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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-08-30 to 2021-11-06 (Only draft report available. Data will be updated upon availability of the final report.)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Only draft report available. Data will be updated upon availability of the final report.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-08-30 to 2021-11-06 (Only draft report available. Data will be updated upon availability of the final report.)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Only draft report available. Data will be updated upon availability of the final report.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed on 2020-04-22
Limit test:
no
Justification for study design:
not applicable
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25 °C), tight closed container
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity and reproductive studies. Wistar rat was selected due to experience with this strain in toxicity and reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
- Females were nulliparous and non-pregnant: yes
- Age (at start of dosing): approx. 12 weeks old
- Age (at mating): 14 weeks old
- Weight (at start of dosing): males: 359 – 435 g; females: 202 – 245 g
- Housing: group-housed, up to 2 animals/sex/cage, with the exception of the mating and gestation/delivery/lactation period, when they will be paired (mating period only) or individually housed (with pups), respectively; cage type: T3H polycarbonate; “SAFE 3/4-S-FASERN” certified wooden chips (J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Cages were arranged in such a way that possible effects due to cage placement were minimised.
- Diet (ad libitum): SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable (manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
- Water (ad libitum): tap water from the municipal supply, as for human consumption
- Acclimation period: 12 days

A pre-exposure period of 14 days was included in the current study. During this period no treatment with the test item occurred.

DETAILS OF FOOD AND WATER QUALITY:
The food is not considered to contain any contaminants that could affect the purpose or integrity of the study.

Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24 °C (target: 22 ± 3 °C)
- Humidity: 36 – 64 % (target: 30 - 70 %)
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 1 % methyl cellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (1% methyl cellulose in distilled water) by mixing in a pestle and mortar, as a visibly stable homogenous suspension at the appropriate concentrations. Formulations were prepared up to 2 days before use (formulation were kept closed, at room temperature until use).

Administration volume: 5 mL/kg

The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

The test item was administered in the morning hours, approximately at similar times each day.

VEHICLE
- Justification for use and choice of vehicle: based on results of trial formulation and a dose range finding toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: CaBenz_s_Kiss_2021_DRF) performed with the test item, 1% methyl cellulose solution was selected as vehicle for this study.

Components of vehicle:
Component 1:
Name: methylcellulosum
Batch number: 2104-4173/8075543
Manufacturer: Parma Product Kft./Shin-Etsu
Expiry date: 2021-11-19/2022-07-10
Storage: room temperature

Component 2:
Name: aqua purificata (distilled water)
Batch number: 2106-5502
Manufacturer: Parma Product Kft.
Expiry date: 2021-12-02
Storage: room temperature
Details on mating procedure:
Mating began after the animals have attained full sexual maturity.
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: females remained with the same male until copulation occurred.
- Proof of pregnancy: a vaginal smear was prepared daily in the morning hours during the mating period and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test item formulations for concentration and homogeneity was performed using an HPLC-DAD method. Representative top, middle and bottom duplicate samples were taken from the test item formulations three times during the study (during the first and last weeks and approximately midway during the dosing period), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

According to an analytical method validation, acceptance criteria of the concentration analysis were set to be at 100 ± 15 % of the nominal value. Acceptance criteria of the homogeneity were that the CV of replicates (top, middle and bottom of test item formulations) must be less than 10%.

Stability of the test item in the vehicle was assessed under the conditions employed in the study during the analytical method validation. In that study, the formulation samples in the 1-200 mg/mL concentration range (using 1% methyl cellulose in distilled water as vehicle) were proven to be being stable for 2 days when stored at room temperature.

All test item formulations were shown to be homogeneous and in the range of 96 to 107 % of nominal concentrations, as shown below. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

1. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 19.14 ± 0.63 mg/mL (96 % of nominal concentration)
60 mg/mL: 63.52 ± 3.65 mg/mL (106 % of nominal concentration)
200 mg/mL: 207.70 ± 10.17 mg/mL (104 % of nominal concentration)

2. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 21.39 ± 0.29 mg/mL (107 % of nominal concentration)
60 mg/mL: 62.54 ± 1.34 mg/mL (104 % of nominal concentration)
200 mg/mL: 212.84 ± 3.21 mg/mL (106 % of nominal concentration)

3. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 20.40 ± 0.42 mg/mL (102 % of nominal concentration)
60 mg/mL: 61.12 ± 2.58 mg/mL (102 % of nominal concentration)
200 mg/mL: 204.60 ± 6.91 mg/mL (102 % of nominal concentration)
Duration of treatment / exposure:
males: 28 days (14 days pre-mating and 14 days mating/post-mating period)
females: circa 53 days (14 days pre-mating, for up to five days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing))
Frequency of treatment:
daily; 7 days/week
Details on study schedule:
not applicable
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a Dose Range Finding (DRF) study (please refer to Section 7.5.1 Repeated dose toxicity: oral: CaBenz_s_Kiss_2021_DRF). Based on the results from the preliminary study, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

- Randomization: before start of dosing, the animals were assigned to their respective dose groups by randomisation based on body weights. It was checked that all animals were within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent body weight. Males and females were randomised separately.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Clinical signs: once a day
Mortality/morbidity: twice daily (at the beginning and end of each working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure, prior to the first dosing (to allow for within-subject comparisons) and at least weekly thereafter, in the morning hours

BODY WEIGHT: Yes
Time schedule for examinations:
- all parental animals: weekly during the pre-exposure period, on the first day of dosing (Day 0, prior to start of dosing), then afterwards at least weekly and prior to the scheduled necropsy (fasted)
- parental females: gestation days 0, 7, 14, 20, and on lactation days 0 (within 24 hours after parturition), 4, 13, and at termination (lactation day 14, fasted)

FOOD CONSUMPTION: Yes
- Time schedule: at least weekly (on body weight measurement days) during pre-mating, mating, pregnancy and lactation.

The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated.

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: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last dose, prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes, pentobarbital anaesthesia
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: total number of erythrocytes, hematocrit value, haemoglobin concentration, mean erythrocyte volume in total sample (MCV), mean haemoglobin volume per red blood cell (RBC) count (MCH), mean hemoglobin concentration of erythrocytes (MCHC), degree of variation in size of the erythrocyte population, reticulocyte (count and percent), total number of leukocytes, neutrophil (count and percent), lymphocyte (count and percent), monocyte (count and percent), eosinophil (count and percent), basophil (count and percent), total number of platelets, mean platelet volume, platelet distribution width, plateletcrit value, activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last dose, prior to scheduled necropsy
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, urea concentration, calcium, cholesterol (total), creatinine, chloride, gamma-glutamyltransferase, glucose, potassium, sodium, inorganic phosphate, total bilirubin, total protein and total bile acids

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: one day after the last dose, prior to scheduled necropsy (samples were taken before 11:30 AM each day, to avoid the diurnal variation of the hormone concentration among animals)
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: all animals
- Parameters checked: thyroxine (T4), triiodothyronine (T3; females only) and thyroid-stimulating hormone (TSH; females only)

URINALYSIS: Yes
- Time schedule for collection of urine: one day after the last dose, prior to scheduled necropsy
- Metabolism cages used for collection of urine: Yes, approximately 16 hours
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: clarity, colour, volume, leukocyte, nitrite, urobilinogen, protein, pH, blood (occult), specific gravity, ketones, bilirubin, glucose, sediment microscopic examination (white blood cell, red blood cell, epithelial cell, crystals, bacteria and amorphous globlets)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- How many animals: 5 males / 5 females
- Battery of functions tested: sensory activity / grip strength /locomotor activity

Animals were subjected to the functional observation battery, including qualitative assessment of fore/hind grip strength and measurement of landing foot splay (hind limbs only).
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed (Irwin, 1968)*.

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 reflex, 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.

