Methyl benzoate

Endpoint summary

Description of key information

Methyl benzoate is not classified for specific target organ toxicity, since the available studies show the potential toxicity above the guidance values (specific target organ toxicity - repeated exposure).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 22, 2022 - January 11, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat was selected as a test system because it has been historically shown to be a suitable model for repeated dose toxicity studies and is recommended in OECD Test Guidelines. The OECD and other regulatory authorities also recommend it. Wistar strain has been chosen for this study because of its widespread use as a rodent model in toxicology studies and well-established historical information available about its genetic and physiologic background. JRF also has extensive in-house historical control data available for this species. Therefore, Wistar rat was selected as the test system to study the effect of the test item. The results of the study may be of value in predicting the toxicity of the test item to human beings.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility (ABF), JRF
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks old
- Housing: groups of 2 and 3 rats/cage/sex during the study period in sterilised polypropylene rat cages. Rat (Animal number 81) with more than 400 g body weight in group housed i.e., 3 rats/cage during the study period, were removed from original cage and housed in a new cage.
- Diet (e.g. ad libitum): Teklad Certified Global 14% Protein Rodent Diet, Envigo, USA, ad libitum
- Water (e.g. ad libitum): filtered drinking water (Reverse Osmosis water filtration system), ad libitum
- Acclimation period: 7 days prior to randomisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 59 to 67
- Air changes (per hr): 17-18
- Photoperiod (hrs dark / hrs light): 12/12; intensity 135 - 263 lux

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was received for each dose level separately. The test item was mixed with the vehicle to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulations were prepared on each day of the dosing and were administered to rats immediately, after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item was miscible with corn oil in the solubility test.
- Amount of vehicle (if gavage): constant dose volume of 4 mL/kg b. wt

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method for stability, active ingredient (a.i.) concentration, and homogeneity of dose formulation was validated at the Department of Chemistry, JRF (JRF study number 228-2-13- 30087). Based on the stability results, the test item was found to be stable up to 8 days in dose formulation at room temperature.
Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle, and lower layers) from each concentration except vehicle control (only one middle aliquot). The sampling was performed prior to initiation of dosing and at monthly intervals thereafter, for dose formulation analysis. One set of samples was used for analyses and the other set of samples was stored in the frozen condition. The second set of samples will be disposed of during report finalization. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis was ±15% from nominal value and %CV < 10.

Duration of treatment / exposure:

main study: 90 d
recovery groups: recovery group animals were maintained untreated for a period of 28 days after the treatment for 90 days

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

originally: 1000 mg/kg b. wt./day; however, high dose was amended based on mortality rate and severity of toxicity
Male: days 1 to 30 and Female: days 1 to 29: 1000 mg/kg b. wt.
Male: days 31 to 63; Female: days 30 to 62: 600 mg/kg b. wt.
Male: days 64 to 90; Female: days 63 to 90: 750 mg/kg b. Wt.

No. of animals per sex per dose:

main groups: 10/sex/dose; recovery groups: 5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three dose levels (low dose – 100, mid-dose - 300 and high dose - 1000 mg/kg b. wt.) were selected based on results of repeated dose 28-day DRF and in consultation with the Sponsor. High dose level was changed intermittently i.e., 1000 mg/kg b. wt. (days 1 to 30), 600 mg/kg b. wt. (days 31 to 63) based on mortality rate, clinical observations, and severity of toxicity and then based on the reduced clinical observations increased to 750 mg/kg b. wt./day for the remaining period of the study.
- Fasting period before blood sampling for clinical biochemistry: fasted overnight (with ad libitum supply of drinking water)
- Rationale for selecting satellite groups: to evaluate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d
- Dose range finding studies: 7 d DRF (JRF Research Number: RES-1-02-30085), 28 d DRF (JRF Research Number: RES- 1-02-30086)

Positive control:

not required by the guideline

Observations and examinations performed and frequency:

MORTALITY AND MORBIDITY
- Time schedule: twice daily


CLINICAL OBSERVATIONS
- Time schedule: twice daily


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment and at weekly intervals, thereafter
- Cage side observations:
Posture: Flattened, limbs may be spread out (Lying on side, Curled up often asleep (Asleep), Sitting but with head hung down (Sitting A) - Sit A, Sitting normally, feet tucked in (Sitting B) - Sit B, Sitting or standing alert, watching (Sitting C) - Sit C) Rearing, Vertical jumping, Writhing (twisting, squirming or contorted motion), Circling
Clonic Convulsion: Absent, Present
Tonic Convulsion: Absent, Present
- Handling Observations:
Ease of removing rat from cage: Very easy (sits quietly); Easy (vocalizations without resistance); Moderately difficult (rears); Difficult (runs around cage); Very difficult (aggressive, attempts to bite with or without vocalizations)
Handling reactivity: Easy (alert, limbs put against the body); Moderately easy (vocalizations without resistance); Difficult (squires, twists, attempts to bite with or without vocalizations); freezes (rigid in hand and totally inactive)
Palpebral closure: Eyelids wide open (EWO); Eyelids slightly closed; Ptosis - drooping of eyelids half; Eyelids completely closed
Lacrimation: None (no external lacrimation); Slight (wetness of lower eyelids); Moderate (wetness of eyelids and its surrounding area); Severe (dropping of tears)
Eye examination: Normal; Abnormal
Piloerection: Absent; Present
Skin examination: Normal; Abnormal
Salivation: None (no external salivation); Slight (wetness of lower mandible); Moderate (wetness of lower mandible and its surrounding areas); Severe (drooling of saliva)
- Open Field Observations:
Gait: Normal; Slightly abnormal; Moderately abnormal; Severely abnormal
Mobility: Normal; Slightly impaired; Moderately impaired; Totally impaired
Arousal: Very low; Low; High; Very high
Vocalizations: Vocalizations (actual number)
Rears: Rears (actual number)
Respiration: Normal; Abnormal
Clonic movement: Absent; Mild clonic tremors of limbs; Chewing, clonus of the jaws; Repetitive clonic tremors of the whole body
Tonic movement: Absent; Tonic contraction or extension of hind limbs; Opisthotonos (head, body and limbs rigidly arched backward); Emprosthotonos (head, body and limbs extended forward)
Urination: Urination (actual number of urine pool in open field)
Defecation: Defecation (actual number of fecal boluses in open field)
Stereotypy: Absent; Present
Bizarre behavior: Absent; Present


BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning of treatment and at weekly intervals, thereafter. Rats from all groups were also weighed on the day of necropsy (fasted body weight) and at death


FOOD CONSUMPTION AND COMPOUND INTAKE:
- A weighed amount of feed was offered weekly. Leftover feed was measured in each cage once at weekly intervals during the study period.


FOOD EFFICIENCY:
- 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: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the commencement of dosing and prior to sacrifices (terminal and recovery). Eye drops of mydriatic agent (Homatropine hydrobromide) were used to dilate pupils approximately 20 minutes before the eye examination. Both eyes of each rat were examined using a direct ophthalmoscope.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment and recovery periods
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters: Haematocrit, Haemoglobin, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Mean Corpuscular Volume, Platelets Count, Erythrocyte Count (Red Blood Cells), Total Leukocyte Count (White Blood Cells), Differential Leukocyte Count, Reticulocyte Count, Prothrombin Time, Activated Partial Thromboplastin Time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment and recovery periods
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters: Alanine Aminotransferase, Albumin, Alkaline Phosphatase, Aspartate Aminotransferase, Bile Acids, Calcium, Creatinine, Creatine Kinase, Gamma Glutamyl Transpeptidase, Glucose, Lactate Dehydrogenase, Inorganic Phosphorous, Total Cholesterol, Total Protein, Total Bilirubin, Triglycerides, Urea, High-density lipoproteins, Low-density lipoproteins, Albumin: Globulin ratio, Globulin, Blood Urea Nitrogen, Chloride, Potassium, Sodium


PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at the end of treatment and recovery periods
- Animals fasted: Yes
- How many animals: all surviving rats

URINALYSIS: Yes
- Time schedule for collection of urine: individually from all surviving rats at the end of the treatment and recovery periods
- Animals fasted: Yes
- Parameters: Appearance, Color, Volume, Sediment evaluation (Microscopic examination), Specific gravity, pH, Protein, Glucose, Ketone, Blood, Bilirubin, Urobilinogen


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the 12th week of treatment (G1 to G4) and last week of the recovery period (G5 and G6) for all surviving rats
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Grip Strength, Foot Splay

IMMUNOLOGY: No


OTHER:
Estrous Cycle Evaluation: On the day of necropsy, a vaginal smear was examined from all surviving female rats to determine the stage of the estrous cycle.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At scheduled terminal and recovery sacrifices, rats were euthanized by carbon dioxide asphyxiation. All rats (surviving and found dead) were subjected to a full gross necropsy under the direct supervision of a veterinary pathologist. All rats were examined carefully for external abnormalities. The cranial, thoracic, and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities

HISTOPATHOLOGY: Yes (see table "Organ weights and microscopic examination")

Statistics:

Data were recorded in standard formats and validated Pristima® software and summarised in tabular form. Data were processed to get group means and standard deviations. All parametric and non-parametric data was analyzed statistically.
Parametric: Body weight, body weight change, food consumption, FOB parameters (grip strength and foot splay), absolute organ weight, relative organ weight, hematology and coagulation, clinical chemistry, thyroid hormones, and some of the urinalysis parameters
Non-parametric: NBO and FOB parameters (urination, defecation, rearing count, vocalization, and motor activity)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats were normal throughout the dosing period in control, low and mid (except male) dose groups.
Mild to moderate salivation was observed intermittently during treatment days 5 to 90 in male of mid dose group and male and female rats of high dose treated group. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al., 2011). Therefore, it was considered that the transient salivation observed in this study was with doubtful or no toxicological significance.
Apart from salivation, there were tremors and piloerection also observed in male and female rats of high dose groups during treatment days 25 to 36. These clinical signs were associated with mortalities and also disappeared on reduction of doses i.e., 1000 mg/kg b. wt./day. Hence, they were considered as effects of test item treatment and adverse in nature.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality or morbidity was observed throughout the treatment and recovery period in male and female rats of control, low and mid dose groups. In the high dose group rats, mortality was observed in both male [on day 24 (Rat N° 93), day 27 (Rat N° 64 and 69). Considering these the dose was reduced to 600 mg/kg b. wt. from day 31 for male rats, and day 30 for the female rats. With no mortality at this dose, as well as no major adverse effects during the clinical observations, dose was further increased to 750 mg/kg b. wt. from day 64 for male rats and day 63 for female rats. Mortalities were observed one female (Rat N° 79) on day 71 and male (Rat N° 61) on day 91. The mortality observed in treatment groups was associated with clinical signs like, tremors, salivation and piloerection. The effects were hence attributed to the test item treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and mean body weight change of male and female rats of low and mid dose groups were comparable with those of vehicle control groups. In high dose groups mean body weight and mean body weight change of female rats were comparable with those of vehicle control groups and male rats having followed statistical variations.
• Significant decrease in mean body weight on days 15, 22, 29, 36, 43 and 90 in male rats of high dose group.
• Significant decrease in mean body weight on days 22, 29, 36, and 43 in male rats of high dose recovery group.
• Significant decrease in mean body weight change on days 8, 15, 22, 29, 36, 43, 50, 85, and 90 in male rats of high dose group.
• Significant decrease in mean body weight change on days 15, 22, 29, 36, and 43 in male rats of high dose recovery group.
Body weight gain reduction of high dose groups was consistent and varies on changing of high doses. Also, it was increased during recovery period compared to treatment period. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Statistically significant decrease (13% decrease than control) was noted in the terminal body weight in the high dose (G4) males. It was recovered at the end of recovery period, as decrease was only 5% and that also without statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption of male and female rats of low and mid dose groups were comparable with those of vehicle control groups except decrease in consumption in male rats of low dose group during days 85-90. In case of high dose groups following statistical changes were noticed.
• Significant increase in mean food consumption in high dose group male rats during days 36-43, 43-50, 50-57, 57-64, 71-78, 78-85 and 85-90 and in female rats during days 1-8, 8-15, 15-22, 22-29, 43-50, 50-57, 64-71 and 71-78.
• Significant increase in mean food consumption in high dose recovery group male rats during days 43-50, 50-57, 57-64, 64-71, 71-78, 78-85 and 85-91 and in female rats during days 1-8, 8-15, 15-22, 22-29, 29-36 and 43-50.
Increased in food consumption of high dose groups was consistent and was without body weight gain. Hence, it was considered as an effect of test item and adverse in nature.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological examination revealed, statistically significant decreases in the eosinophil count in male and female animals from G4 group and in platelet count in G4 males. Values of eosinophil count were below historical range (Male: 0.04 to 0.26; Female: 0.03 to 0.22, 103/µL, 95 percentile, N = 340) for few male and female animals of G4 group, while it was within range for platelets of G4 group. Effects were related to the test item treatment.
Statistically significant increase in reticulocyte count was observed in both sexes of G4 group. It was related to the test item treatment. In the absence of any abnormality in other red blood cell parameters (RBCs, haemoglobin, haematocrit), this increase in reticulocyte count was considered as non-adverse. Moreover, values of all animals of G4 group were within historical ranges (Male: 134.1 to 300.0; Female: 131.5 to 306.2, 109/L, 95 percentile, N = 330).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease was noted in the serum total protein concentration in high dose (G4) males as well as females. It was owed to statistically significant decrease in globulin and albumin, as effect was noted for globulin in high dose (G4) male and female, and mid dose (G3) male, and albumin in high dose (G4) females. Statistically significant increase was noted in albumin: globulin ratio in high dose (G4) and mid dose (G3) males. Alterations were related to the test item treatment.
Statistically significant increase was noted in AST in G4 males and females. It was related to the test item treatment, as values of few animals of G4 males were above historical ranges (Male: 75.36 to 232.86; Female: 65.52 to 234.9, U/L, 95 percentile, N = 300)
Statistically significant decrease was noted in the chloride in high dose (G4) male, and urea and BUN in high dose (G4) and mid dose (G3) females. Effects were related to the test item treatment as values of few animals of high dose group were below historical ranges [Chloride (Male: 103.6 to 111.1; Female: 102.6 to 109.8, mmol/L, 95 percentile, N = 335); Urea: Male: 24.32 to 54.8; Female: 29.12 to 63.62, mg/dL; BUN: Male: 11.36 to 25.59; Female: 13.6 to 29.71, mg/dL, 95 percentile, N = 300].

