Methyl (R)-2-(4-hydroxyphenoxy)propionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 1992 to 09 December 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

EC Number:

411-950-4

EC Name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

Cas Number:

96562-58-2

Molecular formula:

C10H12O4

IUPAC Name:

methyl (2R)-2-(4-hydroxyphenoxy)propanoate

Test material form:

solid

Details on test material:

- Appearance: Tan or brown solid
- Storage conditions: In the dark at ambient temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD (SD) BR strain

Details on species / strain selection:

The rat was chosen because it is a rodent species commonly requested by regulatory authorities and the Sprague-Dawley strain was selected due to the background data available from previous studies performed at the testing laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: The animals had a mean body weight of approximately 200 g for males and 175 g for females.
- Housing: The animals were housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm) and each cage contained two rats of the same sex and group. A metallic tray was placed under each cage and contained autoclaved sawdust
- Diet: ad libitum pelleted diet
- Water: ad libitum access to bottles filled with potable water filtered with a 0.22 micron filter
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 20 % (relative)
- Air changes: About 13 cycles/hour of filtered non recycled air
- Photoperiod: 12 h of light/12 h of darkness

IN-LIFE DATES
- From: 03 November 1992
- To: 09 December 1992

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered by gavage using a glass syringe fitted with a metal probe. The oral route is the preferred route of administration but was selected on the basis of physicochemical properties, results of acute oral and dermal toxicity tests and likely route of human exposure.

Vehicle:

methylcellulose

Remarks:

Aqueous methylcellulose at 1 %

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test material was suspended in the vehicle and homogenised using a magnetic stirrer. The preparations were made for up to 7 days of treatment according to known stability and delivered to the animal room protected from light using aluminium foil.

DOSE VOLUME: A constant dose volume of 5 mL/kg was used

VEHICLE
- Concentration in vehicle: 0, 3, 30 and 200 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF THE PREPARATIONS
Before the study, the homogeneity of the test material suspended in 1 % aqueous methylcellulose was checked. Two preparations at 3 and 200 mg/mL were sampled in duplicate at 3 different levels (top, middle and bottom) and analysed. The stability of the same suspensions was checked. Each preparation was sampled in duplicate the day of preparation and after 1, 4 and 7 or 8 days storage at +4 °C. On weeks 1 and 4 of the study, each preparation (control group included) was sampled and checked for achieved concentration of the test material.
The results of analyses showed good homogeneity and stability for up to 8 days for each suspension.

ASSAY METHOD
The suspension was agitated using a magnetic stirrer. A 1 ml-volume of suspension was sampled into a test tube and diluted 10 times with acetone/water (70/30) solution using a Microlab 1000 Hamilton dilutor. After vortex-mixing, further dilutions were carried out with the mobile phase in accordance with a target concentration of 20 μg/mL. A 20 μL-aliquot was analysed through High Performance Liquid Chromatography with Ultraviolet detection.

HPLC-UV CONDITIONS
Pump: WATERS (LC Module 1
Mobile phase: A = acetonitrile 35 %; B = water 65 %
Flow rate: 1 mL/min
Column: Zorbax C-8rx (Interchim), particle size = 5 µm; length = 25 cm; inner diameter = 4.6 mm
Temperature: Room temperature
Detector: WATERS (LC Module 1); wavelength = 205 nm, sensitivity range= 0.01 AUfs
Injector: WATERS (LC Module 1)
Injected volume: 20 μL
Integrator: SP 4200 (Spectra Physics) or CR-3A (Shimadzu)
Retention time: Approx. 8 mins
Analysis time: Approx. 11 mins

The results of analysis of samples obtained on weeks 1 and 4 revealed that measured concentrations were between 90 and 99 % of nominal.

Duration of treatment / exposure:

4 weeks (29 days)

Frequency of treatment:

Once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Six animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were determined in agreement with the Sponsor based on the results of a 2-week preliminary toxicity study by oral route (gavage) in rats.
- Rationale for animal assignment: Animal groups were formed in order to obtain approximately the same mean body weight and standard deviation between the groups. The stratified body weight procedure and assignment to the treatment groups were performed using a computer-derived randomisation.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations: Clinical signs were observed for each animal at least once a day, at the same approximate daily time. The animals were checked twice a day, including weekends and public holidays for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded for each animal, once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Food consumption for animals in each cage determined: Yes. The quantity of food consumed by the animals of each cage was recorded once a week over a 7-day period during the treatment period. Food intake per animal and per day was calculated using the amount of food given and left in the cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: Approximately 24 hours after treatment, blood samples were taken from the orbital sinus of the animals. The haematology parameters specified were determined on one occasion on week 4 (day 28). The samples were collected into tubes containing the appropriate anti-coagulant; most parameters were determined with EDTA anticoagulant, with the exception of PT, APTT and FIB which were determined in sodium citrate.
- Anaesthetic used for blood collection: Yes, ether anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: All surviving animals
- Parameters examined: Leucocytes (WBC), erythrocytes (RBC), haemoglobin (HB), packed cell volume (PCV), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), thrombocytes (PLAT), differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and monocytes), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 24 hours after treatment, blood samples were taken from the orbital sinus of the animals. The clinical chemistry parameters specified were determined on one occasion on week 4 (day 28). The samples were collected into tubes containing the anti-coagulant lithium heparinate.
- Anaesthetic used for blood collection: Yes, ether anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: All surviving animals
- Parameters examined: Sodium (Na+), potassium (K+), chloride (Cl -), calcium (Ca++), inorganic phosphorus (I.PHOS), glucose (GLUC), urea (UREA), creatinine ( CREAT), total bilirubin (TOT.BIL), total proteins (PROT), albumin (ALB), albumin/globulin ratio (A/G), cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE AND GROSS PATHOLOGY
On completion of the treatment period, after about 18 hours of fasting, all the animals were asphyxiated by carbon dioxide and sacrificed by exsanguination.
All the animals were weighed before necropsy and the following organs weighed wet as soon as possible after dissection: Adrenals, heart, liver, spleen, thymus, epididymides, kidneys, ovaries and testes. Paired organs were weighed separately.
A complete macroscopic examination was performed on all animals. All gross observations were recorded individually.
For all the animals, all macroscopic lesions and the following tissues were preserved in 10 % buffered formalin (except for the eyes and pituitary gland which were fixed in formolsublimate): Adrenals*, aorta, brain (including medulla/ pons, cerebellar and cerebral cortex), caecum, colon, duodenum, eyes with Harderian glands, femoral bone with articulation, heart*, ileum, jejunum, kidneys*, liver*, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary glands, oesophagus, ovaries*, pancreas, pituitary gland, prostate, rectum, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen*, sternum (with bone marrow), stomach, salivary glands (sublingual and submaxillary), testes and epididymides*, thymus, thyroids with parathyroids, trachea, tongue, urinary bladder, uterus (horns and cervix) and vagina.

HISTOPATHOLOGY
All tissues required for microscopic examination were embedded in paraplast, sectioned at approximately 4 microns in thickness and stained with hemalum-eosin.
Microscopic examination was performed on all macroscopic lesions and tissues listed above marked by *for all animals in the high dose and control groups and all macroscopic lesions for all animals in the low and intermediate dose groups.

Statistics:

The following sequence was used for the statistical analysis of body weight, food consumption, haematology, blood biochemistry and organ weight data: The normality of the distribution of the values in each group was checked by Kolmogorov-Smirnov's test. If the distribution was normal, the homogeneity of variances between the groups was assessed by Bartlett's test (more than 2 groups) or Fisher's test (2 groups). If no significant heterogeneity of the variances was established, the comparison between treated and control groups was performed by Dunnett's test. If the variances were heterogeneous, the comparison between treated and control groups was performed by Dunn's test (more than 2 groups) or by Mann Whitney's test (2 groups). If the distribution of values in the groups was not normal, the analysis was repeated after logarithmic transformation of the values (except for organ weights). If this logarithmic transformation failed to normalise the distribution of the values, comparison of treated and control groups was performed by Dunn's test using original values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was observed from day 2 or 3 of treatment in all males and females treated at 1000 mg/kg/day. This clinical sign was considered to treatment related. Regurgitation after dosing was observed on one occasion for one male and one female given 1000 mg/kg/day. Although only noted in the high dose group, these isolated incidents were only noted in two out of the 12 animals in this group and in consequence, they were considered not to be of toxicological importance. Trauma to the right eye (due to blood sampling) was observed in one female given 150 mg/kg/day. This injury was accidental and not treatment-related. There were no treatment-related clinical signs among animals given 15 or 150 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

No mortalities occurred during the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the treatment period, the mean body weight gain of all treated groups was similar to that of respective controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Throughout the treatment period, the mean food consumption of all treated groups was similar to that of respective controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On week 4, minor differences were observed between treated and control groups for some haematological parameters. These included a slightly lower haemoglobin concentration and packed cell volume in males given 1000 mg/kg/day, a slightly lower total white cell count in males given 150 mg/kg/day and a slightly lower prothrombin time in females given 1000 mg/kg/day. However, these differences were minimal and/or not dose-related and the individual values were within the range of the testing facility’s background data. Accordingly, these differences from control were considered not to be of toxicological importance. No perceptible differences were noted between control and treated animals among the other haematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On week 4, a slightly lower plasma inorganic phosphorus concentration was observed in males and females given 1000 mg/kg/day (-20 and -30 %, respectively). A moderately higher (+81 %) plasma triglyceride concentration was also noted in the males of the same group. These differences in inorganic phosphorus and triglyceride concentration were considered to be treatment related. The differences observed between control and treated animals among the other blood biochemical parameters (namely total protein, creatinine and alkaline phosphatase activity) were minor and the individual values were within the range of the testing facility’s background data. Accordingly, they were considered to be of no toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant lower mean relative liver weight was noted for the males of the low and intermediate dose groups, when compared with controls. However, this was not observed in the high dose group. Consequently, this was considered not to be treatment related. No other statistically significant differences from controls were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

