Phosphoric acid, 2-ethylhexyl ester

Endpoint summary

Description of key information

NOAEL = 250 mg/kg bw/d; Dunster & Watson (2012); OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test)
NOAEL = 250 mg/kg bw/d; Astill, D.D. et al. (1996); OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: read across based on metabolism

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study design equivalent to OECD Guideline within sufficient reporting.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no ophthalmology, FOB reported

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Breeding Laboratories, Kingston, New York- Age at study initiation: 36 - 37 days- Weight at study initiation: 105 - 114 g (males), 86 - 97 g (females)- Housing: singly in stainless steel wire cages- Diet (e.g. ad libitum): Kliba rats/mice/hamster maintenance diet "A" 343 Meal (Klingentalmühle AG, Kaiseraugst, Switzerland), ad libitum- Water (e.g. ad libitum): water ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20 - 24°C- Humidity (%): 30 - 70%- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: dispersed in an aqueous solution of Cremophor EL (Polyethylene glycerol triricinoleate)

Details on oral exposure:

Doses were prepared daily by dispersing 2-Ethylhexanol in an aqueous solution of Cremophor EL (5 µg/100 mL, Polyoxyethylene glycerol tricinoleate) by ultra high speed sonication for 1 min. Homogeneity was maintained by magentic stirring throughout dosing. Dose volumes were 10 mL/kg based on weekly body weights. A sufactant (Cremophor) was used to facilitate mixing 2-Ethylhexanol with water. Concentration in the vehicle: 5 µg/100 mLAmount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations and homogeneity were checked by gas chromatographic analysis of samples from each dose level periodically in the study.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily on 5 consecutive days per week

Remarks:

Doses / Concentrations:25 mg/kg bw/dBasis:actual ingested

Remarks:

Doses / Concentrations:125 mg/kg bw/dBasis:actual ingested

Remarks:

Doses / Concentrations:250 mg/kg bw/dBasis:actual ingested

Remarks:

Doses / Concentrations:500 mg/kg bw/dBasis:actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were chosen based on the results of a preliminary 11 days oral toxicity study.Satellite group: Hepatic peroxisome proliferation was determined by measuring the activity of hepatic cyanide-insensitive Palmitoyl Coenzyme A in livers at termination.

Positive control:

No

Observations and examinations performed and frequency:

In-life observations:Animals were inspected twice daily for morbidity or mortality, or once daily on non treatment days. Clinical observations were made daily. Body weights were determined on day - and weekly thereafter. Animals were palpated on weighing. Food consumption was measured weekly.Blood was collected from fasted animals on the morning of days 29 and 84. The following enzyme activity and biochemistry measurements were performed: Alanine aminotransferase, Aspartate transferase, gamma-Glutamyltransferase, Alkaline phosphatase, Glucose, Urea, total protein, Albumin/Globulin, Sodium, Potassium, Calcium, Chloride, Creatinine, Cholesterol, Triglycerides and total Bilirubin.Haematology parameters were Leucocytes, Erythrocytes, Hemoglobin, Hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, Plateles and differential Leucocytes, and Reticulocytes.

Sacrifice and pathology:

Observations at necropsy:Gross Pathology:- Moribund animals were euthanized; dead and euthanized animals were immediately necropsied and assessed grossly.At scheduled termination body and organ weights were measured. Gross and microscopic examinations were made as follows: Adrenals, Brain, Kidneys, Liver, Stomach, Testes and Ovary from all animals were weighed, and with other organs and tissues listed in the U.S. EPA Health Effects Guidelines (1978) fixed in 4% Formalin.Histopathology:- All tissues from high dose and control animals were stained with Hematoxylin-eosin and examined microscopically.Lung, Liver, Spleen, kidneys, Stomach, Sternum, Femur, and Femur bone marrow were examined microscopically at intermdiate dose levels. Skin, Eyes, female Mammary gland, tigh musculatures, and extraorbital lacrymatory glands were not examined in the absence of signs of toxicity. The Liver was also stained with Oil Red for lipid content and examined microscopically.