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, a significant decrease in the serum TSH level in male rats of mid dose group and increase in level in female rats of high dose groups was observed when compared with that vehicle control group.
Statistically, a significant decrease in the serum T3 and T4 levels was observed in male rats of high dose group (G4) and T4 levels in male rats of mid dose group (G3) when compared with that of the vehicle control group. In absence of microscopic changes in thyroid and parathyroid, these changes were considered as not related to test item treatment.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In the home cage, all rats from the treatment (G2, G3, G4 and G6) and vehicle control groups (G1 and G5) showed normal posture, asleep (curled up often asleep), sitting A (Sitting but with head hung down), sitting B (Sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic convulsion were absent in the home cage during NBO. Tremors were noticed during week 5 in male rats of high dose groups and considered as effect of test item treatment.
NBO performed during the handling of rats did not reveal any abnormalities. All rats revealed normal behavior during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rats showed salivation, lacrimation, and piloerection. Eyelids were wide open in all rats. The eye and skin examinations did not reveal any abnormalities in any rat.
In the open field, all rats from treatment and vehicle control groups showed normal gait, mobility, arousal, and respiration during the ‘two-minute observation period’. Clonic and tonic movements, vocalization, stereotypy, and bizarre behavior were absent in this assessment. The rearing, urination, and defecation count of male and female rats from treatment groups were comparable with those of the respective vehicle control groups except the following statistical variations.
• Significant increase in rearing count in male rats of low (on day 46) and high dose (on day 11) groups and decrease in rearing count in female rats of high dose group (on day 46)
• Significant increase in rearing count in during pre-treatment in male rats and on day 81 in female rats and decrease in rearing count on day 25 in male rats of high dose recovery group
• Significant increase in urination count in male (on days 25, 32, 39, and 67) and female (on day 39) rats of high dose recovery group
• Significant increase in defecation count in male (on days 18, 25, 46, and 53) and female (on days 25, 32, 39, and 46) rats of high dose group
• Significant increase in defecation count in male (pre-treatment, on days 4, 18, 25, and 32) rats of high dose recovery group
These findings were intermittently observed and considered incidental in absence of any other supportive findings.
Mean motor activity data of rats from the treatment groups were comparable with those of the respective vehicle control groups.
Sensory reactivity parameters, viz., approach response, touch response, click response, tail pinch response, pupil response, and air righting reflex in rats of the treatment groups were comparable with those of the respective vehicle control groups.
Mean grip strength values (forelimb and hindlimb) of treated rats were comparable with those of the respective vehicle control groups.
Mean hindlimb foot splay values of treated rats were comparable with those of the respective vehicle control groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item treatment led to significant increase in absolute and relative weights of heart and kidneys in male and female rats of G4 when compared to G1. Absolute and relative weights of liver also revealed significant increase (although statistical significance was absent in absolute weights) in G4 female. Increase in relative weights was also observed after recovery period for kidneys and liver in G6 males. Though effects were related to the test item treatment, in absence of treatment related histopathological lesions in liver, kidneys and heart, the effects observed were considered as non-adverse.
Statistically significant decrease was observed in absolute and relative weights of thymus in G4 male rats. Effect was related to the test item treatment and recovered after the recovery period as statistically significant increase was noted in relative weights of thymus in G6 males. Considering the lower incidence and severity of the histopathological lesion (decreased cellularity) and reversal of the effect at recovery sacrifice, this effect was considered as non-adverse.
Statistically significant decrease was also observed in absolute weight of brain in G4 male and female, and relative weight of same in G4 female. Considering the incidence & severity of the histopathological lesions in brain (degeneration of dentate gyrus and grey matter), the decrease in weight was considered as adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Visceral examination of the terminally sacrificed and found dead animals revealed congested blood vessels of brain in animal N° 64 and 69, reddish discoloration of thymus in animal N° 64, 69, 14, 56, 59 and 76 and smaller size of thymus was observed in animal N° 67 and 70. These lesions were considered as incidental/spontaneous. Additionally, smaller size of thymus was observed in animal N° 67 and 70 which could be considered as treatment related finding, as evidenced by histopathological lesion (decreased cellularity of lymphocytes).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed treatment related lesions in brain, sciatic nerve, femur with joint and thymus.
Microscopic examination of brain revealed neuronal degeneration of dentate gyrus and in few animals CA region of hippocampus and cerebellum in G4. In addition to this lesion, degeneration of nerve fibres was also noted in sciatic nerve, which suggests the effect of the test item on nervous system. These lesions in brain and sciatic nerve could be the reason for clinical signs (tremors, piloerection) shown by G4 animals.
Decreased cellularity of lymphocytes was recorded in G4 group animals. Although the incidence and severity were less, this lesion was considered as treatment related, as it was noticed in gross observation also, and the incidence was also out of historical control data range (male: 0.234 to 0.552, mg, 95 percentile, N = 460). This lesion was considered as non-adverse, as it was not observed in lower dose groups and recovered completely.
Microscopic examination also revealed increased trabecular bone in metaphysis area of femur in G4 male and female. This lesion was considered either due to increased bone formation by osteoblasts or decreased resorption by osteoclasts.
The lesions observed in brain (neuronal degeneration), sciatic nerve (degeneration of nerve fibres), thymus (decreased cellularity of lymphocytes) and femur with joint (increased bone) were absent in mid and low dose groups, however, noticed in high dose recovery group except lesions in thymus, which suggest that the recovery wasn't complete within 28 days. There could be a different interpretation in a study with an extended recovery period.
 
Examination of found dead animals (3 males of G4, 1 female of G4 and 1 male of G6) did not reveal any lesion specific to cause of death.

Other effects:

no effects observed

Details on results:

The test item did not show any adverse effects on the estrous cycle of female rats at any dose level. The pattern and number of females showing estrous cycle stages indicates the normal cyclicity of females in all study groups (G1 to G6). This was further supported by normal histopathological observations in the uterus, cervix, and vagina of G1 and G4 females.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (actual dose received)

System:

other: nervous system, musculoskeletal system, immune system

Organ:

bone

brain

neurons

thymus

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

For results, please refer to attached tables below.

Conclusions:

Methyl benzoate at high dose level (1000/600/750 mg/kg b. wt./day) cause the mortalities which were associated with clinical signs like, tremors, salivation and piloerection. Test item treatment at higher dose were also associated with decrease in mean body weight and body weight change. An increase in mean food consumption was noted at higher dose treated rats.
Test item treatment at high dose level led to increases in reticulocyte count, albumin: globulin ratio, AST, weights of heart, kidneys and liver, and decreases in eosinophil, platelet, total protein, albumin, globulin, chloride, urea, BUN, terminal body weight, and weights of thymus and brain. Microscopic examination revealed treatment related histopathological lesions in brain (neuronal degeneration of dentate gyrus and CA region of hippocampus and cerebellum), sciatic nerve (degeneration of nerve fibres), thymus (decreased cellularity of lymphocytes), and femur (increased trabecular bone). Though few alterations were minor, considering mortalities and histopathological alterations, effects were considered as adverse.
At mid dose level (300 mg/kg b. wt./day), test item treatment was associated with decrease in globulin, urea and BUN, and increase in albumin: globulin ratio. These changes are minimal and not supported with any other changes. Hence, these were considered as non-adverse.
The No Observed Adverse Effect Level (NOAEL) of methyl benzoate is thus, concluded as 300 mg/kg b. wt./day under the conditions and procedures followed in this study.

