Propanoic acid, 2-(4-chloro-2-methylphenoxy)-, 2-ethylhexyl ester, (2R)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1998)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

EC Number:

266-358-7

EC Name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

Cas Number:

66423-13-0

Molecular formula:

C18H27ClO3

IUPAC Name:

octyl (2R)-2-(4-chloro-2-methylphenoxy)propanoate

Specific details on test material used for the study:

Stability under test conditions: The stability of the test substance in the diet was confirmed for at least 15 days

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 Horst, The Netherlands
- Age at study initiation: about 5 weeks
- Weight at study initiation (mean): males 148-151 g, females 133-138 g
- Housing: in groups of 2 or 3 animals per cage in Makrolon cages Type IV
- Diet and water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 55
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: fed in diet

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Provimi Kliba 3883.G4.S15

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item in the diet were checked prior to study start and confirmed. Content checks performed three times during the study showed that the test item content agreed with the target concentrations and that the test item was homogeneously distributed in the diet within the defined limits.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

fed in diet, continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

120 ppm

Doses / concentrations 3

Dose / conc.:

600 ppm

Doses / concentrations 4

Dose / conc.:

3 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a subacute pilot study (T2083100). In this study 5 males and females received the test item in dietary concentrations of 0, 1600, 3200 or 6400 ppm over 4 weeks. The dietary concentration of 1600 ppm was not tolerated without effects on the biochemical liver and blood parameters. Starting at 3200 ppm toxic effects were evident, which are expected to result in findings representing or exceeding the maximum tolerated dose (MTD) in a longer study.
Thus, for the subchronic study the dietary concentrations of 0, 120, 600 and 3000 ppm were chosen.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Inspections on mortality and morbidity of the animals were performed twice daily. General clinical observations (in the home cage) were made daily.
DETAILED CLINICAL OBSERVATIONS: Yes, all animals
- Time schedule: Once before the start of treatment and once weekly thereafter animals were clinically examined in detail including observations in a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed.
BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Body weights of animals were determined before the study start and weekly thereafter up to scheduled necropsy. Furthermore, body weights were also recorded immediately before scheduled necropsies for calculation of relative organ weights.
FOOD AND WATER INTAKE : Yes, all animals
Food and water intake was determined per cage at comparable periodical intervals (e.g. weekly). These primary data were then used to calculate the group means for each period of approximately 7 days.
On the basis of these data the following parameters were calculated:
- for each interval: daily food intake per animal, mean daily food intake per animal, mean daily food intake per kg body weight;
- for the total period: measurement of mean food intake per animal and day, mean food intake per kg body weight and day;
- cumulative food intake per animal and cumulative food intake per kg body weight.
Comparable calculations were done for water intake.
OPHTHALMOSCOPIC EXAMINATION: Yes, all animals
- Time schedule for examinations: All animals were subjected to ophthalmological inspection before start of treatment and on day 85.
The pupillary reflex of both eyes was first tested in a darkened room and the anterior regions of the eye were inspected. After dilating the pupils with Mydriaticum Stulln® drops the refractive compartment of the eye as well as iris and fundus were examined using an indirect ophthalmoscope. In addition, the optical media were examined with a ZEISS photo-slit lamp.
HAEMATOLOGY: Yes, all animals
The blood samples were collected in the morning from the retro-bulbar venous plexus of fasting animals anesthetized with CO2/air. The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant). The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
- Time schedule for collection of blood: day 86, 87
- Parameters investigated: Erythrocyte count, Hemoglobin concentration, Hematocrit, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Mean corpuscular volume, Reticulocyte count, Erythrocyte morphology, Thrombocyte count, Thromboplastin time (Hepato-Quick), Leukocyte count, Differential blood count.
CLINICAL CHEMISTRY: Yes, all animals
The blood samples for determination of glucose concentrations were taken from the caudal vein of fasting, non-anesthetized animals. The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of fasting animals anesthetized with CO2/air. The samples for biochemical tests were heparinized. The blood samples for glucose determinations were mixed with perchloric acid (1+10) to precipitate proteins.
- Time schedule for collection of blood: day 86, 87
- Parameters investigated: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma glutamyl transferase, Glucose, Cholesterol, Triglyceride, Creatinine, Urea, Total bilirubin, Total protein, Albumin, Inorganic phosphate, Potassium, Sodium.
URINALYSIS: Yes, all animals
Urine samples were collected at room temperature during a period of 16 hours. During the urine collection periods, water was offered ad libitum but feed was not supplied.
- Time schedule for collection of urine: day 86, 87
- Parameters investigated: Density, Volume, Protein concentration, Protein excretion, Creatinine concentration, Creatinine excretion, Protein/creatinine ratio, Urea concentration, Urea excretion; qualitatively: Blood, Bilirubin, Glucose, Ketone bodies, Urobilinogen, Microscopy of sediment, pH.
NEUROBEHAVIOURAL EXAMINATION: Yes, all animals
- Time schedule for examinations: Functional observations were performed once (not blind)
Functional Observational Battery (FOB) was performed on days 79, 80, 83, 84. The following observations/examinations were performed: Body weights, home-cage observation, observations during handling, open field observations, reflex/physiological observations.
Motor and Locomotor Activity (MA) was performed on day 77-80. The activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA) was examined. Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
Scheduled necropsy: day 93 (males), day 97 (females)
All animals living on the date of their scheduled necropsy and all animals to be killed in moribund state were sacrificed by exsanguination under isoflurane anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination.
At study termination the following organs of animals sacrificed scheduled were weighed before fixation: Brain, Adrenals (both), Heart, Liver, Kidneys (both), Spleen, Thymus, Testes (both), Epididymides (both), Uterus.
HISTOPATHOLOGY: Yes
The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and all treated groups:
Abnormalities, Esophagus, Kidneys, Liver, Thyroid glands (with parathyroids), Trachea.
The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and high dose group:
Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Eyes, Eyelids, Femur (joint, bone marrow), Heart, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Larynx, Lungs, Lymph nodes (mandibular, mesenteric, popliteal), Optic nerves, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulating glands), Skeletal muscle (thigh), Skin (mammary region), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow, Stomach, Testes, Thymus, Trachea, Urinary bladder, Uterus.