Other examinations:

Hepatic peroxisome proliferation: livers were removed at termination and weighed, and cyanide-insensitive pCoA activities and protein concentrations were determined

Statistics:

Means and standard deviations were calculated for body weights, food and water consumption, clinical pathology results, and organ weights. Values for test groups were compared with controls in the main study by ANOVA follwed by Student´s t test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased at 500 mg/kg bw/d

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

increased Reticulocyte count at 500 mg/kg bw/d

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

decrease in serum ALT activity, serum Cholesterol concentration, total protein

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

foci in Forestomach at 500 mg/kg bw/d

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortalities and clinical findings:There were no mortalities or clinical findings differing from controls at any treatment level.Body weights and food consumption:There was decreased weight gain in male and female rats at 500 mg/kg bw/d, starting at week 4 in maless and week 11 in females, amounting to weight losses of 7% in males and 6% in females by week 13. There were no differences from controls at any treatment level in food consumption.Clinical pathology:Differences from controls in treated rats were seen mostly at day 84. Females at 250 and 500 mg/kg bw/d had 30 and 36% decreases in serum ALT activities, respectively. Females at 500 mg/kg bw/d had a 16% decrease in serum Cholesterol concentration and males at 500 mg/kg bw/d had 13% decreases in total protein and Albumin concentrations. There was a 25% increase in Reticulocyte count in male and female rats at 500 mg/kg bw/d. Organ weights:Significant differences from controls were moderate and limited to the Brain, Liver, Stomach and Testes at 250 and 500 mg/kg bw/d. Male rat relative Brain weights increased by 6% at 500 mg/kg bw/d, male kidney weights by 8% at 250 and 16% at 500 mg/kg bw/d, male Liver weights by 8% at 250 and 29% at 500 mg/kg bw/d, male Stomach weights by 11% at 500 mg/kg bw/d, and Testis weights by 5.5% at 250 mg/kg bw/d.Female Kidney weights increased by 5% at 250 and 6% at 500 mg/kg bw/d, female Liver weights by 8% at 250 and 15% at 500 mg/kg bw/d, and female Stomach weights by 6% at 250 and 16% at 500 mg/kg bw/d. Gross observations:Gross lesions differing from controls were seen at 500 mg/kg bw/d. 2/10 males and 4/10 females exhibited single or multiple slightly elevated foci in the Forestomach.Microscopic findings:Dose-related findings were limited to the Forestomach and Liver at 500 mg/kg bw/d. There was a generalized acanthosis of the Forestomach mucosa in 1/10 males with ballooning degeneration of the epithlial wall and acnathosis of the Forestomach mucosa in 2/10 males and 5/10 females. There was a moderate decrease in hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal ß-cell hyperplasia in 3/10 females.Peroxisome proliferation:Increases in hepatic cyanide-insensitive palmitoyl Coenzyme A activity were 6.5-fold in male rats and 3.4-fol in females at 500 mg/kg bw/d.

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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:

body weight and weight gain

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

Based on the effects observed at 500 mg/kg bw/d (reduced body weight, increased relative Liver, Kidney, Stomach and Testes weights, histological changes in the Forestomach) and 250 mg/kg bw/d (slightly increased Kidney, Stomach and Liver weights without histoligical changes) the NOEL is set a 125 mg/kg bw/d. The NOAEL is considered to be 250 mg/kg bw/d.

Executive summary:

It was demonstrated by a metabolism study performed with Phosphoric acid 2 -ethylhexyl ester in rats (Dekant, 2001) that the substance will be quantitatively metabolised to 2 -Ethylhexanol and Phosphate after entering the organism via oral route.

In urine and feces of rats which were dosed with Phosphoric acid 2 -ethylhexyl ester only inorganic Phosphate was detected. No ester was found. Phosphate is known to be an endogenous compound and food additive without reported toxic properties. Therefore, a read across to the second metabolite, 2 -Ethylhexanol, was conducted in order to estimate the toxicological properties in a subchronic oral toxicity study. Since Phosphoric acid 2 -ethylhexyl ester comprises Phosphate and 2 -Ethylhexanol, the metabolite Phosphate accounts for the the molecular weight in the same relation as the second metabolite 2 -Ethylhexanol. Therefore, the effects reported for 2 -Ethylhexanol after 90 d oral application are assumed to be less pronouced in case of the ester, since the weight of alcoholic component is smaller than in case of application of pure 2 -Ethylhexanol at the same dosage.

