Dioctyl phosphonate

Endpoint summary

Description of key information

NOAEL test item (repeated oral toxicity male and females rats treated at least 31 days)= 300 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

OECD TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 30th to July 16th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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)

Version / remarks:

July 29th, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: 7 to 8 weeks old.
- Weight at study initiation: 218 - 231 g for males and 174 - 199 g for females (instead of 200-225 g for males and 175-200 g for females, as indicated in the Study Protocol; this deviation was not considered to have compromised the purpose or integrity of the study).
- Housing: from arrival to mating, animals were housed up to 5 of one sex to a cage, in clear polysulfone cages measuring 59.5×38.0×20.0 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Techniplast –Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. After mating the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week. Recovery animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): a commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study, except before the blood sample.
- Water (e.g. ad libitum): drinking water was supplied ad libitum to each cage via water bottles, except before the urine collection. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 ml/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
- Acclimation period: an acclimatisation period of approximately 21 days was allowed before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY: records of analyses of water and diet are kept on file at testing laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amount of test item was suspended in the vehicle (corn oil). The formulations were prepared at weekly intervals based on stability data obtained from theTest Site (concentrations of 20, 60 and 160 mg/ml). Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): the substance is slightly soluble in water and corn oil was chosen as vehicle (suspension in corn oil).
- Concentration in vehicle: 0 (group 1), 20 (group 2), 60 (group 3) and 160 mg/ml (group 4) of test item
- Amount of vehicle (if gavage): 5 ml/kg body weight based on the most recently body weight.
#Main groups: for the males dose volumes were adjusted once per week for each animal according to the last recorded body weight. For females: dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating; during the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1,4, 7 and 13 postpartum.
#Recovery groups: for both males and females the amount of test item or control item was adjusted once per week for each animal according to the last recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The correct preparation of dose formulation was performed by the laboratory Innovative Environmental Services(IES) Ltd using HPLC-ELSD based on external standards calibration.

#HPLC-ELSD condition:
- HPLC Column: Cosmosil 5C18-AR, 100 Å, 250 x 4.6 mm, 5 μm
- Detector: Detector: Sedere SEDEX 85
- Eluent: eluent A:water and eluent B: tetrahydrofuran
Minutes: 0 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 3 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 8 min; % Eluent A: 25 % ; % Eluent B: 75 %
Minutes: 12 min; % Eluent A: 15 % ; % Eluent B: 85 %
Minutes: 13 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32.1 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 40 min; % Eluent A: 50 % ; % Eluent B: 50 %
- Injection Volume: 20 μl
- Injection Temperature: 45 °C
- Flow Rate: 1.0 ml/minute
- Column Temperature: 45 °C in a thermostatic oven
- Retention Time: approx. 9.8 min.
- Detector Temperature: 50 °C

#RESULTS: The recoveries of test item in test samples from treatment week 1 were found to be within the range of 97 % to 116 %. The % nominals determined for test samples from treatment week 8 were within the range of 87 % to 117 %. This confirms the correct preparation of dose formulation samples at the test facility. The relative standard deviations of the homogeneity samples at the low and high dose of 20 g/l and 160 g/l ranged from 2.1 % to 4.9 %. This shows the homogeneity of the dose formulations. Control samples without test item showed no peak at the retention time of the test item. Thus, the concentrations in these samples were below the limit of detection (LOD) of 2 g/l.

Duration of treatment / exposure:

Main groups: 31 days for males (i.e. 2 weeks prior to paring, during the paring and until the day before necropsy) and for at least 51 days for females (i.e. 2 weeks before paring, during paring, post coitum and post partum periods until day 13 post partum or the day before sacrifice).
Recovery groups: 4 consecutive weeks (total 28 days) for males and 6 consecutive weeks (total 42 days) for females. No treatment was given during the recovery period (4 weeks both for males and for females).

Frequency of treatment:

Once a day, 7 days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 male and 10 female rats for each main group (group 1 to 4) and 5 male and 5 female rats for each recovery (group 5 to 8).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected in consultation with the Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.: Y0270).
- Rationale for animal assignment (if not random): on the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution and/or showing irregular cycle were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed. The rat numbers listed above formed the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number were different for each concurrent study and served to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2.Even numbers were chosen for males and odd numbers for females.
- Rationale for selecting satellite groups: A 4 week treatment-free period was allowed in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. All dose levels included 5 additional animals per sex to be sacrificed after 4 weeks of recovery (Groups 5 to 8).
- Post-exposure recovery period in satellite groups: 4 weeks.