The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution:
Exorbital lacrimal glands, Harderian glands, Head (with skull cap, Nasal Cavity), Intestine (Peyer's patches), Mesentery, Tongue, Ureters, Urethra, Vagina, Zymbal's glands, Physical identifier.

Statistics:

For body weights, body weight gain and absolute organ weights: Dunnett Exact Homogeneous Test; for relative organ weights: Dunnett Exact Homogeneous Test after log. Transformation; for calculated food/water intake per animal: adjusted Mann-Whitney U-tests; for clinical pathology parameters: Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log. Transformation or the Bonferroni/Mann-Whitney U-test.
Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit.
For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett’s tests were performed. Heteroscedastic normally distributed variables were analyzed using appropriately adjusted Dunnett’s tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.
With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance using the SAS procedure PROC CATMOD.
Statistics of MA/LMA were be generated with an evaluation step of the SPADER (=Safety Pharmacology Automated Data Evaluation and Reporting) application.
For statistical evaluations of histopathological findings, if any, the PATHDATA program was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations revealed no toxicological relevant findings up to and including 600 ppm in males and females. At the dose of 3000 ppm, all animals showed increased feces excretion. The finding of gnawed fur on forelegs in several male animals of all groups and in some females in the low and high dose group was considered a chance finding, as it is a known spontaneous finding in rats and did not show a clear dose-dependence.

Mortality:

no mortality observed

Description (incidence):