IMMUNOLOGY: No

FURTHER EXAMINATIONS
- signs of difficult or prolonged parturition were recorded
- on gestation day 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
- abnormal deliveries were noted.
- duration of gestation was recorded.
- dams were observed to record whether they formed a nest from the bedding material and covered their new-borns or not.
- efficiency of suckling was observed by the presence of milk in the pups' stomach.

*Reference:
- Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257, 1968
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the dosing started. Vaginal smears were also checked daily from the beginning of the dosing period until evidence of mating (during the pre-mating and mating periods).

Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which could induce pseudopregnancy.

Sperm parameters (parental animals):
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
One male and one female pup per litter (if possible) were selected for culling for blood sampling on lactation day 4. After that, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. No pups were eliminated when litter size dropped below the culling target (10 pups/litter).

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- after delivery each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), presence of gross abnormalities and abnormal behaviour.
- live pups were counted, sexed, weighed individually within 24 hours of parturition (lactation day 0) and on lactation days 4 and 13
- all the litters were checked and recorded daily for the number of viable and dead pups and any abnormal behaviour or appearance of the pups was also recorded.
- anogenital distance of each pup was measured at the time of the first weighing (lactation day 0; normalized to pup size (the cube root of body weight)).
- number of nipples/areolae in all male pups were recorded on lactation day13.
- one male and one female pup per litter (if possible) were selected for culling for blood sampling on lactation day 4.
- at least two pups per litter were selected for thyroxine (T4) determination in blood samples on lactation day 13

GROSS EXAMINATION OF DEAD PUPS:
Yes, examined externally for gross abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals at the end of dosing period
- Maternal animals: all surviving animals were terminated on lactation day 14

GROSS NECROPSY/MACROSCOPIC EXAMINATION
Necropsy and macroscopic examination were performed on all animals, at the end of the dosing period (after the sample collection for thyroid hormone analysis). The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium)).

After sacrifice, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were 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 were recorded in the females as applicable.

Necropsy and macroscopic examination were performed on all animals, at the end of the dosing period (after the sample collection for thyroid hormone analysis). The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium).

After sacrifice, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were 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 following organs were trimmed of fat and weighed in all adult animals: brain, epididymides, heart, kidneys, liver, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, uterus including cervix, adrenal glands, ovaries and thyroid with parathyroid glands.

Testes, epididymides and the other paired organs were weighed individually, but reported together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

HISTOPATHOLOGY: Yes
On completion of the macroscopic examination, the following tissues and organs were retained from all adult animals: gross findings, adrenals, aorta (thoracic and abdominal), brain (representative regions including cerebrum, cerebellum and medulla/pons), epididymis, eye with the optic nerve, oesophagus, femur with marrow, heart (including both ventricles and atria, septum with papillary muscle), kidney, large intestine (caecum, colon and rectum), extraorbital lachrymal gland, Harderian gland, liver (lobes, left lateral, right medial, caudate), lungs with bronchi (was infused with formalin followed by immersion in fixative; 3 lobes, left, right cranial), lymph node (mandibular and mesenteric), ovary, oviduct, pancreas, pituitary, prostate, salivary gland (including mandibular, sublingual and parotid glands), sciatic nerve, seminal vesicle with coagulating gland, skin (subcutis with mammary gland (inguinal)), skeletal muscle (quadriceps), small intestine (duodenum, ileum and jejunum with Peyer’s patches), spinal cord (cervical, thoracic and lumbar), spleen, sternum with marrow, stomach, testis, thymus, thyroid with parathyroid gland, tongue, trachea, urinary bladder, uterus (horns, body and cervix) and vagina.

The eyes with the optic nerve, testes and epidymides were preserved in modified Davidson’s fixative, all other organs in 10 % buffered formalin solution.

In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:
- on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group),
- all macroscopic findings (abnormalities),
- on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups,
- on the liver of all low and mid dose male and female animals,
- on the liver and kidney of the remaining animals (not processed as per guideline) of the control and high dose groups (males and females),
- on the kidney of low and mid dose male animals.

Special attention was paid to 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.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, and bone marrow).

Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Postmortem examinations (offspring):
SACRIFICE
- all offspring were terminated on lactation day 13

GROSS NECROPSY
Pups euthanized on lactation days 4 or 13 were examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
On lactation day 13, thyroid glands from one male and one female pup from each litter was weight and were preserved in 10% buffered formalin solution.
Statistics:
Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel.

Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:

The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
Reproductive indices:
Male mating index = (Number of males with confirmed mating/Total number of males cohabited) x 100
Male fertility index = (Number of males impregnating a female/Total number of males cohabited) x 100
Female mating index = (Number of sperm positive females/Total number of females cohabited) x 100
Female fertility index = (Number of pregnant females/Number of sperm positive females) x 100
Gestation index = (Number of females with liveborn pups/Number of pregnant females) x 100
Offspring viability indices:
Survival index = (Number of live pups (at designated time)/Number of pups born) x 100
Survival index on lactation day 13 was calculated from number of pups after culling on lactation day 4 instead of number of pups born.

Intrauterine mortality = ((Number of implantations-number of liveborns)/(Number of implantations)) x 100
Total mortality = ((Number of implantations-number of viable pups (at designated time))/(Number of implantations)) x 100
Sex ratio = (Number of female pups (at designated time)/Number of pups (at designated time)) x 100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: test item related effects on body weight and body weight gain were observed in the males of the high dose group. Daily administration of the test item caused reduced mean body weight gain in the male high-dose group in all four observation periods: Days 0 - 7 (-53.4 %; p<0.05), Days 7 - 14 (-40.2 %; p < 0.05), Days 14 - 21 (-36.7 %) and Days 21 - 27 (-19.1 %), the first two attaining statistical significance. Consequently, the mean body weight gain of the male high-dose group was also reduced for the entire observation period (Days 0 - 27) by -42.0 % (p < 0.01). The reduced body weight gain was insufficient to result in a significant reduction of the mean body weight, however in the high dose group, as of the first week the mean body weight decreased in comparison to the control steadily throughout the entire treatment period (-1.8 %, -3.3 %, -3.6 % and -3.8 % for the treatment days 7, 14, 21 and 27) resulting in a significant difference in the terminal (fasted) body weight (-6.5 %; p < 0.01).

Please also refer to section "Overall remarks, attachments".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: a test item-related effect on food consumption of the males of the high dose group was observed. A statistical significantly increased mean food consumption was observed in the high dose male group between Days 7 - 14 (+8.6 %; p < 0.01), and Days 21 - 27 (+9.6 %; p < 0.05) compared to the control. Although in the other treatment weeks the differences did not attain statistical significance, the mean food consumption steadily increased by the duration of the treatment compared to the control (+3.7 %, +8.6 %, +9.4 % and +9.6 % for the weeks 1, 2, 3 and 4) resulting in a significant overall effect for the entire period of Days 0 - 27 (+7.8 % p < 0.01). For the periods with significant differences to the control group, all values of food consumption were within the range of the historical control.

Please also refer to section "Overall remarks, attachments".
Food efficiency:
not examined
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):
Males:
- 300 and 1000 mg/kg bw/day: statistically significant increase of bile-acid levels was noted in the mid-, and high dose treated males (+234.2%; p<0.05 and +357.6%; p<0.01, respectively). The differences to the control in the low dose-treated males and in any of the treatment groups of the females did not attain statistical significance. However, a dose-dependency could be observed in the males and the finding was considered to be test item-related in males.

- 1000 mg/kg bw/day: statistically significantly decrease of the globulin levels (-13.6 %; p < 0.0.1) and consequently an increased albumin/globulin ratio was noted in the high dose-treated male group (+21.8 %; p < 0.05) compared to the control. Furthermore, statistically significant increase of blood urea nitrogen/creatinine ratio was noted in the high dose-treated male group (+31.3 %; p < 0.05). In addition, a statistically significant decrease of total cholesterol was noted in the high dose-treated male group (-24.3 %; p < 0.05) compared to the control. All values were within the historical control data.

