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Repeated dose toxicity: oral

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Administrative data

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:
guideline study
Justification for type of information:
Data is from an experimental study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted: 3rd October, 2008
Principles of method if other than guideline:
The objective of the study was to provide information on the effects of repeated oral administration (2-Methoxyethyl ) benzene (CAS: 3558-60-9) in wistar rats once a day for a minimum period of 28 days with 14 days treatment-free period. According to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2-methoxyethyl)benzene
EC Number:
222-619-7
EC Name:
(2-methoxyethyl)benzene
Cas Number:
3558-60-9
Molecular formula:
C9-H12-O
IUPAC Name:
(2-methoxyethyl)benzene
Test material form:
liquid
Details on test material:
Name: (2-methoxyethyl) benzene
CAS No. 3558-60-9
Other name: : Phenyl ethyl Methyl ether
Molecular Weight: 136.193 g/mol
Molecular Formula: C9-H12-O
SMILES: c1(ccccc1)CCOC
Specific details on test material used for the study:
Name: (2-methoxyethyl) benzene
CAS No. 3558-60-9
Other name: : Phenyl ethyl Methyl ether
Molecular Weight: 136.193 g/mol
Molecular Formula: C9-H12-O
Purity: 99.5024%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Rat is a commonly used species for toxicity studies and is also recommended by the stated regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Male: Minimum: 213.67 g Maximum: 221.65 g
Female: Minimum: 179.27 g Maximum: 181.84 g

- Fasting period before study: No data
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly once during study period. Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material.
- Diet (e.g. ad libitum): A conventional laboratory pellet diet from approved vendor was offered ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: Male and female animals were acclimatised to the test conditions prior to Test Item administration for a period of 7 and 8 days respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.00 and 23.10°C
- Humidity (%): 40.20 to 64.90%
- Air changes (per hr): 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: March 11, 2019 To: May 04, 2019

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected for the dose administration and recommended by the regulatory guideline.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of test items were weighed in the vial. Small quantity of vehicle was added in the vial and mixed well then it was transferred in to a calibrated measuring cylinder. The dose formulation was prepared freshly prior to the dose administration. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration for different dose levels.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil. The test chemical was soluble in corn oil
- Concentration in vehicle: 30, 60 and 90 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated with respect to the parameters covering specificity, linearity, limit of quantification (LOQ), precision (% RSD), Accuracy (% recovery) and Homogeneity.

Specificity: The reference item solution, test item solution, mobile phase, sample diluent(s) and blank test vehicle were injected onto a suitable analytical technique. The result of specificity was reported either no interference observed based on visual comparison or the degree of interference not contributes more than 20 % peak area of the target analyte at the LOQ Level.

Limit of Quantification: The LOQ of the method was the minimum concentration/quantity of a component, which could be quantified precisely and accurately. The LOQ was carried out at the lowest sensitivity of the device under the same instrumental parameters used for the qualified specificity. The LOQ of a compound for a specific method was determined by injecting in duplicate the working solution (s) made in suitable solvent fortified with test item / reference item/reference standard, until it produces a minimum signal to noise ratio ≥ 10:1 for precise quantification.

Linearity: The linearity was carried out by preparing and analyzing minimum five linear concentrations of reference standard with at least one determination. The linear calibration curve was established by plotting the analyte peak response against concentration (mg/L). The linearity was given in the form of equationof the resulting curve and correlation coefficient (r).
Duration of treatment / exposure:
28 Days with 14 days recovery period.
Frequency of treatment:
Once Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (Recovery)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (Recovery)
No. of animals per sex per dose:
Total 35 Males + 35 Females (30 males + 30 females for treatment groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As per the Sponsor's suggestions, the dose levels were 150, 300, and 450 mg/kg for low, mid and high dose respectively.
- Rationale for animal assignment (if not random): Prior to the first day of dosing, male and female animals were separately randomized into four different test groups, based on the most recent body weight, using validated software “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights were set to be within ± 20% of the group means.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection.
- Rationale for selecting satellite groups: To asses the reversal of the effects caused due to the test chemical
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available
Positive control:
No Data Available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs were observed before and after dose administration on the treatment Day 1 and once daily thereafter, preferably at the same time each day and considering the peak period of anticipated effects after dose administration.
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals from main and recovery groups were subjected for detailed clinical examination on treatment Day 1 and weekly thereafter during experimental period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed during randomization, at start of treatment and weekly (± 2 days) thereafter, till the end of experimental period. Fasting body weight of the animals were taken on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Feed Consumption of all animals were determined weekly (± 2 days) once, during the treatment and recovery period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: No Data Available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No Data Available