All male and female animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was not affected at 120 and 600 ppm. Body weight gain was slightly decreased in males and moderately decreased in females at 3000 ppm, so that the body weight at the end of the study was reduced 5.4% in males and 8.1% in females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake was increased in female animals starting at 120 ppm throughout the study. At the low and mid dose this translated into a slightly higher body weight and an increased body weight gain at the mid dose. So this was not considered as a toxicological relevant finding. At the high dose body weight gain was reduced so that there was a mismatch, indicating some disturbances in digestion or metabolism. In males this tendency was not observed.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption showed a transient increase in males dosed at 3000 ppm from day 15 up to day 36 of measurement, but not during the further course of treatment. Therefore, it was considered an adaptive change without toxicological relevance.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few animals in the control and dose groups showed waterklefts, calcification, retrolental opacity or a zone of discontinuity. All of these effects were randomly distributed without dose dependence and are therefore of no toxicological relevance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology revealed no toxicologically relevant changes at the doses of 120 and 600 ppm. At the high dose of 3000 ppm, some minor changes were found in male and female animals in red blood cell parameters and in males also in coagulation. These minor changes were not considered indicative of a toxic effect. However, as similar changes were observed in the previous 4-week toxicity study, they are considered indicative of a compound-related effect. This included a slight reduction in erythrocyte count and in hematocrit and a slight increase in mean corpuscular hemoglobin and in mean corpuscular hemoglobin concentration in both sexes. In addition, slight prolonged Hepato Quick was observed in high dose males. The higher monocytes count found in males starting at 120 ppm was not considered to be toxicologically relevant, as the values of the individual animals were within the range of reference values and since the control group showed low values in comparison to reference values, so that the difference was considered a chance finding due to the control values.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry did not reveal any clear-cut adverse findings. At the high dose of 3000 ppm, a decrease in glucose, a slight increase in alkaline phosphatase and a decrease in cholesterol were observed in the high dose males. In both sexes, the creatinine and protein levels were decreased. Most values were well within the range of historical controls and all differences were slight, so that these alterations were considered to be of minor relevance. Potassium was slightly increased in the high dosed males but even the control animals were in the upper range of the historical control data, therefore this finding was considered to be of minor relevance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis did not reveal any findings, which are considered indicative of a toxic effect. At 600 and 3000 ppm, male animals showed a lower protein excretion and in parallel a reduction in the total amount of excreted protein and in the protein-creatinine ratio. In addition, pH was slightly more acidic than in controls. In females, urinary volume was lower and in parallel, urinary density and excretion of urea were increased and the total amount of excreted creatinine, urea and protein were lower. Most values were well in the range of historical control data, so that they are considered in the physiological range. In addition, such changes were not considered as indicative of any relevant organ toxicity.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related effects in the functional observation battery were observed and no statistically significant effects on motor and locomotor activity were seen.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight evaluation did not reveal any changes at the dose of 120 ppm. At 600 ppm and higher, absolute and relative kidney weight was slightly increased in male animals. In female animals, only the high dose was affected. In addition, at 3000 ppm, relative and absolute liver weights were increased in females. There were further changes in relative organ weights: In high dose males, relative weights of adrenal glands, testes and epididymis were increased. In high dose females, relative weights of brain and adrenal glands were increased. These changes were considered a secondary effect to the reduced body weight since the absolute weights were non-altered. Thus, they were not considered of toxicological relevance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathology revealed no adverse effects at 120 ppm. Nephropathy of the renal P3 segment occurred in females at 600 ppm and above. Tubules in this area appeared to be dilated, flattened and filled with proteinaceous or cellular debris. Males showed minimal nuclear crowding of cortical tubules with an incidence of 1-2-3-5. The relevance of this non-adverse finding remains debatable. Hepatocellular hypertrophy and eosinophilic change were found in all males and females at 3000 ppm, few animals showed minimal single cell necrosis. Females were more affected by incidence and grading than males. Hepatocellular fat storage was decreased in incidence in 3000 ppm males. Diffuse follicular cell hypertrophy of the thyroid was observed in 4/10 females at 3000 ppm. This finding is possibly related to increase liver metabolism of thyroidal hormones and thus represents a secondary, reactive change.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

120 other: ppm (corresponding to 10.8 mg/kg bw and day)

Sex:

male/female

Basis for effect level:

other: Adverse effects on the kidney (nepropathy, organ weight) at next high dose level (600 ppm)

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The subchronic study with the structurally related MCPP-P OE indicated the kidney and liver as target organs for systemic toxicity. The NOAEL for systemic toxicity was 120 ppm, corresponding to 10.8 mg/kg bw/day for males and females.

Executive summary:

A subchronic repeated dose toxicity study with the structurally related MCPP-P OE was conducted according to OECD TG 408 and GLP. The test substance was administered orally via the diet to 10 male and 10 female Wistar rats per dose group in doses of 0, 120, 600 and 3000 ppm up to 97 days. Mortality was not observed during the course of the study. Ophthalmoscopical examinations revealed no evidence of treatment-related effects. No treatment-related effects were observed in the functional observation battery and on motor/locomotor activity. Gross pathology did not reveal any adverse effects. The dose of 600 ppm caused nephropathy in females. In addition, absolute and relative kidney weight was increased in male animals. The dose of 3000 ppm caused increased feces excretion in both sexes, increased food consumption in females, a slight to moderate decrease in body weight gain, minor changes in red blood cell parameters and coagulation, minor changes in plasma in one or both sexes, an increased absolute and relative weight of liver in females and an increased absolute and relative weight of kidneys in females. Microscopically, hepatocellular hypertrophy and eosinophilic change were found in all males and females, few animals showed minimal single cell necrosis. Hepatocellular fat storage was decreased in males. In summary, at 600 ppm and higher, adverse effects were observed on the kidney. At the high dose, liver was also affected. In addition, there are indications on alterations in feces, body weight development, food consumption, clinical chemistry and blood cell parameter. The NOAEL was 120 ppm, corresponding to 10.8 mg/kg bw/day for males and females.