Executive summary:

This Repeated Dose, 90-day Oral Toxicity Study was performed according to OECD guideline 408 (October 03, 2008). The study was conducted to evaluate the potential toxicity of Methyl benzoate when administered orally, through gavage, for a period of 90 consecutive days to male and female Wistar rats (10/sex/dose) at dose levels of 0 (control), 100, 300, and 1000/600/750 mg/kg bw/d in corn oil (dose volume 4 mL/kg bw). 5 additional rats/sex allocated to the recovery groups were maintained untreated for a period of 28 days after the treatment for 90 days.

 

 

Dose Formulation Analysis: The a.i. concentration and homogeneity results of dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable range of ±15% of nominal concentration and %CV < 10, respectively.

Mortality and Morbidity: No mortality or morbidity was observed throughout the treatment and recovery period in male and female rats of control, low and mid dose groups. In high dose group rats, mortality was observed in both male (At 1000 mg/kg bw/day - 3 of 15; at 750 mg/kg bw/day - 1 of 15) and female (at 750 mg/kg bw/day - 1 of 15) rats. These mortalities were associated with clinical sign like, tremors, salivation and piloerection and considered as effect of test item treatment.

Clinical Observations: All rats were normal throughout the dosing period in control and low dose groups. Mild to moderate salivation was observed intermittently during treatment days 5 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al., 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

Apart from salivation, there was tremors and piloerection also observed in male and female rats of high dose groups (1000 mg/kg bw/day) during treatment days 25 to 36. These clinical signs were associated with mortalities and also disappeared on reduction of doses. Hence, they were considered as the effects of the test item treatment and adverse in nature.

Ophthalmological Examination: Ophthalmological examination of all rats did not reveal any abnormalities.

Neurobehavioural Observations: No treatment-related changes were observed in neurobehavioural observations performed in male and female rats from the treatment groups when compared to those respective vehicle control groups except tremors during week 5 in male rats of high dose group (1000 mg/kg bw/day). Tremors were considered as effect of test item treatment.

Functional Observational Battery: No treatment-related changes were observed in functional observational battery parameters performed in male and female rats from treatment groups compared to those respective vehicle control groups.

Body Weight and Body Weight Change: No treatment related changes were observed mean body weight and mean body weight change of male and female rats of low and mid dose groups compared to that of vehicle control group. In high dose groups, statistically significant reduction in body weight gain observed at different interval in male rats. These changes were consistent and varies on changing of high doses. Also, it was increased during recovery period compared to treatment period. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Food Consumption: No treatment related changes were observed mean food consumption of male and female rats of low and mid dose groups compared to that of vehicle control group. Statistically significant increase in food consumption was observed at different interval in male and female rats of high dose groups when compared to those of vehicle control groups. These effects were consistent and without body weight gain. Hence, it was considered as an effect of test item and adverse in nature.

Hematology and Coagulation: A statistically significant decreases in the eosinophil count in male and female rats and platelet count in male rats were observed in high dose group. These changes were considered as adverse effect of treatment as values of eosinophil count were outside historical range for few male and female rats.

Clinical Chemistry: Statistically significant decrease in level of total protein (High dose - males and females), globulin (High dose – males and females; Mid dose - males) and albumin (High dose – females) were observed. Also increase in level of albumin: globulin ratio was noted in male rats of high dose and mid dose groups. Apart from these effects, statistically significant increase in the levels of AST (High dose – males and females) and decrease in level of chloride (High dose – males), urea (High dose – females; Mid dose - females) and BUN (High dose – females; Mid dose - females) were noted. Values of these parameters were outside the historical control data and considered as effect of test item and adverse in nature.

Hormone Analysis: No treatment-related changes were observed in serum level of T3, T4 and TSH of rats from treatment groups compared to those respective vehicle control groups indicating no major adverse effects on the thyroid functions.

Organ Weight: A treatment related decrease in terminal body weight and increase in weight of heart, kidney and liver was observed in the high dose treated rats. However, there were no treatment related histopathological changes observed in these organs, hence, effect was considered as non-adverse. Also, a significant decrease in weight of thymus (males) and brain (male and female) were noticed in high dose groups rats. In case of thymus incidence and severity of the histopathological lesion (decreased cellularity) were considered lower and reversible. Hence, these effects were considered as non-adverse. Considering the incidence & severity of the histopathological lesions in brain (degeneration of dentate gyrus and grey matter), the decrease in weight was considered as adverse effects.

Macroscopic Examination: Smaller size of thymus was observed in rat N° 67 and 70 which could be considered as treatment related finding, as evidenced by histopathological lesion (decreased cellularity of lymphocytes).

Microscopic Examination: Microscopic examination revealed treatment related lesions in brain (neuronal degeneration of dentate gyrus and in few animals CA region of hippocampus and cerebellum), sciatic nerve (degeneration of nerve fibres), femur with joint (increased bone) and thymus (decreased cellularity of lymphocytes) were observed in rats treated with high dose. Lesions found in brain and sciatic nerve could be cause of clinical signs like tremors and piloerection. Decreased cellularity of lymphocytes was recorded in the high dose animals. Although incidence and severity were low, the incidence was out of the historical control range and, therefore considered as treatment related. However, this lesion was considered as non-adverse, since it was not observed in lower dose groups and recovered completely. Lesion observed in femur was considered either due to increased bone formation by osteoblasts or decreased resorption by osteoclasts.