Please also refer to section "Overall remarks, attachments".
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: a statistically significant decrease of the T4 level was noted in the high dose-treated males (-45.2%; p<0.01) compared to the control, with a mean level (29 nmol/L) below the range of historical controls (31 to 106 nmol/L).

Please also refer to section "Overall remarks, attachments".
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 1000 mg/kg bw/day: there was a statistical significant decrease in the pH of the urine in the high dose male group compared to the control group (control group: pH= 7.0; treatment group: pH = 5.8; p < 0.01).

2) Females
- 100, 300 and 1000 mg/kg bw/day: there was a statistical significant decrease in the pH of the urine in the low dose-, (pH=5.8; p<0.01), in the mid dose-, (pH=5.7; p<0.05) and in the high dose-treated female groups (pH=5.7; p<0.01) compared to the control group (pH = 6.4).

Please also refer to section "Overall remarks, attachments".

Overall, a reduction of the pH is a well-known effect of the benzoic acid and benzoates and therefore this can be regarded as a test item-related effect, but is without toxicological relevance (Lenis et al.,1998b and EC SCAN, 2020)*.

*References:
- Lenis NP, van Diepen JTM, van der Pasch BLCP, Jongbloed AW and Kogut J, 1998b, Dose-response relationship of dietary benzoic acid and buffering capacity on urinary pH in growing pigs, unpublished Report ID-DLO No. 98-77, , Department of Nutrition of Pigs and Poultry, Institute for Animal Science and Health, Lelystad, The Netherlands
- EC SCAN, Opinion of the Scientific Committee on Animal Nutrition on the use of benzoic acid in feedingstuffs for pigs for fattening (2020) https://ec.europa.eu/food/system/files/2020-12/sci-com_scan-old_report_out100.pdf

Please also refer to section "Overall remarks, attachments".
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1) Males
Liver:
- 300 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 4/12 of mid dose-treated male animals. This phenomenon, observed on the histological picture of haematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

- 1000 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 10/12 male of high dose-treated. This phenomenon, observed on the histological picture of haematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

In the present study, this phenomenon in the liver – based on the incidence – seems to be a test item related lesion. This could be considered as a slight reversible lesion in the liver.

Kidney:
- 300 mg/kg bw/day: histological examination revealed slight focal lymphocytic and histiocytic infiltration in the kidney in 1/12 mid dose male.

- 1000 mg/kg bw/day: histological examination revealed slight focal lymphocytic and histiocytic infiltration in the kidney in 3/12 high dose male animals.

The kidney finding could indicate the initial phase of chronic progressive nephropathy (CPN) of laboratory rats.

2) Females
- 1000 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 9/12 female of high dose-treated. This phenomenon, observed on the histological picture of haematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

In the present study, this phenomenon in the liver – based on the incidence – seems to be a test item related lesion. This could be considered as a slight reversible lesion in the liver.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg bw/day: in the high dose group, the gestation index was 60%, as four out of ten pregnant animals had stillborn pups only. This effect was ascribed as test item related. The gestation index was 100% in all the other groups (control, low and mid dose).

Please also refer to section "Overall remarks, attachments".
CLINICAL SIGNS
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: during the general clinical and detailed clinical observations, no clinical signs were observed in the current study.

MORTALITY
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: no mortality was seen during the study.

BODY WEIGHT AND WEIGHT CHANGES
1) Males:
- 100 and 300 mg/kg bw/day: no test item related changes were observed in body weight and body weight gain of the low and mid dose group animals compared to control data.

2) Females:
- 100 and 300 mg/kg bw/day: no test item related changes were observed in body weight and body weight gain of the low and mid dose group animals compared to control data.
- 1000 mg/kg bw/day: the female animals of the high dose group showed no statistically significant change in the mean body weight, and body weight gain if compared to the control in any of the observation periods with the exception of the lactation period 0-13 where the mean body weight gain of 39.2g was 23.32% above of the control group (p <0.01). In view of the fact, that one animal of the control group was a clear outlier (body weight gain of -6 g/d, instead of the average body weight gain of +35.6 g of the rest of this dose group) and as the mean body weight gain of 39.2 g is within the range of historical control data for the same period (15 to 68 g), this change is regarded as toxicologically not relevant.

Please also refer to section "Overall remarks, attachments".

FOOD CONSUMPTION
1) Males
- 100 mg/kg bw/day: statistical significant differences to control were observed between Days 21 - 27 (-6.5 %; p < 0.05) in low dose males. As these differences were below 10% and were without dose dependenc and caused no statistical significant changes in the mean body weights or body weight gains, these food intake differences can be regarded as incidental and not related to the test item.

2) Females:
- 100, 300 and 1000 mg/kg bw/day: statistical significant differences to control were observed occasionally, such as between Days 0 - 7 (-4.1 %; p < 0.05) in low dose females and between Days 0 - 7 (+9.6 %; p < 0.01), between Days 7 - 14 (+6.2 %; p < 0.05) and consequently between Days 0-14 (+7.9%; p <0.01) as well as lactation days 7 - 13 (+13.0 %; p < 0.05) in mid dose females and between Days 0 - 7 (+4.7 %; p < 0.01) and Days 0 - 14 (+5.7 %; p < 0.05) in high dose females. As these differences were below 10% and were without dose dependency and caused no statistical significant changes in the mean body weights or body weight gains, these food intake differences can be regarded as incidental and not related to the test item.

- 1000 mg/k bw/day: statistically significant lower mean food consumption was observed in the high dose female group between lactation days 0 - 7 (-24.4 %; p <0.05) and between lactation days 7 - 13 (-20.2 %; p < 0.05) and consequently between lactation days 0 - 13 (-22.1 %; p < 0.05) compared to the control. The reduced food intake of these dams correlates with the smaller mean litter size and reduced mean body weight of the pups in this group.

Please also refer to section "Overall remarks, attachments".

HAEMATOLOGICAL FINDINGS
1) Males
- 100, 300 and 1000 mg/kg bw/day: no test item-related effects were observed for haemtaology at any dose level compared to the control group.

- 100 mg/kg bw/day: compared to the control, the haematocrit and haemoglobin were elevated by +4.9 % (p<0.05) and +4.9 % (p < 0.05), respectively. Because the statistical significance is observed only in the low dose group compared to the control group and due to the lack of dose dependency as well as these values were with the range of the historical data, these changes can be regarded as incidental.

- 1000 mg/kg bw/day: compared to the control, a statistically significant increase of white blood cells was noted (+67 %; p < 0.05) in high dose males. Due to the broad distribution of the individual data of the control animals and because the mean values were within the range of historical control, these changes can be regarded as incidental and not test item related.

In the high dose-treated males, the relative (RETIC %) and absolute number of reticulocytes (RETIC) were increased by +53.6 % (p < 0.01) and +52.0 % (p < 0.01) compared to the control. An increased reticulocyte number (reticulocytosis) is an indicator of potential blood loss, but in lack of any significant change in the red blood cell parameters (erythrocytes, heamatocrit and heamoglobin) and the lack of similar effect in the female animals and since the mean RETIC values of 4.30 (relative value) and 354.16 (absolute value) are well within the range of the historical control data (2.6 to 4.5 (relative value) and 207.0 to 408.4 (absolute value)) the increased RETIC [%] and RETIC [K/µL] values can be regarded as of no toxicological relevance.

In the high dose-treated males, the platelet distribution width was statistical significantly elevated (+8.4 %, p < 0.05) compared to the control. In view of the lack of dose dependency and lack of changes in the other platelet-related parameters (total number of platelets, mean platelet volume and plateletcrit value) and since the platelet distribution width value of 8.74 is well within the range of the historical control (7.2 to 9.6), the increased platelet distribution width value can be regarded as incidental and not test item related.

2) Females:
- 100, 300 and 1000 mg/kg bw/day: no test item-related effects were observed for haemtaology at any dose level compared to the control group.

