total eight females from control, nine females from the 60 mg/kg bw/day dose group and ten females from the 125 and 250 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

OFFSPRING LITTER SIZE AND VIABILITY

No toxicologically significant effects were detected.

No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

OFFSPRING GROWTH AND DEVELOPMENT

No toxicologically significant effects were detected.

There were no significant differences in litter, offspring weights or surface righting reflex. Statistical analysis of the data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, no milk in stomach, cold, weak, pale, no tail and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.

NECROPSY:

Offspring:

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Conclusions:
The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats by gavage, at dose levels of 0, 60, 125 and 250 mg/kg bw/day, resulted in some effects in animals of either sex from all treatment groups, none of which were considered to be due to adverse systemic toxicity. The histopathological changes seen in the forestomach may be a result of local irritation of the test item rather than a true effect of systemic toxicity. The NOAEL for systemic toxicity was therefore determined to be 250 mg/kg bw/day. Additionally, no treatment related effects were detected in any of the reproductive parameters examined and therefore the NOEL for reproductive/developmental toxicity was greater than 250 mg/kg bw/day.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and are compatible with the requirements of the OECD TG 422.

This study was also designed to be compatible with the 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 twice daily by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 60, 125 and 250 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food 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 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post-partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post-partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality: There were no unscheduled deaths.

Clinical Observations.

Episodes of increased salivation were evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 125 mg/kg bw/day during the treatment period. An isolated incident of noisy respiration was also evident in one female treated with 250 mg/kg bw/day on Day 6. No such effects were detected in females treated with 125 mg/kg bw/day or animals of either sex treated with 60 mg/kg bw/day. Such effects on salivation are likely due to the palatability of the test substance which is a known skin irritant.

Behavioural Assessment.

There were no toxicologically significant changes in the behavioural parameters measured.

Functional Performance Tests.

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Body Weight.

Males treated with 250 mg/kg bw/day showed a reduction in body weight gain during Weeks 1, 2 and during the final week of treatment. No such effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 125 or 60 mg/kg bw/day.

Food Consumption.

Males treated with 250 mg/kg bw/day showed a slight reduction in overall food consumption when compared to controls. No such effects were detected in females treated with 250 mg/kg bw/day or in animals of either sex treated with 125 or 60 mg/kg bw/day.

Water Consumption.

No toxicologically significant effect on water consumption was detected.

Reproductive Performance:

Mating and Fertility: There were no treatment-related effects on mating or conception rates for treated animals.

Gestation Lengths.

There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability: Of the litters born, litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. There were no significant intergroup differences in sex ratio.

Offspring Growth and Development.

Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post-partum were comparable to controls.

Offspring Observations.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Laboratory Investigations:

Haematology: There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry: There were no toxicologically significant effects detected in the blood chemical parameters measured.

Pathology:

Necropsy: Sloughing on the non-glandular and/or glandular regions of the stomach were evident in a number of animals treated with 250 and 125 mg/kg bw/day and in one male treated with 60 mg/kg bw/day. No such effects were detected in females treated with 60 mg/kg bw/day.

Organ Weights.

No toxicologically significant effects were detected in the organ weights measured.

Histopathology.

Stomach: Focal to multifocal squamous hyperplasia with hyperkeratosis, submucosal inflammation, and sometimes erosion in the forestomach were noted in a dose-related manner in treated animals of all dose groups. The minimally increased incidence of mucosal glandular dilation in the glandular stomach of females from intermediate and high dose groups were considered secondary to the locally irritant effects in the forestomach.

Conclusion.

The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats by gavage, at dose levels of 0, 60, 125 and 250 mg/kg bw/day, resulted in some effects in animals of either sex from all treatment groups, none of which were considered to be due to adverse systemic toxicity. The histopathological changes seen in the forestomach may be a result of local irritation of the test item rather than a true effect of systemic toxicity. The NOAEL for systemic toxicity was therefore determined to be 250 mg/kg bw/day. Additionally, no treatment related effects were detected in any of the reproductive parameters examined and therefore the NOEL for reproductive/developmental toxicity was greater than 250 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric Acid, linear and branched Octyl Esters [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
For the Source substance, there is evidence from a screening for reproduction and developmental toxicity study (OECD 422). The study had NO(A)EL of 125 (125 or 250 for males/females), and 250 mg/kg bw/day (highest dose tested) for reproduction and development. In the Source study, 250 mg/kg bw/day was the highest dose tested; the NOEL at 125 mg/kg bw/day was due to local irritancy (stomach) and was most probably not due to systemic toxicity. Such effects in the rat may be considered as not relevant to man. The NOAEL can therefore be considered to be 250 mg/kg bw/day. Reproduction (mating and fertility) and in utero development were unaffected.
The data suggest that this type of substance may be predicted to have no appreciable effects on reproduction and development and to be of low to moderate systemic toxicity at relatively high doses. It may be predicted that such a study would allow read-across to the Target substance which shares several common characteristics with the Source substance.
4. DATA MATRIX
Please refer to attached justification.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse toxicity seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse toxicity seen
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No adverse effects on any parameters of the reproductive cycle were evident, therefore no classification is required.