Methyl 2-benzoylbenzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2014-February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-benzoylbenzoate

EC Number:

210-112-3

EC Name:

Methyl 2-benzoylbenzoate

Cas Number:

606-28-0

Molecular formula:

C15H12O3

IUPAC Name:

methyl 2-benzoylbenzoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): RCX 14-672
- Chemical name: methyl-2-benzoylbenzoate
- Substance type: organic
- Physical state: white to light yellowish powder
- Analytical purity: >99%
- Impurities (identity and concentrations): unknown
- Lot/batch No.: N14003
- Expiration date of the lot/batch: 30 November 2015
- Stability under test conditions: stable
- Storage condition of test material: controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity

Specific details on test material used for the study:

Identification: RCX 14-672
Chemical name: methyl-2-benzoylbenzoate
Batch no.: N14003
CAS no.: 606-28-0
EC no.: 210-112-3
Molecular formula: C15H12O3
Molecular mass: 240.3 g/mol
Description: white to light yellowish powder
Purity: >99% (gas chromatography)
Water solubility: 117.7 mg/l
Test item storage: at room temperature, protected from light
Stability: stable under storage conditions
Expiry date: 30 November 2015

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:WI rats
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: young adult rats, approximately 11 weeks old at starting and 13 weeks at mating.
- Weight at study initiation: males: 349-389 g, females: 230-271 g.
- Housing: standard laboratory conditions
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed
- Water (e.g. ad libitum): tap water from municipal supply from 500 ml bottle ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 35 - 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours light, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2 September 2014 to 25 October 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (polyethylene glycol 400), as a visibly stable homogenous formulation at the appropriate concentrations according to the selected dose level and volume, in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared in up to 8-day intervals and stored at room temperature, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed in the study. Analysis of RCX 14-672 formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration. The formulation was considered stable for 8 days at room temperature.

VEHICLE
The vehicle was selected based on the formulation and analytical trials.
- Name: Poly(ethylene glycol) 400
- Lot/Batch number: BCBL5307V
- Manufacturer: Sigma-Aldrich Co.
- Expiry/Retest Date: January 2015
- Storage: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was performed on 3 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the
Group 1 (control) solution to confirm the absence of test item.

Duration of treatment / exposure:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals.

Dosing of both sexes began after 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy.

Males were dosed for up to 30 days (14 days pre-mating and up to 16 days mating/post-mating) and then euthanized and subjected to necropsy examination. Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

31.25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat. The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on high liver weight changes at 1000 mg/kg bw/day (>60% above control), the highest dose levels selected was 500 mg/kg bw/day. The oral route was selected as it is a possible route of exposure to the test item in humans.

- Rationale for animal assignment:
All parental (P) animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable. Detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weights of the female animals were additionally taken on gestational Days GD10 and 17 in order to give accurate treatment volumes but these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7, then at least weekly (on the days of body weight measurements).

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling three samples were taken from each selected animal (subgroup B, 5 males and 5 or 6 females/group), one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry

- Parameters checked: see tables in Report.

URINALYSIS: Yes
Parameters chekced: leukocytes, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood eythrocytes, specific gravity, sediment, volume, colour/appearance.

NEUROBEHAVIOURAL EXAMINATION: Yes
Five males and 5 females/group: Assessment of potential test item related neurotoxicity was performed in the morning and prior to dosing, during the last exposure week (males on Day 27; females on PPD 4). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength (manual and instrumental) and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessments of motor activity were performed.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured.
Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip
support. The results were tabulated with individual and mean data.

Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was tested. A modified Irwin test was performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an
untreated animal.

Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an openfield for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Obtained data were evaluated for
distance travelled in 5-minute segments.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the females as applicable. Small liver samples of approximately 1 g were collected from from all surviving animals, was snap frozen and stored at approximately -80 C in case of further analysis.

HISTOPATHOLOGY: Yes
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution.

The following tissues and organs were retained from all animals:
Lungs with bronchi, skeletal muscle (quadriceps), adrenal gland, lymph node, small intestine, animal identification, ovary, spinal cord, aorta, oviduct,spleen, brain, pancreas, sternum with marrow, epididymis. pituitary, stomach, eye with the optic nerve, prostate, testis, oesophagus. salivary gland (including mandibular, sublingual and parotid glands), thymus, femur with marrow , thyroid with parathyroid gland, heart, tongue, kidney, sciatic nerve, trachea, large intestine, seminal vesicle with coagulating gland, urinary bladder, extraorbital lachrymal gland, uterus, Harderian gland, skin subcutis with mammary gland (inguinal), vagina, liver.