- 1000 mg/kg bw/day: compared to the control, a statistically significant decrease of white blood cells was noted (-52.3 %; p < 0.05) in high dose females. Due to the broad distribution of the individual data of the control animals and lack of dose dependency and because the mean values were within the range of historical control, these changes can be regarded as incidental and not test item related.

In the high dose-treated females, the absolute number of neutrophils was decreased by -55.5 % (p < 0.05) compared to the control. In the lack of dose dependency and since the decrease in the relative number of neutrophils (%) was statistically not significant and since the mean value was within the historical control range, this change is regarded as toxicologically not relevant.

In the high dose-treated females, the platelet count and the total platelet volume (paletecrit) were found to be decreased by -22.7 % (p < 0.05) and -19.4 % (p < 0.01), respectively, compared to the control. Furthermore the prothrombin time was increased by +8.3 % (p < 0.05). As the mean values of these parameters were well within historical controls, these changes are regarded as toxicologically not relevant.

Please also refer to section "Overall remarks, attachments".

CLINICAL BIOCHEMISTRY FINDINGS
Females
- 100 and 1000 mg/kg bw/day: at the 1000 mg/kg bw/day dose level, globulin level was decreased by -7.7 % (p < 0.05) without statistically significant change in the albumin/globulin ratio compared to the control. The finding was not considered to be test item -related. Furthermore, a statistically significant decrease of alkaline phosphatase levels were noted in the low and high dose-treated female groups (-29.5 %; p < 0.05 and -30.2 %; p < 0.01, respectively). Since there is a lack of dose-dependency, no statistical significance in the mid dose group and all values were within the historical control data, these changes can be regarded as incidental and not related to the test item. Overall, a decreased level of alkaline phosphatase is not considered to be of toxicological relevance. Lastly, statistically significant increase of the mean natrium level and a decrease of the mean potassium level was noted in the low dose-treated female (+2.4%; p<0.05 and -21.9%; p<0.01, respectively) compared to the control, leading to an increased Na+/K+ ratio in this group (+28.8%; p<0.01). In addition, a statistically significant increase of the mean Na+ level was noted in the high dose female group (+2.4%; p<0.05). As the differences attained statistical significance only in the low dose group and/or no clear dose dependency was observed, these changes can be regarded as incidental and not related to the test item.

Please also refer to section "Overall remarks, attachments".

ENDOCRINE FINDINGS
- 100, 300 and 1000 mg/kg bw/day: the thyroid hormone levels (T3, T4 and TSH ) of the female animals (dams) of any of the dose groups were statistically not different to the control group.

URINALYSIS FINDINGS
Males and females
- 100, 300 and 1000 mg/kg bw/day: no test item related changes were observed in the urinalysis parameters clarity, colour, volume, leukocyte, nitrite, urobilinogen, protein, blood (occult), specific gravity, ketones, bilirubin, glucose and sediment microscopic examination (white blood cell, red blood cell, epithelial cell, crystals, bacteria and amorphous globlets) of males and females of any dose groups compared to the control group.

BEHAVIOUR
Males and females:
- 100, 300 and 1000 mg/kg bw/day: the modified Irwin test did not reveal any test item-related effect for males and females at any dose level. Furthermore, there was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity. The only statistical significant findings was made during the measurement of forelimb grip strength of the low dose result measured during the first trial with the forelimb of the low dose males (first trail only; -14.56%, p<0.05), which can be regarded as incidental.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20 - 30 minutes. The some isolated, statistically significant changes (males of the low and high dose groups at the 40 to 45 minute meaurement: -42.3 % (p < 0.05) and -32.2 % (p < 0.05) compared to the control, respectively; females of the mid dose group at the 25 to 30 minute measurement: -61.5 % (p < 0.05) compared to the control group; females of the high dose group at the 0 to 5 minute and 10 to 15 minutes measurements: +27.9 % (p < 0.05) and +35.2 % (p < 0.05) compared to the control, respectively) were without a clear trend or dose response, and the pattern of the movements’ intensity under the whole duration of the test was similar in all dose groups, these changes were considered toxicologically irrelevant, and not test item related. Furthermore, these values were all within the historical control data range.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS
1) Males
Fasted body weight:
- 1000 mg/kg bw/day: a statistical significant lower mean absolute and brain weight normalized, fasted body weight was noted in high dose-treated males (-6.5%; p<0.01 and -7.2%; p<0.01, respectively) compared to the control.

Heart:
- 1000 mg/kg bw/day: a statistical significant higher mean absolute, body weight-, and brain weight normalized, heart weights were noted in high dose-treated males (+12.0%; p<0.01 and +20.0%; p<0.01 and +11.3%; p<0.01, respectively) comapred to the control. All values of males were within the range of the historical control data and no findings were made during histopathology of the heart. The findings of the heart were not considered to be of toxicological relevance.

Brain:
- 1000 mg/kg bw/day: higher mean body weight adjusted brain weights were found in high-dose treated males (+7.8%; p<0.01) compared to the control. As the absolute values of the brain weights were almost identical to the control (+0.8%), this body weight-normalized increase is attributable to the reduced body weight of these animals. No significant changes in brain weights of the females were noted.

Epididymis:
- 100 mg/kg bw/day: statistically significant changes were observed for the absolute and body weight-normalized epididymis weights of the males of the low dose group (+6.5%; p<0.05 and+8.7%; p<0.01, respectively) compared to the control. In lack of dose dependency and since these values were within the range of historical control data, these differences can be regarded as incidental.

Thymus:
- 1000 mg/kg bw/day: statistically significant changes were observed for the brain weight-normalized thymus weights of males of the high dose group (-14.1%; p<0.05) compared to the control. In lack of dose dependency and since this value was within the range of historical control data, the difference can be regarded as incidental.

2) Females
Liver:
- 1000 mg/kg bw/day: a statistical significant higher mean body weight-normalized, liver weight (+10.9 %; p < 0.05) were noted in high dose-treated females compared to the control. Since the body weight of the females was decreased in the high dose group compared to the control group (not statistically significant), the difference in liver weight normalized to the body weight between the high dose group and control group was observed and, therefore, this finding was not considered to be test item-related.

Heart:
- 1000 mg/kg bw/day: statistical significant higher mean body weight-, and brain weight normalized, heart weights (+7.9 %; p < 0.05 and +4.6 %; p < 0.01, respectively) were noted compared to the control. In addition, an increased body weight-normalized heart weight was noted in the low dose-treated females (+7.5 %; p < 0.05) compared to the control. All values of females were within the range of the historical control data and no findings were made during histopathology of the heart. The findings of the heart were not considered to be of toxicological relevance.

Uterus:
- 1000 mg/kg bw/day: statistically significant increased uterus weights were noted in the high dose-treated females in the absolute and body weight-normalized values (+16.1%; p<0.01 and +20.4%; p<0.01, respectively) compared to the control. This finding was not considered to be test item-related due to the lack of a dose-dependency.

Thyroid and parathyroid:
- 100 and 1000 mg/kg bw/day: statistically significant increased thyroid and parathyroid weights were noted in the high dose-treated females in the absolute and body weight-, and brain weight-normalized values (+11.8%; p<0.01 and +14.9%; p<0.01 and +12.7%; p<0.05, respectively) and in the absolute and brain-weight normalized values of the low dose-treated females (+12.4%; p<0.01 05 and +12.3%; p<0.05, respectively) compared to the control. No significant change in the thyroid and parathyroid weights of any of the male treatment groups were noted.The findings in the femals can be considered to be not test item-related due to the lack of a dose-dependency. Furthermore, all values of the females were within the historical control data.

Please also refer to section "Overall remarks, attachments".

GROSS PATHOLOGICAL FINDINGS
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: no external findings were made in males and females of any dose group as well as control group during the macroscopic examination.