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was conducted prior to exposure (during Acclimatization period) of all the animals and during week 4th for main group animals and on week 6th for recovery group animals.
- Dose groups that were examined: The examination was conducted in the high dose and control group animals for main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Clinical Pathology evaluation of all surviving rats were performed on the termination day, just prior to necropsy.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All animals from all the dose group was examined.
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical Pathology evaluation of all surviving rats were performed on the termination day, just prior to necropsy.
- Animals fasted: Yes
- How many animals: All animals from all the dose group was examined.
- Parameters checked in table were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the last week of the treatment and recovery periods, all surviving rats of main groups and recovery groups were housed in metabolic cages overnight, (Filtered water was provided ad libitum) and urine samples were collected.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurobehavioural examinations was performed in week 4th for main group animals and in week 6th for recovery group animals.
- Dose groups that were examined: All animals from all the dose group was examined
- Battery of functions tested: sensory activity, grip strength and foot splay were observed.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all surviving animals from main group were sacrificed on Day 29 and from recovery group were sacrificed on recovery Day 15. All surviving animals (main and recovery groups) were subjected to necropsy and detailed gross pathology evaluation. Animals were fasted overnight before necropsy. Animals were weighed, euthanized by CO2 asphyxiation and examined externally. All orifices and the cranial, thoracic and visceral cavities were opened and examined macroscopically.

HISTOPATHOLOGY: Yes, the following tissues were preserved in 10 % Neutral Buffered Formalin (NBF) [except eyes and testes; which were fixed using Modified Davidson’s Fluid] for subsequent histopathological examination.
Adrenals, Pancreas, Aorta, Peyer's Patches, Bone (femur) with joint, Pituitary (weighing after fixation), Brain (cerebrum,cerebellum,mid brain), Prostate and Seminal vesicle with coagulating glands as a whole, Cecum, Rectum, Colon, Salivary glands, Duodenum, Sciatic Nerve, Epididymides, Skeletal muscle, Eyes with optic nerve, Skin, Gross lesion (if any), Spinal Cord (cervical, mid-thoracic and lumbar), Heart, Spleen, Ileum, Sternum with marrow, Jejunum, Stomach, Kidneys, Testes, Liver, Thymus, Lung, Thyroid Parathyroids (weighing after fixation), Mammary glands, Trachea, Mesenteric and Mandibular lymph node, Urinary Bladder, Oesophagus, Uterus with Vagina and Ovaries.
Other examinations:
Oestrus Cycle: At termination, the oestrus cycle of all females were determined by taking vaginal smears.