For the adult animals, a detailed histological examination was performed as follows:
• on the selected list of retained organs in the control and high dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• reproductive organs of all animals of the control and high dose group and all females (uterus, cervix, ovary, oviduct and vagina for females) that failed to deliver healthy pups.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Detailed histological examination was performed on all retained organs in the control and high dose groups and any macroscopic findings (abnormalities) observed in all animals.

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

As test item related histopathology findings were noted in the liver and kidneys of high dose animals, additional histopathology investigations were requested. The liver of all mid and low dose males and all mid dose females and the kidney of all mid and low dose animals was embedded in paraffin wax, processed to slides for histopathology investigation and examined.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used
and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In clinical chemistry analysis, the albumin concentration was higher than the control in both sexes (by ~11% at the high dose). Consequently, total protein concentration was also higher and attained statistical significance in both high dose male and female treated groups. These changes could be associated with the organ weight changes and the microscopic liver findings

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total travelled distance in the test groups was comparable to the control groups for both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item related differences were found in liver and kidney weights at 500 mg/kg bw/day in both sexes and at 125 mg/kg bw/day in males. The absolute and relative liver weight was higher than the control in all dosed male group and in high dose females attaining statistical significance, except the absolute and brain weight related values in the low dose males. The absolute liver weight was higher by approximately 71% in males and by 20% in females. The changes in mid and high dose males and high dose females correlated with the clinical chemistry parameters (increased albumin and total protein) and with the microscopic findings. Compared to control, higher kidney weights were recorded for both sexes in the high dose group and for males in the mid dose group. The differences were approximately 10-30% (absolute value) and 11-33% (relative values), and attaining statistical significance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Test item related microscopic findings were noted in the liver and kidney at dose levels of 125 and 500 mg/kg bw/day. These microscopic changes correlated with increased liver and kidney weights related brain weights. Minimal to mild centrilobular hypertrophy of the liver was noted in 5/5 males and 4/5 females from the high dose groups. In the mid dose groups, minimal hypertrophy was only observed in 4/12 males. No hypertrophy was detected in the mid and low dose females. In addition, no hypertrophy was present when the liver sections from the low dose males were examined. This non-adverse alteration was characterized by increased size of hepatocytes with centrilobular pattern of distribution, without concurrent degeneration and/or necrosis. The hepatocellular hypertrophy was considered to be an adaptive response. In the kidneys, test item related changes were observed in the tubules (cortical and/or corticomedullary junction). In the high dose group, minimal to mild bilateral focal/multifocal cortical tubule degeneration were seen in 2/5 males and 1/5 female. At the mid dose level, minimal focal unilateral tubule degeneration in 1/12 male and 1/12 female were noted. There was no evidence of tubule degeneration in the kidneys at the low dose level. Additionally, the irregular incidence of proteinaceous tubular casts recorded only in treated animals (1/12 low, 0/12 mid and 3/5 high dose males; 3/12 low, 1/12 mid and 1/5 high dose females) indicated that these changes were not treatment-related, therefore were considered to be of no toxicological significance.

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the study, one adult high dose female died during the gavage procedure on Day 35. Pathological changes in the lungs indicated that misdosing was the cause of death. No test item related adverse effects or systemic clinical signs were noted during the study.

BODY WEIGHT AND WEIGHT GAIN
No test item related effects were noted on the mean body weight and body weight gain values following daily administration of RCX 14-672 at dose levels up to and including 500 mg/kg bw/day. Occasional statistically significant differences were regarded as incidental and without toxicological significance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls.
Occasional statistically significant differences were regarded as incidental and of no toxicological significance.

HAEMATOLOGY
When compared to the controls, there were no differences that could be considered toxicologically significant in the treated animals. There was no effect of treatment on coagulation parameters investigated, i.e. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT).

CLINICAL CHEMISTRY
Changes considered related to RCX 14-672 administration were noted in the clinical chemistry parameters examined under the conditions of this study. On Day 28, Albumin (Alb.) concentration was slightly higher than control in all dose groups of males. The differences attained statistical significance in low and high dose males. Consequently total protein concentration (Tot.Prot) was also higher with attaining statistical significance in dosed males. On PPD5, the Albumin (Alb.) and consequently the total protein (Tot.Prot) concentrations were higher than the control in females. The differences attained statistical significance in all dosed groups. However, the clinical chemistry changes at the low and mid doses were of minor magnitude and were considered to be of no toxicological significance. These changes could be associated with the organ weight changes and the microscopic liver findings. The phosphorous level in high dose females was statistically higher (by 24%, p<0.01) than control value. The individual values were higher than the control and were outside the historical control range. This finding could be associated with the microscopic kidney findings. Other clinical chemistry parameters in males and females were occasionally statistically significant. However, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

URINALYSIS
There was no effect of treatment noted during urinalysis. Slight differences which attained statistical significance were record in urine pH (p<0.05) and gravity in low dose males (p<0.05). However, these findings were regarded as minor variations and to be of no toxicological importance.