Males:
Kidney:
- 1000 mg/kg bw/day: in 1/12 high dose-treated male animal, bilateral multifocal pale discoloration of the kidney was observed. Furthermore, unilateral/bilateral pelvic dilation of the kidney was observed in 2/12 high dose-treated males.

Stomach:
- 1000 mg/kg bw/day: red/dark red multifocal discoloration of the glandular mucosa of the stomach was observed in in 4/12 male high dose animals.

Seminal vesicle with coagulating gland
- 100 mg/kg bw/day: in 1/12 low dose male, bilateral, small seminal vesicle with coagulating gland was noted.

Females:
Stomach:
- 0, 100, and 300 mg/kg bw/day: red/dark red focal or multifocal discoloration of the glandular mucosa of the stomach was observed in 2/11 female control, 2/10 female low dose, 1/11 female mid dose and in 4/12 male high dose animals.

Kidney:
- 300 and 1000 mg/kg bw/day: unilateral/bilateral pelvic dilation of the kidney was observed in 1/11 mid dose-treated female and in 1/10 high dose-treated female.

Uterus:
- 100 and 300 mg/kg bw/day: bilateral dilatation with clear fluid of the body and horns of the uterus observed in 1/2 non pregnant low dose female and in 1/1 mid dose female animal.

Please also refer to section "Overall remarks, attachments"

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
1) Males
Stomach:
- 1000 mg/kg bw/day: focal/multifocal red discoloration in the mucous membrane of the stomach (glandular region) was observed in 4/12 high dose males. Histological examination revealed focal congestion (2/4) or focal erosion (2/4). The focal congestion or focal erosion in the stomach in treated rats is most likely in connection with the treatment procedure (gastric tube) without toxicological significance.

Reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands):
- 0 and 1000 mg/kg bw/day: in the male animals belonging to the high-dose and control groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in all cases.

Kidney:
- 0, 100, 300 and 1000 mg/kg bw/day: pyelectasia in the kidneys (one side) (Control: 1/12, 100 mg/kg bw/day: 2/12; 300 mg/kg bw/day: No. 2/12; 1000 mg/kg bw/day: 2/12) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.

Thyroid:
- 0 and 1000 mg/kg bw/day: histological examination did not reveal pathological lesion (atrophy degeneration, pigmentation, mineralization, amyloidosis, inflammation, proliferation, follicular hyperplasia, C-cell hyperplasia, adenoma, and /or carcinoma) in the investigated thyroid glands of experimental animals belonging to the control and 1000 mg/kg bw/day treated groups.
No morphological difference was detectable in the histological picture of thyroid glands between the different treated and control groups.

2) Females
Stomach:
- 0, 100 and 300 mg/kg bw/day: focal/multifocal red discoloration in the mucous membrane of the stomach (glandular region) was observed in 2/12 control, 2/12 low dose and 1/12 mid dose female animals. Histological examination revealed focal congestion (300 mg/kg bw/day: 1/1) or focal erosion (Control No.: 2/2; 100 mg/kg bw/day: No.: 2/2). The focal congestion or focal erosion in the stomach in treated rats is most likely in connection with the treatment procedure (gastric tube) without toxicological significance.

Reproductive system (ovaries, uterus, cervix, vagina ):
- 0 and 1000 mg/kg bw/day: in the female animals belonging to the high-dose and control groups the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well. The endometrium of nursing mothers was normal as well. No degenerative, inflammatory or other histopathological lesions were detectable, including the high dose treated animals.

- 100 and 300 mg/kg bw/day: in two cases (one animal in the low and mid dose group each) the dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.

Kidney:
The pyelectasia in the kidneys (one side) (Control: 2/12; 300 mg/kg bw/day: 1/1; 1000 mg/kg bw/day: 1/12) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.

Thyroid:
- 0 and 1000 mg/kg bw/day: histological examination did not reveal pathological lesion (atrophy degeneration, pigmentation, mineralization, amyloidosis, inflammation, proliferation, follicular hyperplasia, C-cell hyperplasia, adenoma, and /or carcinoma) in the investigated thyroid glands of experimental animals belonging to the control and 1000 mg/kg bw/day treated groups. In one case of one high dose female single cyst formation (one side) was observed, which can be regarded as incidental.
No morphological difference was detectable in the histological picture of thyroid glands between the different treated and control groups.

Please also refer to section "Overall remarks, attachments"

REPRODUCTIVE FUNCTION: OESTROUS CYCLE
- 100, 300 and 1000 mg/kg bw/day: no effect of the test item on oestrus cycles was noted.
Each female selected for the treatment showed acceptable cycles (mean cycle length was in the range of 4.1-4.4 days for each group) before starting the treatment period. There was no effect of test item on the oestrus cycle of females (mean cycle length was in the 4.1-4.5 days for each group) after starting of the treatment). No indication of test item-related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods.

- 100 mg/kg bw/day: prolonged diestrus (presumably pseudopregnancy) was recorded for one low dose female, but then the cycles of the animal became normal and the animal successfully mated and became pregnant. This is considered as being a normal finding, not being a test item-related effect.

REPRODUCTIVE FUNCTION: SPERM PARAMETERS
- 0 and 1000 mg/kg bw/day: the various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all cases as well, with the exception of one animal (100 mg/kg bw/day). In the seminal vesicles of this animal decreased amount of secrete was seen, without degeneration, inflammation or other pathological finding. This phenomenon could be considered as incidental disorder without toxicological significance.

REPRODUCTIVE PERFORMANCE
- 100, 300 and 1000 mg/kg bw/day: test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears) occurred within 5 days of pairing (cohabitation). The mean duration of mating was 2.3, 2.6, 2.4 and 2.3 days in the control, low, mid and high dose groups, respectively, and the values are within the historical control data.

- 100, 300 and 1000 mg/kg bw/day: there were no differences between the control and test item-dosed groups in the mating and fertility index that could be ascribed to test item administration. The mating index was 100% in all groups (males and females). The male and female fertility index was 92%, 83%, 92% and 83 % in the control, low, mid and high dose groups, respectively.

- 100, 300 and 1000 mg/kg bw/day: there was no effect of treatment during the gestation period. The mean duration of pregnancy was comparable in the control and test item-treated groups and the results were within the historical control data. As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.

- 100, 300 and 1000 mg/kg bw/day: the mean number of implantation sites was comparable to the control mean in all dose groups.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg bw/day: treatment with the test item caused statistically lower mean number of live born pups (-52.2%; p˂0.05) and statistically higher mean percentage of dead pups on postnatal day 0 (p˂0.01) and between postnatal days 1-4 (+5231.7%; p˂0.01). These resulted in statistically significant higher mean percentage of prenatal mortality (+599%; p˂0.05), higher mean percentage of total mortality on postnatal day 0 (+266.2%, p<0.05), on postnatal day 4 (+404.8%, p<0.01) and on postnatal day 13 (+268.4%, p<0.01) in this group compared to the control. The decrease in number of live born pups and the increase in mortality observed in the 1000 mg/kg bw/day dose group can be considered to be test item-related.

Four out of ten pregnant high dose female animals had stillborn pups only. One female had one live born pup (in addition to 10 stillborn pups), but this one pup was hypothermic on postnatal day 0 and was found to be cannibalized on postnatal day 1. Furthermore, another two pups were cannibalized on the day of birth and 11 pups were found to be cannibalized between postnatal days 1 and 4. No pups died from postnatal day 5 until the end of the observation period (postnatal day 13).

Please also refer to section "Overall remarks, attachments"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg bw/day: test item-related decrease in the mean pups body weight and body weight gain was observed in the high dose group for both sexes at postnatal days 0, 4 and 13.

At postnatal day (PND) 0, runts (pups with body weights smaller than the mean-2xSD of the body weights of controls) were observed with a frequency of 0/115, 2/117, 0/121 and 18/50 in control, low, mid and high dose treated groups, respectively.