4/6 females of the high dose group showed serous contents in the uterine horns; dilatation of the uterine horns was noted in 2 of these animals. These findings were considered to be of spontaneous nature as they are related to the pro-oestrous phase of the sexual cycle. Therefore, this finding was considered to be of no toxicological significance. The few other macroscopic findings noted were those which are commonly recorded as spontaneous changes in laboratory rats of this strain. Accordingly, they were considered to bear no relationship to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Slight to marked dilation of the uterine horns was noted in 4/4 examined females of the high dose group. In the ovaries of these females, as well as for 3/6 control females, marked follicular development was noted together with the presence of corpora lutea of different generations. All these morphological changes in the ovaries and the uterus are indicative of the pro-oestrus phase of the sexual cycle. Accordingly, they were considered not to be treatment-related. The few other microscopic findings noted were those which are commonly recorded as spontaneous changes in laboratory rats of this strain. Furthermore, their incidence, severity and morphological characteristics were similar between treated and control groups. Accordingly, they were considered to bear no relationship to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The daily oral administration of the test material was well tolerated, inducing at 1000 mg/kg/day only ptyalism and minor changes in plasma inorganic phosphorus and triglyceride concentrations. In isolation, these effects are of doubtful toxicological importance.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects noted at this dose

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

other: Only ptyalism and minor changes in plasma inorganic phosphorus and triglyceride concentrations were seen at 1000 mg/kg/day. In isolation, these effects are of doubtful toxicological importance.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Summary of selected Clinical Chemistry Parameters

Parameter		Males				Females
		Dose (mg/kg/day)
		0	15	150	1000	0	15	150	1000
I.PHOS (mmol/L)	Mean SD N	3.62 0.463 6	3.64 0.305 6	3.35 0.339 6	2.90* 0.509 6	2.75 0.161 6	2.79 0.194 6	2.61 0.253 6	2.11** 0.172 6
CREAT (µmol/L)	Mean SD N	43 5.1 6	42 2.0 6	42 2.4 6	38* 1.2 6	45 3.6 6	48 3.9 6	49 2.2 6	48 3.6 6
PROT (g/L)	Mean SD N	66 1.9 6	64 1.9 6	65 1.8 6	61** 2.8 6	67 1.7 6	67 3.2 6	69 2.9 6	64 2.4 6
TRIG (mmol/L)	Mean SD N	0.81 0.352 6	0.62 0.304 6	1.02 0.254 6	1.47** 0.249 6	0.28 0.101 6	0.36 0.057 6	0.30 0.059 6	0.36 0.079 6
ALP (IU/L)	Mean SD N	412 77.2 6	422 62.9 6	373 39.1 6	327* 44.1 6	243 49.3 6	249 32.2 6	339 51.8 6	206 19.2 6

Significance of the difference between treated and control groups (Dunnett test):

*P<0.05

**P<0.01

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the No Observable Adverse Effect Level was 1000 mg/kg/day (males and females).

Executive summary:

The potential for the test material to cause repeated dose toxicity was investigated in accordance with the standardised guidelines OECD 407 and EU Method B.7 under GLP conditions.

Four groups of 6 male and 6 female Sprague-Dawley rats received the test material daily by gavage for 4 weeks at doses of 0 (control), 15, 150 or 1000 mg/kg/day. The vehicle was aqueous methylcellulose at 1 %. The animals were checked at intervals each day for clinical signs and mortality. Food consumption was measured weekly during the treatment period and body weights were determined weekly from allocation to experimental groups throughout to termination. Haematological and blood biochemistry investigations were performed on day 28. On day 30, all the animals were sacrificed and a macroscopic examination performed. Designated organs were weighed and a range of tissues preserved for microscopic examination.

Ptyalism was observed from day 2 or 3 of treatment in all males and females given 1000 mg/kg/day. This clinical sign was considered to be treatment-related. No mortalities occurred during the course of the study. The food consumption and body weight gain of all treated groups was similar to that of respective controls during the study. There were no treatment related findings among the haematological parameters. A slightly lower plasma inorganic phosphorus concentration was observed in males given 1000 mg/kg/day, with a slightly higher plasma triglyceride concentration in males of the same group. These findings were considered to be treatment-related. There were no treatment-related effects on the weights of those organs determined. None of the macroscopic and microscopic findings noted were considered to be treatment-related.

The daily oral administration of the test material to rats for 4 weeks at doses of 15, 150 or 1000 mg/kg/day was well tolerated inducing at 1000 mg/kg/day only ptyalism and minor changes in plasma inorganic phosphorus and triglyceride concentrations. In isolation, these effects are of doubtful toxicological importance. The No Observable Effect Level is 150 mg/kg/day but the No Observable Adverse Effect Level of the test material is 1000 mg/kg/day.

Under the conditions of this study, the No Observable Adverse Effect Level was 1000 mg/kg/day (males and females).