Bone Marrow Smear Examination: Bone marrow smear (from femur) were prepared at the time of necropsy. Since no treatment-related effects were noted in Hematological or Histopathological evaluation, bone marrow smears were not examined.
Statistics:
Raw Data were processed using Statistical Software Sigma Plot 14.0. The mean and Standard Deviation were calculated using the software and all data were summarized in tabular form. All continuous data (body weight, percent change in body weight with respect to day 1, feed consumption, functional observation battery/neurobehavioral observation, foot splay record, grip strength, motor activity, haematology, clinical chemistry, absolute and relative organ weights, etc.) were checked for their normality and for homogeneity, and analysed using one-way ANOVA for comparison of means. Heterogneous data was analysed using Dunnett’s test. In case of recovery groups, means were compared using t-test for homogeneous data and Mann Whitney’s test for heterogeneous data. The significane threshold for all tests (p value) was set at 0.05. Significant differences obtained from the statistical analysis have been indicated at the relevant places in this report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were noted upto 450.0 mg/kg body weight in either sex.
Mortality:
no mortality observed
Description (incidence):
No morbidity or mortality were detected in animals of any of the experimental groups throughout the duration of the experiment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related decrease was noted in males in body weight and body weight gain (percent change in body weight with respect to Day 1) upto 450 mg/kg when compared to concurrent control animals in either sex. In high dose recovery female, statstically significant (P<0.05) decrease were noted at 450 mg/kg body weight on day 8 and 22.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related effect on food consumption was noted up to 450.0 mg/kg in either sex.
Food efficiency:
not specified
Description (incidence and severity):
No Data Available
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological abnormalities were detected in the control (G1) and high-dose (G4) groups at week 4 in either sex.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted in hematology parameters upto 450 mg/kg in either sex. Statistically significant (P<0.05) decrease in male was noted in Prothrombin Time at 300 mg/kg. In high dose recovery group, statistically significant (P<0.05) increase were noted in Neutrophils in male and RBC, Hematocrit and Hemoglobin in female at 450 mg/kg. The observed variations in hematology parameters at the end of treatment and treatment free period, considered as an inconsistence with dose related response in either sex, reversed in recovery group, minimal in nature and it was further not evidenced by histopathological observation
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted in clinical biochemistry parameters upto 450 mg/kg in either sex.
Statistically significant (P<0.05) increase of Sodium (Female: G3 and G4 at 300 mg/kg body weigh, 450 mg/kg respectively), Chloride (Female: G3 at 300 mg/kg body weight) whereas statistically significant (P<0.05) decrease in Aspartate amino transferase (Male: G2 at 150 mg/kg body weight) and Albumin (Male: G2, G3 and G4 at 150, 300 and 450 mg/kg) were observed. During treatment free recovery period showed statistically significant (P<0.05) increase in Calcium and Phosphorus (Male: G4-R at 450 mg/kg body weight) while statistically significant (P<0.05) decrease in Creatine Kinase (CK) (Male: G4-R at 450 mg/kg body weight) was observed. The observed variations in biochemical parameters at the end of treatment and treatment free period, considered as an inconsistence with dose related response in either sex, reversed in recovery group, minimal in nature and it was further not evidenced by histopathological observation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related changes were noted in urine analysis upto 450 mg/kg body weight in either sex of treated and treatment free groups.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in functional observational battery/neurobehavioral observation upto 450 mg/kg. Mean foot splay of all dose groups was also found comparable to the control mean.
Motor activity measurements revealed statistically (P<0.05) significant decreases in male in Resting Time (RT) and Ambulatory Time (AT) at 450 mg/kg. In female, statistically (P<0.05) significant decrease in Stereotypic Time (ST) at 300 and 450 mg/kg and statistically significant (P<0.05) increase in Ambulatory Time (AT) at 300 and 450 mg/kg and Counter clockwise rotation (CCR) at 150 mg/kg. This changes were not related to treatment, as it was inconsistent, not dose dependent manner.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted in absolute and relative organ weight (relative to body weight and brain weight) upto 450 mg/kg in either sex.
Statistically significant (P<0.05) increase was observed in absolute weight of Adrenals (Male: G2, G3 at 150, 300 mg/kg of body weight). Observed weight variation in the organ did not showed dose dependency and are minor in nature, so could be considered as spontaneous.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted in gross pathological examination upto 450 mg/kg in either sex. Except minimal reduced size of biilateral testes in one animal G2 (150 mg/kg body weight), mild bilateral reduced size in G3 (one animal at 300 mg/kg body weight), and moderate bilateral reduced size in G1-R (one animal at 0 mg/kg body weight) and G4-R (one animal at 450 mg/kg body weight ) were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological findings were observed in vehicle control group and in high dose group animals. Microscopic examination revealed varying degree of different pathological changes in various organs belonging to control and treatment groups as follows: Liver: focal minimal lymphocytic infiltrate (Male: G4: 1/5); multifocal minimal Bile duct hyperplasia (Male : G4: 1/5); Kidneys (Unilateral): focal minimal perivascular lymphocytic infiltrate (Male: G4: 1/5; Stomach : Squamous Cyst (Female: G4: 1/5). Histopathology was not extended to lower dose groups and recovery group, as there is no test item related changes were noted in control and high dose groups.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Formulation Analysis: The concentration and homogenity assessment of the dose formulation of (2-Methoxyethyl ) benzene (CAS: 3558-60-9) were performed on Day 01 and Day 22. The results of the analyzed formulation were 30.075, 57.247 and 89.977 mg/ml for Day 01 and 28.914, 55.978 and 87.189 mg/ml for Day 22 for low, mid and high dose concentration verification were within the acceptance limits ± 15% of the nominal concentrations for all the dose formulation analysis.