Please also refer to section "Overall remarks, attachments"
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Results still pending
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
CLINICAL SIGNS
- 0, 100, 300 and 1000 mg/kg bw/day: except that one hypothermic pup in the high dose group (postnatal day (PND) 0), all surviving pups were externally normal and were symptom free from PND 0 up to and including on PND 13 in all dose groups.

MORTALITY/VIABILITY
- 100 and 300 mg/kg bw/day: no statistically significant effect on mortality was observed.

- 0, 100, 300 and 1000 mg/kg bw/day: no statistically significant differences was noted in the survival indices on postnatal days 0, 4 and 13 in any of the control and test item treated groups, however it was 36.8% lower than control on postnatal day 4 in the high dose group, and was out of the historical control range.

Please also refer to section "Overall remarks, attachments"

BODY WEIGHTS AND WEIGHT CHANGES
- 100 and 300 mg/kg bw/day: there were no test item-related differences in the offspring body weights or body weight gains in the low and mid dose test item treated groups when compared to the control. The mean litter body weight of the low dose treated group on postnatal days 4 and 13 was -9.6% (p<0.05) and -7.4% (p<0.05) lower than the control. As this decrease was not observed in the mid dose group, and as the sex-wise evaluation of the same parameter did not attain statistical significance, and since the observed body weight values were well within the range of historical control, this difference can be regarded as incidental without toxicologically relevance.

Please also refer to section "Overall remarks, attachments"

ANOGENTIAL DISTANCE
- 100, 300 and 1000 mg/kg bw/day: no test item related effect was observed on anogenital distance in any of the treated groups.

Please also refer to section "Overall remarks, attachments"

NIPPLE RETENTION IN MALE PUPS
- 100, 300 and 1000 mg/kg bw/day: no test item effect was observed on nipple retention in any of the treated groups.

Please also refer to section "Overall remarks, attachments"

ORGAN WEIGHTS AND WEIGHT CHANGES
- 100 and 300 mg/kg bw/day: no test item related differences were observed in the absolute or relative thyroid weights and in the mean T4 level of postnatal day 13 pups in the low and mid dose group compared to control.

- 1000 mg/kg bw/day: compared to the control, statistically lower mean absolute thyroid weight (-4.4%, p<0.05) and statistically higher mean relative thyroid weight (+15.4%, p<0.05) was noted in the high dose group. As the mean T4 level was similar to the control group, this effect could be ascribed to the low mean terminal body weight (-17.1%, p<0.01) of these pups.

Please also refer to section "Overall remarks, attachments"

SEX RATIO
- 100, 300 and 1000 mg/kg bw/day: the sex ratio of female pups was comparable to the control at all time points in the low, mid and high dose groups on postnatal day 0. The sex ratio of female pups was significantly lower (p˂0.05) compared to the control on postnatal day 4 (-40.8 %; p˂0.05) and 13 (-40.8 %; p˂0.05) in the high dose group. Considering that because of the high mortality on postnatal day 0 (30.08%), the number of pups in this group became relatively low by postnatal day 4 and 13, and as the mean value was within the historical control range, this sex ratio difference cannot be regarded as test item related effect.

ENDOCRINE FINDINGS
- 100, 300 and 1000 mg/kg bw/day: no test item related differences were observed in the mean T4 level of postnatal day 13 pups in the low, mid and high dose group compared to control.

Please also refer to section "Overall remarks, attachments"

OTHER FINDINGS
- 0, 100, 300 and 1000 mg/kg bw/day: evidence of suckling on lactation day 0 was recorded for all live born pups of all dose groups and control group in the study.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422, calcium dibenzoate in 1 % methyl cellulose solution was administered via gavage to groups of male and female Han:WIST rats (n = 12 animals/sex/group) at dose levels of 100, 300 and 1000 mg/kg bw/day. A vehicle control group was run concurrently. The administration occurred daily on 7 days/week. The substance was administered to males for 14 days pre-mating and 14 days mating/post-mating period (28 days in total) and to females for 14 days pre-mating, for up to five days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing)(up to 53 days in total). Besides an investigation of the parental generation, the development of the F1 offspring was observed until 13 days post-partum.

No test item related effects were observed in male and female animals for clinical signs, mortality, detailed clinical examination, haematology, behavioural examination, and gross pathology. However, test item related effects on body weight and body weight gain were observed in the males of the 1000 mg/kg bw/day dose group. Daily administration of the test item caused statistically significant reduced mean body weight gain in the males in all four observation periods: Days 0 - 7, Days 7 - 14, Days 14 – 21 (not significant) and Days 21 - 27 (not significant). Consequently, the mean body weight gain of the males was also statistically significant reduced for the entire observation period (Days 0 - 27) by -42.0 %. The reduced body weight gain was insufficient to result in a significant reduction of the mean body weight, however in the 1000 mg/kg bw/day dose group, as of the first week the mean body weight difference to the control steadily decreased throughout the entire treatment period (-1.8 %, -3.3 %, -3.6 % and -3.8 % for the treatment days 7, 14, 21 and 27) resulting in a statistically significant difference in the terminal (fasted) body weight. In addition, a test item-related effect on food consumption of the males of the 1000 mg/kg bw/day dose group was observed. A statistical significantly increased mean food consumption was observed in the males between Days 7 - 14, and Days 21 - 27 compared to the control. Although in the other treatment weeks the differences did not attain statistical significance, the mean food consumption steadily increased by the duration of the treatment compared to the control (+3.7 %, +8.6 %, +9.4 % and +9.6 % for the weeks 1, 2, 3 and 4) resulting in a statistically significant overall effect for the entire period of Days 0 - 27.

A statistically significant increase of bile acid levels was noted in the males of the 300 and 1000 mg/kg bw/day dose groups. The differences to the control in the 100 mg/kg bw/day dose-treated males and in any of the treatment groups of the females did not attain statistical significance. However, a dose-dependency could be observed in the males and the finding was considered to be test item-related in males. Also, at the 1000 mg/kg bw/day dose level a statistically significantly decrease of the globulin levels and a statistically significant increased albumin/globulin ratio was noted in males compared to the control. Furthermore, statistically significant increase of blood urea nitrogen/creatinine ratio was noted in the high dose-treated male group. In addition, a statistically significant decrease of total cholesterol was noted in the high dose-treated male group compared to the control. All values were within the historical control data abd are therefore of questionable biological relevance.

A statistically significant decrease of the T4 level was noted in the 1000 mg/kg bw/day dose-treated males compared to the control, with a mean level (29 nmol/L) below the range of historical controls (31 to 106 nmol/L). This finding was considered to be test item related. Also, a test item related effect was observed regarding the pH of urine. The pH of females of any dose level and males of the 1000 mg/kg bw/day dose level were statistically significantly reduced. Overall, a reduction of the pH is a well-known effect of the benzoic acid and benzoates and therefore this can be regarded as a test item-related effect, but is without toxicological relevance (Lenis et al.,1998b and EC SCAN, 2020)*.

In males, a statistically significant higher liver and kidney weights (absolute, relative body-weight-normalised, relative brain-weight-normalized) were noted in males of the 1000 mg/kg bw/day dose group compared to the control. The findings of the liver in the males of the 1000 mg/kg bw/day dose group were considered as test item-related. In addition, a statistically significant higher kidney weight (absolute, relative body-weight-normalised, relative brain-weight-normalized) were noted in the females of the 1000 mg/kg bw/day dose group and also in females of the 100 mg/kg bw/day dose group compared to the control. In the females of the 300 mg/kg bw/day dose group, the enlargement of the kidney attained statistical significance in the absolute and body weight-normalized values.