NEUROBEHAVIOUR
There were no treatment related effects. There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total travelled distance in the test groups was comparable to the control groups for both sexes.

ORGAN WEIGHTS
Test item related differences were found in liver and kidney weights at 500 mg/kg bw/day in both sexes and at 125 mg/kg bw/day in males. The absolute and relative liver weight was higher than the control in all dosed male group and in high dose females attaining statistical significance, except the absolute and brain weight related values in the low dose males. The absolute liver weight was higher by approximately 71% in males and by 20% in females. The changes in mid and high dose males and high dose females correlated with the clinical chemistry parameters (increased albumin and total protein) and with the microscopic findings. Compared to control, higher kidney weights were recorded for both sexes in the high dose group and for males in the mid dose group. The differences were approximately 10-30% (absolute value) and 11-33% (relative values), and attaining statistical significance.

GROSS PATHOLOGY
No test item related macroscopic findings were observed. Enlargement or pale discoloration of the liver in 1/12 control female, enlarged spleen in
1/12 low dose female, single ulcers of the stomach glandular mucosa in 1/12 low dose, 1/12 mid dose and 2/11 high dose female rats, were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item related microscopic findings were noted in the liver and kidney at dose levels of 125 and 500 mg/kg bw/day. These microscopic changes correlated with increased liver and kidney weights related brain weights. Minimal to mild centrilobular hypertrophy of the liver was noted in 5/5 males and 4/5 females from the high dose groups. In the mid dose groups, minimal hypertrophy was only observed in 4/12 males. No hypertrophy was detected in the mid and low dose females. In addition, no hypertrophy was present when the liver sections from the low dose males were examined. This non-adverse alteration was characterized by increased size of hepatocytes with centrilobular pattern of distribution, without concurrent degeneration and/or necrosis. The hepatocellular hypertrophy was considered to be an adaptive response.

In the kidneys, test item related changes were observed in the tubules (cortical and/or corticomedullary junction). In the high dose group, minimal to mild bilateral focal/multifocal cortical tubule degeneration were seen in 2/5 males and 1/5 female. At the mid dose level, minimal focal unilateral tubule degeneration in 1/12 male and 1/12 female were noted. There was no evidence of tubule degeneration in the kidneys at the low dose level. Additionally, the irregular incidence of proteinaceous tubular casts recorded only in treated animals (1/12 low, 0/12 mid and 3/5 high dose males; 3/12 low, 1/12 mid and 1/5 high dose females) indicated that these changes were not treatment-related, therefore were considered to be of no toxicological significance.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

31.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

2

Key result

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

dose level: 500 mg/kg bw

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

The measured concentrations of RCX 14-672 evaluated for each test item-dose group varied between 95 % and 99 % of the nominal contents. No test item was detected in the control samples. These results were within acceptable ranges (85% - 115%) and are acceptable for the study purposes.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this OECD 422 study, the no observed adverse effect level (NOAEL) for RCX 14-672 is considered to be 31.25 mg/kg bw/day for the parental/adult generation.

Executive summary:

A combined repeated dose toxicity study/reproduction/developmental toxicity screening test (OECD 422) in rats was conducted to obtain information on the toxicity of the test item RCX 14-672. Repeated daily administration of RCX 14-672 by oral gavage to Wistar rats at dose levels of 31.25, 125, or 500 mg/kg bw/day during the treatment period under the conditions of this study did not result in test item related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation or urinalysis parameters. In clinical chemistry analysis, the albumin concentration was higher than the control in both sexes (by ~11% at the high dose). Consequently, total protein concentration was also higher and attained statistical significance in both high dose male and female treated groups. These changes could be associated with the organ weight changes and the microscopic liver findings. Test item related differences were found in liver and kidney weights at 500 mg/kg bw/day in both sexes and at 125 mg/kg bw/day in males, in correlation with the histopathological changes. The test item administration was associated with hepatocellular hypertrophy of the liver, and degeneration in the cortical/corticomedullary tubules of the kidney in the mid and the high dose in both sexes. The incidence and severity suggested dose relationship in both alterations. Hypertrophy occurred in the liver with centrilobular zonation, minimal and mild severity in both sexes at the high dose, and with minimal intensity in the mid dose males. This change was regarded as non-adverse adaptive response. No hypertrophy was observed in the mid and low dose females and in the low dose males. In the kidney, test item related degeneration altered tubules were noted in high dose animals with minimal to mild intensity and mainly bilateral distribution, in one mid dose male and one mid dose female as unilateral with minimal intensity. There was no evidence of tubule degeneration in the kidneys at the low dose level.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for RCX 14-672 is considered to be 31.25 mg/kg bw/day for the parental/adult generation.