Observations and examinations performed and frequency:

MORTALITY (All groups)
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CAGE SIDE OBSERVATIONS (All groups)
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Data are reported until Week 4 (males of main and recovery groups),Week 6 (females of main and recovery groups) and Week 4 of recovery (recovery groups).

DETAILED CLINICAL OBSERVATIONS (All groups)
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day.

BODY WEIGHT (All groups)
- Main groups: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.
- Recovery groups: each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION (All groups)
- Main groups: the weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.
- Recovery groups: the weight of food consumed by each cage of rats was recorded at weekly intervals starting from Day 1 of dosing.
Note: the test item was administrated by oral gavage and not in diet.

HAEMATOLOGY:
- Time schedule for collection of blood: just before sacrificial procedure.
- Anaesthetic used for blood collection: under isofluorane anaesthesia from the retro-orbital sinus
- Animals fasted: yes
- How many animals: 5 males and 5 females randomly selected from each group during sacrificial procedure.
- Parameters checked:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells)
Platelets
Coagulation (Prothrombin time and Activated partial thromboplastin time)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: just before sacrificial procedure.
- Animals fasted: yes.
- How many animals: 5 males and 5 females randomly selected from each group during sacrificial procedure.
- Parameters checked:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride
Inorganic phosphorus

URINALYSIS:
- Time schedule for collection of urine: collection during the last week of treatment.
- Animals fasted: yes. The urine analysis was performed on the same male 5 animals selected for the clinical pathology investigations.
- Parameters checked:
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes was examined microscopically for:
Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule and dose groups for examinations: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), for assessment of grip strength and for motor activity measurement (for approximately 5 minutes using activity recording device) .
For the females the tests were performed on Day 13 post partum. Measurements were performed using a computer generated random order (for the main groups). Once during Week 4 of recovery, these evaluations were also performed in all animals.
- Battery of functions tested: sensory activity / grip strength / motor activity.

BIOANALYSIS - Thyroid hormone determination (T3, T4 and TSH)
As a part of the necropsy procedure approximately 0.8 ml of blood samples were taken from all parental males and females of the main groups and from animals of the recovery groups. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and thyroid stimulating hormone (TSH) by a multiplex assay. The determination was restricted as samples from all parental males from all main groups.

OTHER: other analysis such as vaginal smears (main groups), mating (main groups), parturition and gestation length (main groups), data on pups (e.g.anogenital distance and nipple count), blood collection for thyroid hormone determination (T3, T4, TSH) for pups were performed during the study. These data are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).

Sacrifice and pathology:

Parental animals and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
- Main groups: the males were killed after the mating of all females after 31 days of treatment period. The females with live pups were killed on Day 14 post partum. One female with total litter loss on Day 5 post partum was killed on Day 6 post partum.
- Recovery groups: all males and females of recovery groups were killed after 4 weeks of recovery.


GROSS PATHOLOGY (all groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain(cerebrum,cerebellum,medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus (where present)
Thyroid gland
Uterus – cervix
The ratios of organ weight to body weight were calculated for each animal.

HISTOPATHOLOGY:
The histopathological examination was performed for the following tissues on 5 males and on 5 females of the control and high dose groups (already selected for clinical pathology evaluation) killed at term:
Adrenal glands
Bone marrow (from sternum)
Brain
Clitoral gland
Caecum
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem brionchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary glands (males and females)
Mammary area
Optic nerves
Ovaries
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
All abnormalities were examinated for all animal of all main groups.

OTHER: Specific analysis on parental females (i.e. number of visible implantation sites and number of corpora lutea) and on pups (i.e. external examination, sex confirmation and nipple count, thyroid weight) were performed during the study. These data are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant clinical signs were observed throughout the study in all groups of both sexes, during treatment and recovery phases

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Main groups: no relevant differences in body weight and body weight gain were noted between control and treated animals throughout the study. No toxicological relevance was attributed to the slight but statistically significant decrease in body weight gain of mid-dose females on Day 14 post coitum due the absence of dose-relation.
- Recovery groups: means of body weight and body weight gain were comparable between controls and treated groups, both in males and females throughout the treatment and the recovery phases.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in main and in recovery groups was unaffected by treatment in both sexes during the study.
Note: the test item was administrated by oral gavage and not in diet.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Main groups: male no. X0920072 (800 mg/kg/day) showed increased neutrophils. Compared with controls, the increment was 6.1 fold. Since the incidence was minimal, the relation with the test item was excluded. The statistically significant differences of mean corpuscular volume and mean corpuscular haemoglobin observed between controls and females dosed at 800 mg/kg/day were of slight severity (6% above controls, both), therefore they were considered to be of no toxicological relevance.
- Recovery groups: the statistically significant difference of platelets recorded between controls and females receiving 300 mg/kg/day was not dose-related, therefore it was considered to be incidental.