Oestrus Cycle: No any abnormalities detected in the oestrus cycle in any of the treatment group at termination.

Bone Marrow examinations: No treatment-related effects were noted in Hematological or Histopathological evaluation, hence bone marrow smears were not examined.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
other: Oestrus cycle
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on all the observations and results, it was concluded that the test chemical did not induce significant local and systemic toxicity at all the tested doses and thus the NOAEL for the test chemical was observed to be 450 mg/kg bw.
Executive summary:

A short term repeated dose toxicity study, according to OECD Guideline 407 (2008) was conducted using male and female Wistar rats. The test chemical, (2 -Methoxyethyl)benzene (CAS No.: 3558 -60 -9) was administered to male and female Wistar rats once a day for a minimum period of 28 days with 14 days treatment-free period. A total number of 60 Wistar rats (30 males and 30 females) were randomly allocated to six different dose groups of 5 animals/sex/group. The animals allocated to Group G2, G3 and G4/G4-R received 150, 300and 450mg/kg body weight of the test chemical respectively, whereas the animals of Group G1/G1-R, received vehicle alone [Corn oil] for 28 consecutive days. Observations comprised of mortality/morbidity, clinical signs, detailed clinical observation, body weight, body weight gain, feed consumption, ophthalmoscopic examination, functional observational battery/neurobehavioral observation, hematology, clinical biochemistry, urinalysis, gross pathology and histopathology (vehicle control group and high dose group).

No treatment related mortality/morbidity were noted in either sex upto 450 mg/kg body weight. No treatment related clinical signs were noted upto 450 mg/kg body weight in either sex. No abnormalities were detected during ophthalmological examination at 450 mg/kg body weight in either sex, when compared with control group. No treatment related decrease in body weight and body weight gain (percent change in body weight with respect to Day 1)was noted in males upto 450 mg/kg when compared to concurrent control animals in either sex. No treatment related changes were noted in feed consumption upto 450 mg/kg body weight in either sex. No treatment related changes were noted in functional observational battery/neurobehavioral observation, foot splay, grip strength and motor activity assessment upto 450 mg/kg body weight in either sex. No treatment related changes were noted in hematology parameters upto 450 mg/kg in either sex. Statistically significant (P<0.05) decrease in male was noted in Prothrombin Time at 300 mg/kg. In high dose recovery group, statistically significant (P<0.05) increase were noted in Neutrophils in male and RBC, Hematocrit and Hemoglobin in female at 450 mg/kg. The observed variations in hematology parameters at the end of treatment and treatment free period, considered as an inconsistence with dose related response in either sex, reversed in recovery group, minimal in nature and it was further not evidenced by histopathological observation. No treatment related changes were noted in clinical biochemistry parameters upto 450 mg/kg in either sex. Statistically significant (P<0.05) increase of Sodium (Female: G3 and G4 at 300 mg/kg body weigh, 450 mg/kg respectively), Chloride (Female: G3 at 300 mg/kg body weight) whereas statistically significant (P<0.05) decrease in Aspartate amino transferase (Male: G2 at 150 mg/kg body weight) and  Albumin (Male: G2, G3 and G4 at 150, 300 and 450 mg/kg) were observed. During treatment free recovery period showed statistically significant (P<0.05) increase in Calcium and Phosphorus (Male: G4-R at 450 mg/kg body weight) while statistically significant (P<0.05) decrease in Creatine Kinase (CK) (Male: G4-R at 450 mg/kg body weight) was observed. The observed variations in biochemical parameters at the end of treatment and treatment free period, considered as an inconsistence with dose related response in either sex, reversed in recovery group, minimal in nature and it was further not evidenced by histopathological observation.

No treatment related changes were noted in urine analysis upto 450 mg/kg body weight in either sex. No any abnormalities detected in the oestrus cycle in any of the treatment group at termination. No treatment related gross (external and internal) pathlogical changes were noted upto 450 mg/kg body weight in either sex. No treatment related histopathological exmanination were noted in any of the tissue/organ at 450 mg/kg body weight in either sex. Based on all the observations and results, it was concluded that the test chemical, (2 -Methoxyethyl)benzene (CAS No.: 3558 -60 -9) did not induce significant local and systemic toxicity at all the tested doses and thus the NOAEL for the test chemical was observed to be 450 mg/kg bw. This study was performed according to GLP.

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