Histopathological examination showed test item related effects in male and female animals. At the 300 mg/kg bw/day dose level, histological examination of the liver revealed vacuolation in the hepatocytes in 4/12 male animals. The same effect was observed in 10/12 males and 9/12 female of the 1000 mg/kg bw/day dose group. Histological examination of the kidneys revealed slight focal lymphocytic and histiocytic infiltration in the kidneys of 1/12 and 3/12 males of the 300 and 1000 mg/kg bw/day dose levels, respectively, which also considered to be test item related. The kidney finding could indicate the inital phase of chronic progressive nephropathy (CPN) of laboratory rats, a common disease in aging rats.

No test item related effect was determined in the parental animals for reproductive function (oestrous cycle and sperm measures). However, the substance influenced the reproductive performance. At the 1000 mg/kg bw/day dose level, the gestation index was 60%, as four out of ten pregnant animals had stillborn pups only. This effect was ascribed as test item related. The gestation index was 100% in all the other groups (control, 100 and 300 mg/kg bw/day dose groups).

Regarding the F1 generation, no test item related effects were observed for anogenital distance, nipple retention (male pups only), thyroid weights, sex ratio, T4 level (pups on postnatal day 13 only) and suckling behaviour. Except that one hypothermic pup eas found in the 1000 mg/kg bw/day dose group on postnatal day (PND) 0, all surviving pups were externally normal and were symptom free from PND 0 up to and including on PND 13 in all dose groups. However, treatment with the test item caused statistically significant lower mean number of live born pups and statistically significant higher mean percentage of dead pups on postnatal day 0 and between postnatal days 1-4 at the 1000 mg/kg bw/day dose level. These resulted in statistically significant higher mean percentage of prenatal mortality, higher mean percentage of total mortality on postnatal day 0, on postnatal day 4 and on postnatal day 13 in this group compared to the control. The decrease in number of live born pups and the increase in mortality observed in the 1000 mg/kg bw/day dose group can be considered to be test item related. No test item related mortality was observed at the 100 and 300 mg/kg bw/day dose levels.

At the 1000 mg/kg bw/day dose level, four out of ten pregnant females had stillborn pups only. One female had one live born pup (in addition to 10 stillborn pups), but this one pup was hypothermic on postnatal day 0 and was found to be cannibalized on postnatal day 1. Furthermore, another two pups were cannibalized on the day of birth and 11 pups were found to be cannibalized between postnatal days 1 and 4. No pups died from postnatal day 5 onward until the end of the observation period (postnatal day 13).

Lastly, test item-related decrease in the mean pup body weight and body weight gain was observed in the 1000 mg/kg bw/day dose group for both sexes at postnatal days 0, 4 and 13. No test item related effects on body weight and body weight gain of the F1 generation were observed at the 100 and 300 mg/kg bw/day dose levels. At PND 0, runts (pups with body weights smaller than the mean-2xSD of the body weights of controls) were observed with a frequency of 0/115, 2/117, 0/121 and 18/50 in control, low, mid and high dose treated groups, respectively.

Based on the histopathological findings in the liver (vacuolation of hepatocytes) and increased kidney weight (relative and absolute) at 1000 mg/kg bw/day, the No observed adverse effect level (NOAEL) for systemic toxicity is 300 mg/kg bw/day for female rats. Furthermore, based on decreased body weight gain/fasted body weight, increased food consumption, increased bile acid, decreased T4 levels, increased liver and kidney weights as well as histopathological findings in the liver (vacuolation of hepatocytes) and kidney (slight focal lymphocytic and histiocytic infiltration) at 1000 mg/kg bw/day, the NOAEL for systemic toxicity is 300 mg/kg bw/day for male rats.

Although there are histopathological findings in the liver (vacuolation of hepatocytes; 4/12 males) and the kidney (slight focal lymphocytic and histiocytic infiltration; 1/12) at the 300 mg/kg bw/day dose level, the NOAEL of the male rats is derived at 1000 mg/kg bw/day. The finding in liver at the 300 mg/kg bw/ day dose level is considered to indicate an adaptive response of the liver due to the low frequency of occurrence.

Furthermore, the NOAEL for reproductive toxicity is 300 mg/kg bw/day based on decreased gestation index at the 1000 mg/kg bw/day dose level. Lastly, the NOAEL for developmental toxicity is 300 mg/kg bw/day based on decreased number of live born pups, increased prenatal mortality, increased total mortality of pups, and decreased body weight and body weight gain at the 1000 mg/kg bw/day dose level.

*References:
- Lenis NP, van Diepen JTM, van der Pasch BLCP, Jongbloed AW and Kogut J, 1998b, Dose-response relationship of dietary benzoic acid and buffering capacity on urinary pH in growing pigs, unpublished Report ID-DLO No. 98-77, , Department of Nutrition of Pigs and Poultry, Institute for Animal Science and Health, Lelystad, The Netherlands
- EC SCAN, Opinion of the Scientific Committee on Animal Nutrition on the use of benzoic acid in feedingstuffs for pigs for fattening (2020) https://ec.europa.eu/food/system/files/2020-12/sci-com_scan-old_report_out100.pdf
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
dose range finding study for an OECD 442 study (combined repeated dose toxicity study with reproduction/developmental toxicity screening test)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2021-07-15 to 2021-07-30
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: dose-range finding study without following a guideline explicitly
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the current dose range finding study conducted prior to a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, groups of five males and five females were administered daily calcium dibenzoate in 1 % methyl cellulose solution via gavage at dose levels of 100, 300 and 1000 mg/kg bw/day for duration of 14 days. A vehicle control group was run concurrently. The following parameters were investigated: clinical signs, mortality, body weight, food consumption, haematology, clinical chemistry, gross pathology and organ weights.
GLP compliance:
no
Remarks:
The study was not conducted under GLP, since it is a preliminary dose range finding study for a OECD guideline 422 study.
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25 °C), tight closed container
Species:
rat
Strain:
Wistar
Remarks:
Han/WIST
Details on species / strain selection:
The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
- Females were nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks old
- Weight at study initiation: males: 341 – 386 g; females: 199 – 231 g
- Housing: 2 or 3 animals/cage; cage type: T3H/T4 polycarbonate; housing/enrichment: group housed by same sex. “SAFE 3/4-S-FASERN” certified wooden chips (J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Cages were arranged in such a way that possible effects due to cage placement are minimised.
- Diet (ad libitum): SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable (manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
- Water (ad libitum): tap water from the municipal supply, as for human consumption
- Acclimation period: at least 15 days

DETAILS OF FOOD AND WATER QUALITY:
The food is not considered to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24 °C (target: 22 ± 3 °C)
- Humidity: 40 – 64 % (target: 30 - 70 %)
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is the most relevant route of human exposure.
Vehicle:
other: 1 % methyl cellulose solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (1% methyl cellulose solution), as a visibly stable homogenous formulation at the appropriate concentrations. Formulations were prepared daily and were stirred continuously until the end of treatment.

Administration volume: 5 mL/kg bw

The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were dosed concurrently with the vehicle only.

VEHICLE
- Justification for use and choice of vehicle: based on results of trial formulation performed with the test item, 1% methyl cellulose solution was selected as vehicle for this study. After mixing for approx. 20 minutes, the test item formulation seemed to be homogenous (based on visual assessment).

Components of vehicle:
Component 1:
Name: methylcellulosum
Batch number: 8075546
Manufacturer: Shin-Etsu Chemical Co. Ltd.
Expiry date: 2022-07-10
Storage: room temperature

Component 2:
Name: aqua purificata (distilled water)
Batch number: 2104-5527
Manufacturer: Parma Produkt Kft.
Expiry date: 2021-11-27
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 using a HPLC-DAD method. Representative top, middle and bottom duplicate samples were taken from test item formulations once during the study, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration. Acceptance criteria of the homogeneity were that the CV of replicates (top, middle and bottom of test item formulations) must be less than 10%.