All the above findings are limited to high dose only and not observed in rats treated with mid and low dose groups.

Examination of found dead animals (3 males of G4, 1 female of G4 and 1 male of G6) did not reveal any lesion specific to cause of death.

 

Conclusion:

Methyl benzoate at high dose level (1000/600/750 mg/kg bw/day) cause the mortalities which were associated with clinical signs like, tremors, salivation and piloerection. Test item treatment at higher dose were also associated with decrease in mean body weight and body weight change. An increase in mean food consumption was noted at higher dose treated rats.

Test item treatment at high dose level led to increases in reticulocyte count, albumin: globulin ratio, AST, weights of heart, kidneys and liver, and decreases in eosinophil, platelet, total protein, albumin, globulin, chloride, urea, BUN, terminal body weight, and weights of thymus and brain. Microscopic examination revealed treatment related histopathological lesions in brain (neuronal degeneration of dentate gyrus and CA region of hippocampus and cerebellum), sciatic nerve (degeneration of nerve fibres), thymus (decreased cellularity of lymphocytes), and femur (increased trabecular bone). Though few alterations were minor, considering mortalities and histopathological alterations, effects were considered as adverse.

At mid dose level (300 mg/kg bw/day), test item treatment was associated with decrease in globulin, urea and BUN, and increase in albumin: globulin ratio. These changes are minimal and not supported with any other changes. Hence, these were considered as non-adverse.

The No Observed Adverse Effect Level (NOAEL) of Methyl Benzoate is thus, concluded as 300 mg/kg bw/day under the conditions and procedures followed in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

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

7 d dose range finding study

 

A study was conducted to evaluate the potential toxicity of Methyl benzoate when administered orally, through gavage, for a period of 7 consecutive days to Wistar rats (5 rats/sex/group).

 

Prior to the repeated dose 7-days toxicity phase, a Maximum Tolerated Dose (MTD) phase was conducted to select the dose levels. In this dose escalation phase, groups of 3 male and 3 female rats/set were dosed once at dose levels of 800, 1200 and 2000 mg/kg bw in corn oil. Dosing of each set was performed with gap of 24 h between doses. Rats of each set were observed for 72 h post dosing and then subjected to gross examination. No mortality, morbidity and clinical signs were observed during the observation periods after single administration of the test item. Therefore, dose levels of 0 (control), 250, 500, and 1000 mg/kg bw/d in corn oil were selected for the 7 d DRF study.

 

No mortality or morbidity was observed during the treatment period in male and female rats of control and treatment groups.

All rats were normal, throughout the observation period in control and treatment groups.

Mean body weight and body weight change of male and female rats of treatment groups were comparable with that of the vehicle control group.

No treatment related change was observed in food consumption of rats from treatment groups when compared with that of the vehicle control group, except for a statistically significant increase in mean food consumption which was observed during days 4-7 in male rats of the high dose group. This variation cannot be interpreted conclusively in absence of changes in body weight of same rats and of clinical or histological data.

Mean organ weight, and relative organ weight of rats from treatment groups were comparable with that of the vehicle control group except for a significant increase in the absolute and relative weights of kidneys in male and female rats in high dose group as compared to control group. Change in weight of kidneys was considered as effect of test item.

Furthermore, a significant decrease was observed in absolute and relative thymus weight in females of high dose group when compared with that of the vehicle control group. This decrease in the weight of thymus was considered as an effect of the test item, especially since a slighter entity of the same effect, even though without statistical significance, was also observed in high dose group of male rats.

Gross examination did not reveal any treatment related findings.

No treatment related changes were observed for in-life parameters as well as terminal parameters of male and female rats at dose levels 250, 500 and 1000 mg/kg bw/day, except for a significant increase in the absolute and relative weight of kidneys in male and female rats, a significant decrease in the absolute and relative weight of the thymus in female rats and a significant increase in food consumption on days 4-7 in male rats at the dose level of 1000 mg/kg bw/day.

Based on the results, it is concluded that Methyl benzoate produces systemic effects (increase in absolute and relative weights of kidneys in male and female rats, decrease in the absolute and relative weight of thymus in female rats and increase in food consumption on days 4-7 in male rats) at dose level 1000 mg/kg bw/day when administered orally, through gavage, for 7 consecutive days in rats

under the conditions and procedures followed in this experiment.

 

28 d dose range finding study

 

A Repeated Dose, 28-day Oral Toxicity Study was performed similar to OECD guideline 407 (October 03, 2008) and designed as Dose Range Finding Study for the subsequent 90 d repeated dose toxicity study. The study was conducted to evaluate the potential toxicity of Methyl benzoate when administered orally, through gavage, for a period of 28 consecutive days to male and female Wistar rats (5/sex/dose) at dose levels of 0 (control), 250, 500, and 1000 mg/kg bw/d in corn oil (dose volume 4 mL/kg bw).

 

No mortality or morbidity were observed in male and female rats of any group, throughout the treatment period.

All rats were normal, throughout the observation period in vehicle control and treatment groups except for mild salivation which was observed intermittently during the study period only post dosing, but not in the morning in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al., 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

No treatment related change was observed in neurobehavioral observations performed in male and female rats from treatment groups when compared with that of the control group.

Mean body weight and body weight change of male and female rats from treatment groups were comparable with that of the vehicle control group except for a statistically significant decrease in mean body weight on days 22 (12.03%), and 28 (14.1%) and body weight change on days 15, 22 and 28 which were observed in male rats of high dose when compared with that of the vehicle control group. The reduction in the body weight of males in the high dose group was more than 10% of control group and considered as effect of test item treatment.

