2-ethyl-2-[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate

Endpoint summary

Description of key information

A NOAEL of 1000 mg/kg bw/d was determined for the test item based on effects observed in a combined repeated dose / reprotox screening test (OECD422).

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conducted in according to OECD guideline and GLP without deviation

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 176 to 200 g for males and 151 to 175 g for females
- Allocation: The rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights
- Housing: 5 of one sex to a cage, in clear polisulphone solid bottomed cages measuring 59.5⇥38⇥20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. 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 post partum. Thereafter individual dose volumes remained constant.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the present study in the range from 20 to 200 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspension (r > 0.98; accuracy 90-110%; precision CV < 5%).
Samples of the formulations prepared on Day 1 and Last Week were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (85-115%).

Duration of treatment / exposure:

Males: 5 weeks; females: 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, gestation and post partum periods up to Day 3 post partum.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Each group comprised 10 male and 10 female rats

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

MORTALITY: all animals were checked early in each working day and again in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Once before com- mencement of treatment (data not tabulated) and daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

NEUROBEHAVIOURAL EXAMINATION:
- FUNCTIONAL OBSERVATION BATTERY TESTS: 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.
- GRIP STRENGTH AND SENSORY REACTIVITY TO STIMULI: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected (computer generated random order) from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. For males the tests were performed on Day 30 of study and for females on Day 3 post partum.
- MOTOR ACTIVITY ASSESSMENT: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (60 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the test was performed on Day 30 of study and for females on Day 3 post partum.

BODY WEIGHT: Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination. Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY INVESTIGATIONS: As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation. At the same time interval, individual overnight urine samples were also collected from the same male animals under the same conditions. 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. After collection of the urine samples, the water bottles were supplied again to the animals.

HAEMATOLOGY: 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 TEST: Prothrombin time

CLINICAL CHEMISTRY: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, Bile acids, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: 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

Sacrifice and pathology:

Terminal studies:
- Euthanasia: Parental animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
- Necropsy: The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables 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 the Williams test. 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 mortality occurred throughout the study and no treatment-related signs were noted.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study and no treatment-related signs were noted.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both sexes during the study.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No remarkable changes were noted at post mortem examination in treated animals, when compared with controls.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

HAEMATOLOGY: Lymphocytosis was recorded in a number of males dosed with 300 and 1000 mg/kg/day. Lymphocytes mean group values were 36% and 41%, respectively, above controls. The other statistically significant differences between control and treated animals (mean corpuscular haemoglobin concentration in males dosed with 100 mg/kg/day, erythrocytes in females of the same group and basophils in females dosed with 1000 mg/kg/day) were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.
Coagulation: No changes were recorded in the coagulation parameters.

CLINICAL CHEMISTRY: Statistically significant differences between control and treated animals were observed, such as: decrease of triglycerides and chloride in males dosed with 300 mg/kg/day, decrease of albumin in those dosed with 100 mg/kg/day, increase of alkaline phosphatase and decrease of total protein in females treated with 100 mg/kg/day, decrease of globulin in those receiving 100 and 1000 mg/kg/day, increase of albumin/globulin ratio and decrease of creatinine in those treated with 1000 mg/kg/day. Due to the direction of changes and/or the absence of dose-relation, these were considered incidental.

URINALYSIS: Reduced diuresis was recorded in males dosed with 100 mg/kg/day. In addition, urinary haemoglobin, associated with the presence of erythrocytes in the urinary sediment, was recorded in one control animal and one male dosed with 100 mg/kg/day. Due to the absence of dose-relation, the above changes were considered unrelated to treatment.

NEUROBEHAVIOUR:
- Clinical observations (Functional Observation Battery Tests): Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test
item.
- Motor activity, grip strength and sensory reaction to stimuli: Motor activity and sensory reaction to stimuli measurements recorded at the end of treatment were comparable between control and treated groups in animals of both sexes. Variations recorded in mean grip strength in Group 4 males receiving 1000 mg/kg bw/day and mean landing foot splay in males receiving 300 and 1000 mg/kg bw/day were considered incidental, since they were observed in one sex only and without correlation with the dose.

HISTOPATHOLOGY: The histopathological changes reported in control and treated animals, such as mucosal erosion in the stomach, lymphoid depletion in the thymus, lymphoid hyperplasia in the spleen or malignant nephroblastoma, seen in a control male, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

SPERMATOGENIC CYCLE: A detailed qualitative examination of the testes was performed in five control and high dose group males. Seminiferous tubules were evaluated with respect to their stage in the sperma- togenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

Based on the results of the present study, the NOAEL for general toxicity was considered to be the highest dose of 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate information on toxic effects on rats after repeated oral dosing of the test item. Experimental procedures were based on the OECD Guideline no 422 and the study was performed according to GLP.

Both male and female rats were treated for approximately 5 weeks. Three different doses were tested (100, 300 and 1000 mg/kg bw/day) and compared to a control group that received only the vehicle (corn oil).

No differences in body weights and food consumption were observed in treated animals compared to the control group.

No clinical signs were observed during the study. No adverse findings were recorded in clinical pathology investigations (haematology, clinical chemistry and urine analysis) apart for lymphocytosis in mid- and high dose groups in males. No relevant differences were recorded in the absolute and relative organ weights of treated animals. No treatment-related changes were noted at macroscopic and microscopic observations.

Based on the results the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be the highest dose of 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is one study available that assesses the possible toxic effect of the test substance after repeated oral dosing. The study was performed according to GLP and internationally accepted guidelines. The experimental procedures were based on the OECD guideline 422, in which both female and male rats were treated during 5 weeks. Three doses were tested (100, 300 and 1000 mg/kg bw/day) and compared to a control group that received the vehicle only.

No differences in body weights, food consumption or clinical signs were observed in treated animals compared to the control group. Next, no adverse findings were recorded in clinical pathology investigations (haematology, clinical chemistry and urine analysis) nor differences in the absolute and relative organ weights of treated animals were recorded. Also, no treatment-related changes were noted at macroscopic and microscopic observations.

Based on these results the NOAEL was considered to be the highest dose of 1000 mg/kg bw/day.

There are no test reports available that address the repeated dose toxicity of the test substance when exposure occurs via the inhalation or the dermal pathway. However, based on the results of the oral repeated dose toxicity study this is not deemed necessary.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well documented study according to GLP and internationally accepted guidelines.

Justification for classification or non-classification

Based on the results of the repeated dose toxicity testing the test item should not be classified for repeated-dose toxicity or Specific Target Organ Toxicity following Repeated Exposure (STOT-RE) according to the criteria described in

Directive 67/548/EEC (Dangerous Substances Directive) and the EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) respectively.