#Coagulation
- Main group: activated thromboplastin time was lower than controls in females receiving 100 mg/kg/day (9%). Due to the absence of dose-relation, this finding was considered to be incidental.
- Recovery phase:the statistically significant increase of prothrombin time recorded in males dosed at 800 mg/kg/day (8% above controls) was not observed at the end of treatment, therefore it was considered to be unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Main groups: compared with controls, males dosed at 800 mg/kg/day showed a statistically significant increase of cholesterol (54%), protein (6%) and albumin (6%), females from the same treated group showed increase of glucose (47%) and decrease of sodium (1%). Changes were of limited severity, therefore they were not considered to be adverse. The other statistically significant changes recorded (urea in males, chloride, phosphorus, bile acids and potassium in females) were not dose-related, therefore they were considered to be incidental. In addition, sporadic changes were recorded in animals, such as: high value of gamma-glutamyltransferase in one female receiving 800 mg/kg/day (no. X0920077) and slight to moderate increase of both transaminases enzymes in male no. X0920036 and female no. X0920053 (dosed at 100 and 300 mg/kg/day, respectively). Due to the absence of dose relation and/or to the minimal incidence, these findings were considered unrelated to treatment.
- Recovery groups: compared with controls, bile acids were increased in some treated males (2.5 to 2.8 fold). Since this finding was not observed during at the end of dosing, it was considered to be unrelated to treatment. The statistically significant differences of cholesterol, globulin and sodium recorded between controls and females receiving 300 mg/kg/day were not dose-related, therefore they were considered to be incidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Main groups: no relevant changes were observed. The augmented diuresis recorded in males dosed at 300 mg/kg/day (56% above controls) was considered to be incidental.
- Recovery groups: no changes were recorded.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Main and recovery groups: No toxicological relevance was attributed to the statistically significant increase in motor activity and sensory reactivity to stimuli of treated animals because it was related to the individual low control values measured (when compared to all other control values in the study).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes were observed on absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared to the controls. The sporadic statistically significant changes observed in some organs such as the increased relative spleen weight (14%) or absolute and relative thyroid weight (30% for relative weight) of high dose treated females sacrificed at the end of recovery were not considered toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main groups: the most relevant changes observed at necropsy of mid- and high dose rats, mainly in males, sacrificed at the end of treatment period, were thickening of non glandular region of the stomach, associated with oedematous consistency in one mid-dose treated male sacrificed at the end of treatment period. All other observed changes, such as enlarged adrenals, were considered spontaneous and incidental, also observed in untreated animals under our experimental conditions and/or characteristically seen in untreated Sprague Dawley SD rats of the same age.
- Recovery groups: no relevant changes were observed, after 4 weeks of recovery from treatment, in animals sacrificed at the end of the recovery period. All observed changes were considered spontaneous and incidental, also observed in untreated animals under our experimental conditions and/or characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Main and recovery groups: Neurotoxicity assessment (removal of animals from the home cage and observations in open arena) did not reveal changes attributable to the test item in treated animals of both sexes, when compared to the control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Main groups: treatment-related changes were observed in the non glandular squamous epithelium of the stomach (forestomach) of mid- and high dose animals of both sexes. Mid- and high dose males and females showed minimal to marked hyperplasia of the non glandular squamous epithelium associated or not with minimal to mild mucosal ulceration or mucosal erosion of the forestomach. The hyperplasia of the non glandular squamous epithelium of the stomach observed in mid- and high dose animals of both sexes was in general associated to hyperkeratosis, presence of inflammatory cells, oedema in the submucosa, although the distribution and severity of the lesions were in general higher in males than in females.
The apparently increased incidence of cortical vacuolation of adrenals (zona fasciculata) observed in some high dose males, after the extension of histopathological evaluation to the remaining control, low, mid- and high dose group males, was seen comparable to control animals.
The remaining sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males.
- Recovery groups: after a 4-week treatment-free period, minimal squamous epithelial hyperplasia associated with hyperkeratosis was still present in two males previously dosed at 800 mg/kg/day (high dose), however, this finding showed a trend of recovery in high dose males and a complete recovery in high dose females. All other reported findings such as cortical vacuolation of adrenals (zona fasciculata) are suggested to be incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Other effects:

no effects observed

Description (incidence and severity):

Parental animals: no relevant changes were recorded.