Each 10 male and 10 female rats were treated with the test substance at dosages of 0, 25, 125, 250 or 500 mg/kg bw/d for 13 weeks. During the treatment period animal observations were made for general health, mortality, food consumption and body weight. Blood samples were taken at treatment on days 29 and 84 for hematological and biochemical investigations. After 13 weeks the surviving animals were killed and were subjected to macroscopic and microscopic investigations.

Mortalities or clinical findings were not different from controls. Clinical changes were reported at 500 mg/kg bw/d (decreased total protein and Albumin in males; decreased serum Cholesterol in females). Reticulocyte count was increased at high dose. The toxicological relevance of these findings is unclear. At 500 mg/kg bw/d there was reduced body weight gain (6% in males, 7% in females), increased relative Liver weight (29% in males, 15% in females), Kidney weight (16% in males, 6% in females), Stomach weight (11% in males, 16% in females), and Testes weight (6%) and moderate gross and microscopic changes in the Liver and forestomach. There were no behavioral effects or effects on the Spleen or Thymus. Histopathology revealed only findings in high dose aninmals. The changes observed in the Forestomach are attributed to the irritant properties of 2 -Ethylhexanol and therefore considered to be a local effect.

Based on the above mentioned considerations the NOEL of 2 -Ethylhexanol is considered to be 125 mg/kg bw/d. The NOAEL might be derived at 250 mg/kg bw/d.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 16 September 2011 and 02 May 2012. The in-life phase of the study was conducted between 20 September 2011 (first day of treatment) and 14 November 2011 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 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).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 oral 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.

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.

Remarks:

Doses / Concentrations:60 mg/kg bw/dayBasis:actual ingested

Remarks:

Doses / Concentrations:125 mg/kg bw/dayBasis:actual ingested

Remarks:

Doses / Concentrations:250 mg/kg bw/dayBasis:actual ingested

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.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.

Positive control:

No positive control.

Observations and examinations performed and frequency:

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 behaviourUrination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, LachrymationFUNCTIONAL 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 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 assessedProthrombin 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)

Sacrifice and pathology:

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:AdrenalsProstateBrainSeminal vesiclesEpididymidesSpleenHeartTestesKidneysThymusLiverThyroid (weighed post-fixation with Parathyroid)OvariesUterus (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:AdrenalsOvariesAorta (thoracic)PancreasBone & bone marrow (femur including stifle joint)Bone & bone marrow (sternum)PituitaryProstateBrain (including cerebrum, cerebellum and pons)OesophagusCaecumRectumCoagulating glandSalivary glands (submaxillary)ColonSciatic nerveDuodenumSeminal vesiclesEpididymides•Skin (hind limb)Eyes*Spinal cord (cervical, mid-thoracic and lumbar)Gross lesionsHeartSpleenIleum (including peyer’s patches)StomachJejunumThyroid/parathyroidKidneysTracheaLiverTestes• Lungs (with brochi)ThymusLymph nodes (cervical and mesenteric)Urinary bladderMammary glandUterus/CevixMuscle (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 fluidAll 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.

Other examinations:

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 pairingii)Date of matingiii)Date and time of observed start of parturitioniv)Date and time of observed completion of parturitionLITTER 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 bornii)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 partumiv)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.

Statistics:

The following parameters were subjected to statistical analysis:Quantitative functional performance dataBody weight and body weight changeFood and water consumptions during gestation and lactationPre-coital interval and gestation lengthLitter size and litter weightsSex ratioCorpora lutea and implantation sitesImplantation losses and viability indicesOffspring body weight and body weight changeOffspring surface rightingHaematology, blood chemistry, adult absolute and body weight-relative organ weights

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

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.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effect on food consumption or food efficiency was detected for females during the pre-mating, gestation or lactation phases of the study.

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:

no effects observed

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:

no effects observed

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):

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

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 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.

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:

not examined

Details on 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 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. 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: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.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. See table of revelant findings in forestomach in any other information on results incl.tables.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.

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food efficiency

Dose descriptor:

NOEL

Remarks:

(reproductive/developmental toxicity)

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Histopathology:

The following related microscopic findings were detected.