Mean food consumption of male and female rats from low dose and mid dose groups were comparable with that of the vehicle control group except in male rats of mid dose group where a significant increase in mean food consumption was observed during days 22 to 28 (about 20 %). Similarly, statistically significant increase in food consumption was observed during days 22 to 28 (about 15 %) in male rats and during days 8 to 28 (ranging from 21 to 25 %) in female rats of high dose group when compared with that of the vehicle control group. These changes could be considered due to the test item treatment.

Statistically significant increase in the levels of AST in male and female, ALT in female, and albumin: globulin ratio in male was observed in rats of high dose group when compared with the vehicle control group. Lipid profile tests showed statistically significant increases in the level of HDL in (G4) male, triglyceride in (G4) female, cholesterol, and HDL in (G3 and G4) female rats. Significant decrease in the level of electrolytes parameter (potassium and chloride) were observed in male rats and (sodium, potassium, and chloride) in the female rats of high dose group when compared with that of the control group. Statistically significant decrease in the level of urea and blood urea nitrogen were observed in the female rats of mid dose and high dose groups when compared with that of the vehicle control group. Effects/alterations in clinical chemistry parameters were considered as effect due to test item treatment in mid dose and high dose group.

Terminal body weight was significantly reduced by 15 % in males compared to control, while no change in terminal body weight was observed in female rats.

The relative weight of heart and brain in male rats of the high dose group was significantly increased when compared with that of the vehicle control group. A statistically significant increase in the absolute weight of the heart of males in G3 was observed. These changes may be related to the decrease in the terminal body weight of high dose group in males. Similarly, a statistically significant decrease was noted in the absolute weight of thymus and prostate with seminal vesicles and coagulating gland in male rats of high dose group when compared with that of the vehicle control group. Effects could be related to the test item treatment.

Statistically significant increase in weight of kidneys (relative weight in both sexes and absolute weight in females) and liver (relative weight in males and absolute weight in females) was observed in the rats of the high dose group when compared with that of vehicle control group. These changes are likely to be related to enzymatic changes and electrolyte level. Hence, it was considered as related to the test item treatment.

Gross examination of all rats did not reveal any treatment related finding.

Microscopic examination of liver and kidneys in the mid dose and high dose group did not reveal any treatment related effect.

 

Test item treatment at the dose level of 1000 mg/kg bw/day led to a decrease in mean body weight on days 22 and 28, decrease in body weight change on days 15 to 28 in male rats, increased food consumption during days 22 to 28 in male rats and during days 8 to 28 in female rats.

Test item treatment at dose level 1000 mg/kg bw/day led to an increase in the level of AST in both sexes, ALT in females, albumin: globulin ratio in male, and lipid profile (HDL in both sexes, triglyceride, and cholesterol in female) tests. Similarly, treatment at high dose also led to decreases in electrolyte parameters (potassium and chloride in both sexes, and sodium in females) and BUN and urea in females. In organ weight, significant changes were noted in absolute and/or relative weights of heart, brain, thymus, prostate and seminal vesicles with coagulating gland in male rats and kidneys and liver in both sexes.

Test item treatment at 500 mg/kg bw/day led to an increase in food consumption in male rats, and an increase in the level of HDL and cholesterol and a decrease in the level of BUN and urea in the female rats.

No adverse effects were observed at 250 mg/kg bw/d.

Therefore, the No Observed Adverse Effect Level (NOAEL) of Methyl Benzoate is 250 mg/kg bw/day when administered orally through the gavage up to 28 days in Wistar rats, under conditions and procedure followed in this study.

 

90 d repeated dose toxicity study

A Repeated Dose, 90-day Oral Toxicity Study was performed according to OECD guideline 408 (October 03, 2008). The study was conducted to evaluate the potential toxicity of Methyl benzoate when administered orally, through gavage, for a period of 90 consecutive days to male and female Wistar rats (10/sex/dose) at dose levels of 0 (control), 100, 300, and 1000/600/750 mg/kg bw/d in corn oil (dose volume 4 mL/kg bw). 5 additional rats/sex allocated to the recovery groups were maintained untreated for a period of 28 days after the treatment for 90 days.

 

Dose Formulation Analysis: The a.i. concentration and homogeneity results of dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable range of ±15% of nominal concentration and %CV < 10, respectively.

Mortality and Morbidity: No mortality or morbidity was observed throughout the treatment and recovery period in male and female rats of control, low and mid dose groups. In high dose group rats, mortality was observed in both male (At 1000 mg/kg bw/day - 3 of 15; at 750 mg/kg bw/day - 1 of 15) and female (at 750 mg/kg bw/day - 1 of 15) rats. These mortalities were associated with clinical sign like, tremors, salivation and piloerection and considered as effect of test item treatment.

Clinical Observations: All rats were normal throughout the dosing period in control and low dose groups. Mild to moderate salivation was observed intermittently during treatment days 5 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al., 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

Apart from salivation, there were tremors and piloerection also observed in male and female rats of high dose groups (1000 mg/kg bw/day) during treatment days 25 to 36. These clinical signs were associated with mortalities and also disappeared on reduction of doses. Hence, they were considered as the effects of the test item treatment and adverse in nature.

Ophthalmological Examination: Ophthalmological examination of all rats did not reveal any abnormalities.

Neurobehavioural Observations: No treatment-related changes were observed in neurobehavioural observations performed in male and female rats from the treatment groups when compared to those respective vehicle control groups except tremors during week 5 in male rats of high dose group (1000 mg/kg bw/day). Tremors were considered as effect of test item treatment.