Details on results:

The results related to reproductive parameters (i.e oestrous cycle and spermatogenic cycle), pairing combination, mating performance, implantation, pre-birth loss, gestation length) and data on pups (e.g. sex ratio, anogenital distance, clinical signs of pups and thyroid weight of pups, thyroid hormone determination) are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Based on results in male and female Sprangue Dawley rats exposed to dose levels of 100, 300, and 800 mg/kg bw/day of test substance for at least 31 days, no observed adverse effect level (NOAEL) for general toxicity is 300 mg/kg bw/day for both males and females.

Executive summary:

The toxicity in Sprague Dawley rat after repeated dosing with test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and and early lactation of the offspring were investigated.

A 4 week treatment-free period was allowed in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. The vehicle was corn oil. All doses (0, 100, 300, 800 mg/kg bw/day) were administered at a constant volume of 5 ml/kg body weight.

The test item was administrated according to the following doses for the main groups: 0 mg/kg bw/day (group 1), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 800 mg/kg bw/day (group 4). Each main group was composed by 10 males and 10 females.

The test item was administrated according to the following doses for the recovery groups: 0 mg/kg bw/day (group 5), 100 mg/kg bw/day (group 6), 300 mg/kg bw/day (group 7) and 800 mg/kg bw/day (group 8). Each recovery group was composed by 5 males and 5 females.

 

Main groups

Males of the main groups were treated for 2 weeks prior to pairing and during pairing until the day before necropsy, for a total of 31 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). Two not pregnant females were dosed up to the day before necropsy. The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry in five animals/sex/group randomly selected and urinalysis in 5 males/group randomly selected), litter data, sex ratios, thyroid hormone determination, macroscopic observations and organ weights. Clinical signs, anogenital distance, external and/or internal examination were recorded for pups.

Histopathological examination was performed in the first instance in control and high dose groups (five animals/sex/group randomly selected), as well as on all abnormalities detected during post mortem observation at the end of treatment period. Subsequently, on the basis of treatment-related findings the histopathological evaluation was extended to the stomach of all low and mid-dose groups and to the remaining control and high dose group animals of both sexes, whereas the histopathological evaluation of adrenals was extended to all low and mid-dose groups and to the remaining control and high dose group males sacrificed at the end of the treatment and recovery period. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups.

 

Recovery groups

Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery animals (females) were treated for up to 6 weeks, when most of the main group females were killed, and killed after 4 weeks of recovery period.

The following parameters were evaluated in these animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), body weight, food consumption, macroscopic observations and organ weights.

 

Results:

- Mortality and fate of females: no mortality occurred throughout the study. Two females, one in the mid-dose group and one in the high dose group, were found not pregnant at necropsy. In addition, one female in the high dose group had total litter loss on Day 5 post partum. The number of females with live pups on Day 14 post partum was: 10 in the control and in the low dose group , 9 in the mid-dose group and 8 in the high dose group.

- Clinical signs: Main and recovery groups: No significant clinical signs Sprague Dawley rat were observed throughout the study in all groups of both sexes.

- Clinical observations (Functional Observation Battery Tests) , motor activity, grip strength and sensory reactivity to stimuli: Main and recovery groups: Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment), as well as, motor activity, grip strength and sensory reactivity to stimuli did not show differences between control and treated groups, both for main and recovery groups.

- Body weight and body weight gain: Main and recovery groups: No relevant differences in body weight and body weight gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.

- Food consumption: Main and recovery groups: No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

- Haematology:

# Dosing phase: The changes observed in some haematological parameters in animals dosed at 800 mg/kg/day were of slight severity, therefore they were considered to be of no toxicological relevance.

# Recovery phase: The statistically significant difference of platelets recorded between controls and females receiving 300 mg/kg/day was not dose-related, therefore it was considered to be incidental.

- Coagulation:

# Dosing phase: Activated thromboplastin time was lower than controls in females receiving 100 mg/kg/day (9%). Due to the absence of dose-relation, this finding was considered to be incidental.

# Recovery phase: The statistically significant increase of prothrombin time recorded in males dosed at 800 mg/kg/day (8% above controls) was not observed at the end of treatment, therefore it was considered to be unrelated to treatment.