Groups	1 (control)		2 (60 mg/kg bw/day)		3 (125 mg/kg bw/day)		4 (250 mg/kg bw/day)
Tissues examined	5 M	5 F	6 M	5 F	5 M	5 F	8 M	7 F
Squamous cell hyperplasia:                     Mean severity	0 0	0 0	2 2.0	2 2.0	5 1.4	5 2.0	8 2.6	7 3.3
Total Affected	0	0	2	2	5	5	8	7
Hyperkeratosis:                 Mean severity	0 0	0 0	2 2.0	2 2.0	4 1.5	5 1.6	8 2.4	7 3.0
Total Affected	0	0	2	2	4	5	8	7

 

Submucosal inflammation:                 Mean severity	0 0	0 0	1 2.0	2 1.5	5 1.0	3 1.7	8 1.6	7 2.9
Total Affected	0	0	1	2	5	3	8	7
Erosion:                         Mean severity	0 0	0 0	0 0	0 0	0 0	1 2.0	2 2.0	3 2.7
Total Affected	0	0	0	0	0	1	2	3

Reproductive Performance

Mating

There were no treatment-related effects on mating performance.

Fertility

No treatment-related effects on fertility were detected for treated animals 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.

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.

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 60, 125 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex from all treatment groups. The microscopic changes identified in the stomach may be considered to be adverse however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 250 mg/kg bw/day for females and 125 mg/kg bw/day for males.No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 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 Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

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 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.

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.

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.

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 60, 125 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex from all treatment groups.

The microscopic changes identified in the stomach may be considered to be adverse, however, they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 250 mg/kg bw/day for females and 125 mg/kg bw/day for males.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 250 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Dunster & Watson (2012) - OECD 422

The oral administration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate to rats for a period of up to eight weeks (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of up to 250 mg/kg bw/day resulted in treatment related effects detected in animals of either sex from all treatment groups.

Clinical signs were detected in animals of either sex treated with 250 mg/kg bw/day during the study. Incidents of increased salivation were reported together with noisy respiration in one 250 mg/kg bw/day female. At 125 mg/kg/day observations of increased salivation were also detected in males from Day 12 onwards. 

The physical condition of males treated with 250 mg/kg bw/day was also affected with reductions in body weight development during Weeks 1, 2 and 6 of treatment. Subsequently a slight reduction in overall body weight gain and a slight reduction in food consumption and food efficiency during Weeks 1 and 2 was evident in these males. Observations of this nature are often reported when a test item formulation is unpalatable or irritant and can be associated with gastric irritancy rather than attributable to systemic toxicity. This was supported macroscopically with sloughing on the non glandular and/or glandular regions of the stomach 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. Microscopic stomach changes were identified as focal to multifocal squamous hyperplasia with hyperkeratosis, submucosal inflammation, and erosion in the forestomach of treated animals from all treatment groups. Minimally increased incidence of mucosal glandular dilation in the glandular stomach of females treated with 250 and 125 mg/kg bw/day were considered secondary to the locally irritant effects in the forestomach. Based on the extent of histopathology changes in the stomach the findings would be considered evidence of a significant adverse finding however they were considered to be the result of local irritancy of the test item and therefore cannot be considered indicative of true systemic toxicity.

There were no toxicologically significant effects observed during the weekly open field arena observations, the haematological parameters measured or the blood chemical parameters measured.

There were no treatment-related effects detected in the reproductive parameters observed.

Astill, D.D. et al. (1996)

Based on the effects observed at 500 mg/kg bw/d (reduced body weight, increased relative Liver, Kidney, Stomach and Testes weights, histological changes in the Forestomach) and 250 mg/kg bw/d (slightly increased Kidney, Stomach and Liver weights without histoligical changes) the NOEL is set a 125 mg/kg bw/d. The NOAEL is considered to be 250 mg/kg bw/d.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This study on 2-ethylhexanol, is based on read-across to phosphoric acid, 2-ethylhexyl ester, as it is the main metabolite that is produced in vivo within a 24 h period. This read-across approach allows an estimation of the toxicological properties of phosphoric acid, 2-ethylhexyl ester in a 90-day repeated dose study. The NOAELs are the same as that observed in the submitted OECD 422 study, with no major difference in observed systemic effects.

Justification for classification or non-classification

The substance does not meet the criteria for any specific target organ toxicity classification, based on the data presented in this dossier. Additionally the substance is considered to have low potential for systemic toxicity since severe irritancy / corrosivity will limit exposure in the workplace.

As such the appropriate classification for the substance is considered to be that based on its’ corrosive properties.