Functional Observational Battery: No treatment-related changes were observed in functional observational battery parameters performed in male and female rats from treatment groups compared to those respective vehicle control groups.

Body Weight and Body Weight Change: No treatment related changes were observed in mean body weight and mean body weight change of male and female rats of low and mid dose groups compared to that of vehicle control group. In high dose groups, statistically significant reduction in body weight gain was observed at different interval in male rats. These changes were consistent and vary on changing of high doses. Also, it was increased during recovery period compared to treatment period. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Food Consumption: No treatment related changes were observed in mean food consumption of male and female rats of low and mid dose groups compared to that of vehicle control group. Statistically significant increase in food consumption was observed at different interval in male and female rats of high dose groups when compared to those of vehicle control groups. These effects were consistent and without body weight gain. Hence, it was considered as an effect of test item and adverse in nature.

Hematology and Coagulation: Statistically significant decreases in the eosinophil count in male and female rats and platelet count in male rats were observed in high dose group. These changes were considered as adverse effect of treatment as values of eosinophil count were outside historical range for few male and female rats.

Clinical Chemistry: Statistically significant decrease in level of total protein (High dose - males and females), globulin (High dose – males and females; Mid dose - males) and albumin (High dose – females) were observed. Also increase in level of albumin: globulin ratio was noted in male rats of high dose and mid dose groups. Apart from these effects, statistically significant increase in the levels of AST (High dose – males and females) and decrease in level of chloride (High dose – males), urea (High dose – females; Mid dose - females) and BUN (High dose – females; Mid dose - females) were noted. Values of these parameters were outside the historical control data and considered as effect of test item and adverse in nature.

Hormone Analysis: No treatment-related changes were observed in serum level of T3, T4 and TSH of rats from treatment groups compared to those respective vehicle control groups indicating no major adverse effects on the thyroid functions.

Organ Weight: A treatment related decrease in terminal body weight and increase in weight of heart, kidney and liver was observed in the high dose treated rats. However, there were no treatment related histopathological changes observed in these organs, hence, effect was considered as non-adverse. Also, a significant decrease in weight of thymus (males) and brain (male and female) were noticed in high dose groups rats. In case of thymus incidence and severity of the histopathological lesion (decreased cellularity) were considered lower and reversible. Hence, these effects were considered as non-adverse. Considering the incidence & severity of the histopathological lesions in brain (degeneration of dentate gyrus and grey matter), the decrease in weight was considered as adverse effect.

Macroscopic Examination: Smaller size of thymus was observed in rat N° 67 and 70 which could be considered as treatment related finding, as evidenced by histopathological lesion (decreased cellularity of lymphocytes).

Microscopic Examination: Microscopic examination revealed treatment related lesions in brain (neuronal degeneration of dentate gyrus and in few animals CA region of hippocampus and cerebellum), sciatic nerve (degeneration of nerve fibres), femur with joint (increased bone) and thymus (decreased cellularity of lymphocytes) were observed in rats treated with high dose. Lesions found in brain and sciatic nerve could be cause of clinical signs like tremors and piloerection. Decreased cellularity of lymphocytes was recorded in the high dose animals. Although incidence and severity were low, the incidence was out of the historical control range and, therefore considered as treatment related. However, this lesion was considered as non-adverse, since it was not observed in lower dose groups and recovered completely. Lesion observed in femur was considered either due to increased bone formation by osteoblasts or decreased resorption by osteoclasts.

All the above findings are limited to high dose only and not observed in rats treated with mid and low dose groups.

Examination of found dead animals (3 males of G4, 1 female of G4 and 1 male of G6) did not reveal any lesion specific to cause of death.

 

Methyl benzoate at high dose level (1000/600/750 mg/kg bw/day) cause the mortalities which were associated with clinical signs like, tremors, salivation and piloerection. Test item treatment at higher dose were also associated with decrease in mean body weight and body weight change. An increase in mean food consumption was noted at higher dose treated rats.

Test item treatment at high dose level led to increases in reticulocyte count, albumin: globulin ratio, AST, weights of heart, kidneys and liver, and decreases in eosinophil, platelet, total protein, albumin, globulin, chloride, urea, BUN, terminal body weight, and weights of thymus and brain. Microscopic examination revealed treatment related histopathological lesions in brain (neuronal degeneration of dentate gyrus and CA region of hippocampus and cerebellum), sciatic nerve (degeneration of nerve fibres), thymus (decreased cellularity of lymphocytes), and femur (increased trabecular bone). Though few alterations were minor, considering mortalities and histopathological alterations, effects were considered as adverse.

At mid dose level (300 mg/kg bw/day), test item treatment was associated with decrease in globulin, urea and BUN, and increase in albumin: globulin ratio. These changes are minimal and not supported with any other changes. Hence, these were considered as non-adverse.

The No Observed Adverse Effect Level (NOAEL) of Methyl Benzoate is thus, concluded as 300 mg/kg bw/day under the conditions and procedures followed in this study.

Justification for classification or non-classification

- Repeated dose toxicity, oral:

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure of Methyl benzoate, the compound does not need to be classified according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

 

-Repeated dose toxicity, dermal:

As no data of the specific target organ toxicity potential after repeated dermal exposure of Methyl benzoateis available a classification is not possible according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

 

-Repeated dose toxicity, inhalation:

As no data of the specific target organ toxicity potential after repeated dermal exposure of Methyl benzoateis available a classification is not possible according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.