- Clinical chemistry:

# Dosing phase: Changes observed in some biochemical parameters in animals dosed at 800 mg/kg/day compared with controls (cholesterol, protein and albumin in males; glucose and sodium in females) were considered of no toxicological relevance due to the minimal severity. The other statistically significant changes recorded in animals dosed at 300 mg/kg/day (urea in males, chloride, phosphorus, bile acids and potassium in females) were not dose-related, therefore they were not considered to be adverse.

# Recovery phase: The statistically significant differences of cholesterol, globulin and sodium recorded between controls and females receiving 300 mg/kg/day were not dose-related, therefore they were

considered to be incidental.

- Urinalysis:

# Dosing phase: No relevant changes were observed. The augmented diuresis recorded in males dosed at

300 mg/kg/day (56% above controls) was considered to be incidental.

# Recovery phase: No changes were recorded.

- Thyroid hormones: Parental animals: No relevant changes were recorded.

- Macroscopic observations:

# Final sacrifice: The most relevant changes observed at necropsy of mid- and high dose rats of both sexes, sacrificed at the end of treatment period, were thickening of non glandular region of the stomach, associated with oedematous consistency in one mid-dose treated male sacrificed at the end of treatment period.

# Recovery sacrifice: No relevant changes were observed, after 4 weeks of recovery from treatment, in animals sacrificed at the end of recovery period.

- Microscopic observations:

#Final sacrifice: Treatment-related changes were seen in the forestomach of mid- and high dose males and females, when compared to the controls. The findings consisted of squamous epithelial hyperplasia associated or not with mucosal ulceration or erosion, hyperkeratosis, presence of inflammatory cells, or oedema in the submucosa and were seen in all high dose males and most of females and, in some instances, in mid-dose groups of both sexes. The severity and distribution of the lesions were higher in males than in females. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males.

# Recovery sacrifice: After a 4-week treatment-free period, minimal squamous epithelial hyperplasia associated with hyperkeratosis, although still present in two males previously dosed at 800 mg/kg/day (high dose), showed a trend of reversibility in the forestomach of high dose males and a complete recovery in the high dose females.

 

Conclusion:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study conducted according to internationally accepted testing guidelines.

System:

gastrointestinal tract

Organ:

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 was performed on Dioctyl phosphonate. The test item was administered by oral gavage to Sprague Dawley rats (10 males and 10 females for each main group) using corn oil for vehicle at dose levels of 0, 100, 300 e 800 mg/kg bw/day. The assessment of delayed toxicity or persistence of adverse effects was performed on recovery groups (5 males and 5 females for each dose group) . Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 31 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and postpartum periods until Day 13 post partum (for at least 51 days). Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery animals (females) were treated for up to 6 weeks and killed after 4 weeks of recovery period. During the in vivo phase of the study, nor mortality neither effects treatment related (i.e. clinical signs including neurotoxicological evaluation, difference in body weight, food consumption, effects on haematology, clinical chemistry parameters, hormone analysis or urinalysis) were observed both for main groups and for recovery groups respect to control group. However at macroscopic and microscopic observations, treatment-related changes were seen in the forestomach of mid- and high dose males and females, when compared to the controls. At necropsy, after 4 weeks of recovery period, the treatment-related changes seen in the forestomach of treated animals showed a complete recovery in the high dose females and a trend of reversibility in high dose males, although still present in two males previously dosed at 800 mg/kg/day (high dose). Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and females.

Justification for classification or non-classification

According to the CLP Regulation (EC n.1272/2008), classification in category 1 for repeated dose toxicity applies to substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure. Substances are classified in category 1 for target organ toxicity (repeat exposure) on the basis of:

— reliable and good quality evidence from human cases or epidemiological studies; or

— observations from appropriate studies in experimental animals in which significant and/or severe toxic effects, of relevance to human health, were produced at generally low exposure concentrations.

As for classification in category 2, it applies to substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations.

 

For studies of 28 -day duration, classification in category 1 applies when toxicity by oral route is seen at concentrations below 30 mg/kg bw/day, while category 2 applies for toxicity at doses between 30 and 300 mg/kg bw/day.

 

Based on the results related to repeated toxicity obtained by Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 on the test item (i.e. NOAEL oral repeated toxicity male/female rats treated at least 31 days = 300 mg/kg bw/day), no classification after repeated exposure by oral route is suggested for Dioctyl phosphonate according to the CLP Regulation (EC n.1272/2008).