Results:
Based on the results, all test item formulations were shown to be homogeneous and they were found to be in the range of 95 to 104% of nominal concentrations. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

1) 100 mg/kg bw/day
Nominal concentration: 20 mg/mL
Mean measured concentration: 20.71 mg/mL
Measured/nominal concentration (%) ± RSD: 103.6 ± 4.7 %

2) 300 mg/kg bw/day
Nominal concentration: 60 mg/mL
Mean measured concentration: 60.56 mg/mL
Measured/nominal concentration (%) ± RSD: 100.9 ± 2.6 %

3) 1000 mg/kg bw/day
Nominal concentration: 200 mg/mL
Mean measured concentration: 190.42 mg/mL
Measured/nominal concentration (%) ± RSD: 95.2 ± 5.3 %
Duration of treatment / exposure:
14 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.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in aqueous solution, calcium dibenzoate dissociates to one Ca2+-cation and two benzoate-anions. In the dissociated form, it can be assumed that the toxicological behavior of all types of nontoxic cation-containing benzoates (including benzoic acid) are likely to show a similar toxicological effect (i.e. benzoic acid, Na-benzoate, K-benzoate, Ca-dibenzoate, etc.). Therefore
the toxicological data of these compounds could be used to predict the potential toxicological effect of such benzoate salts.

1. LD50 of Na-Benzoate in an acute toxicity study was determined to be 3´140mgkg bw (Loeser E. 1977 SIDS)
2. In a prenatal developmental toxicity study (Onodera et al., Eisei Shikenjo Hokoku (1978) 96: 47-55), the dietary application of doses of 1´306 mg/kg bw of Na-benzoate had no toxic effect to the mother and to the foetuses.
3. A two year-long carcinogenicity study with Fischer rats (Sodemoto Yukio and Enomoto Makoto, Journal of Environmental Pathology and Toxicology (1979) 4: 87 - 95.), the dietary application of Na-benzoate (1 and 2%) showed a NOAEL of the equivalent of 1´000mg/kg bw
4. A four-generation toxicological study with a dietary-applied top-dose of 750 mg/kg bw benzoic acid produced no adverse effects (Kieckebusch, W. & Lang, K., Arzneim.-Forsch. (1960) 10: 1001-1003)

Although the above repeated-dose studies used the dietary route for the application, the data provide enough evidence to assume, that the oral gavage application of the top-allowed dose of 1´000mg/kg bw is reasonable from the animal welfare point of view.

The aim was to use a maximum of 1000 mg/kg bw/day at the highest dose level to induce toxic effects, but no death or suffering, and to have a NOAEL at the lowest dose level. Lower dose levels were set with a factor of approximately 3. Based on these, doses of 1000, 300, and 100 mg/kg bw/day were selected for this study.

- Randomization: before start of dosing, the animals were assigned to their respective dose groups by randomisation based on body weights. It was checked that all animals were within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent body weight. Males and females were randomised separately.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Clinical signs: once a day at approx. the same time with minor variations at the end of the working day
Mortality/morbidity: twice daily (at the beginning and end of each working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule (morning hours): prior to the first dosing (Day 0) and then weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation (pre-dosing period), on the
first day of dosing (Day 0, prior to start of dosing), then weekly, including on Day 13 (last dosing day) and prior to the scheduled necropsy (fasted), on Day 14.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Time schedule: the determination of food consumption was performed for all groups on Days 7 and 13. The remaining non-consumed food was weighed with a precision of 1 g.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no

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: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last dosing, prior to scheduled necropsy on Day 14
- Anaesthetic used for blood collection: Yes, pentobarbital anaesthesia
- Animals fasted: Yes, overnight fasting
- How many animals: all surviving animals of all groups
- Parameters checked: erythrocytes (total number), haematocrit, haemoglobin concentration, mean erythrocyte volume, mean haemoglobin volume, mean haemoglobin concentration, reticulocyte, leukocytes (total number), neutrophil (percent and count), lymphocyte (percent and count), monocyte (percent and count), eosinophil (percent and count), basophil (percent and count), platelets (total number), mean platelet volume, platelet distribution width and plateletcrit value.

Also, the degree of variation in size of the erythrocyte population was recorded.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last dosing, prior to scheduled necropsy on Day 14
- Animals fasted: Yes, overnight fasting
- How many animals: all surviving animals of all groups
- Parameters checked: albumin, alkaline phosphatase, alanine aminotransferase, blood urea nitrogen, calcium, cholesterol (total), creatinine, chloride, gamma-glutamyltransferase, glucose, potassium, sodium, inorganic phosphate, bilirubin (total), protein (total) and urea concentration

PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy and macroscopic examination were performed on all animals, at the end of the dosing period on Day 14. The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium)).

After exsanguinations, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs were observed macroscopically. All abnormalities were recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

Organ eights:
The following organs were trimmed of fat and weighed in all animals: brain, epididymides, heart, kidneys, liver, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, uterus including cervix, adrenal glands, ovaries and thyroid with parathyroid glands.

Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

HISTOPATHOLOGY: No
Optional endpoint(s):
not applicable
Other examinations:
not applicable
Statistics:
Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel. Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:

The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests shows significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis is required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
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:
not examined
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):
Males
- 300 and 1000 mg/kg bw/day: compared to the controls, higher alanine aminotransferase and alkaline phosphatase level was observed in the test-item treated males, attained statistical significance in high dose (alanine aminotransferase; p<0.05) or in mid dose animals (alkaline phosphatase; p<0.05). No similar pattern was observed in the females, however the alanine aminotransferase level was 50% higher than control group mean value in the high dose group without attaining statistically significance.

Males:
Alkaline phosphatase
Control group: 117.2 ± 12.05 U/L
300 mg/kg bw/day: 152.8 ± 28.26 U/L

Alanine aminotransferase
Control group: 75.4 ± 26.31 U/L
1000 mg/kg bw/day: 165.6 ± 69.63 U/L

Females:
Alanine aminotransferase
Control group: 55.2 ± 32.28 U/L
1000 mg/kg bw/day: 82.8 ± 46.61 U/L

Please also refer to the section "Overall remarks, attachments" below.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) Males
- 1000 mg/kg bw/day: compared to control group, statistically significantly higher absolute liver weight was observed in high dose males (14.5%, p˂0.05).
Control group: 10.78 ± 1.23 g
1000 mg/kg bw/day: 12.35 ± 0.77

2) Females
- 300 and 1000 mg/kg bw/day: compared to control group, statistically significantly higher absolute liver weight was observed in mid dose and in high dose females (9.8% and 33.1%, p˂0.05 and p˂0.01 respectively). In females, in the high dose group, it was associated also with a statistically significant higher relative liver weight (28.0%; p<0.01 when adjusted to the body weight) and in the mid and high dose group (11.1%; p<0.05 and 33.2%; p<0.01 respectively) when adjusted to the brain. All statistically significantly higher liver weights (absolute and relative) were above the range of the historical control data.

Absolute liver weight
Control group: 6.33 ± 0.06 g
300 mg/kg bw/day: 6.95 ± 0.39 g
1000 mg/kg bw/day: 8.43 ± 0.87

Relative liver weight (adjusted to body weight)
Control group: 2.849 ± 0.182 %
300 mg/kg bw/day: 3.070 ± 0.181 % (not significant)
1000 mg/kg bw/day: 3.646 ± 0.313 %

Relative liver weight (adjusted to brain weight)
Control group: 315.92 ± 19.57 %
300 mg/kg bw/day: 351.09 ± 27.23 %
1000 mg/kg bw/day: 420.94 ± 53.07 %

These changes in liver weight (male and females), associated with changes noted in alanine aminotransferase may be related to the treatment.

Please also refer to the section "Overall remarks, attachments" below.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND DETAILED CLINICAL SIGNS
- 0, 100, 300 and 1000 mg/kg bw/day: no clinical signs were observed during the study.

MORTALITY
- 0, 100, 300 and 1000 mg/kg bw/day: there was no mortality during the study.

BODY WEIGHT AND WEIGHT CHANGES
- 100, 300 and 1000 mg/kg bw/day: there were no effects on body weighs or on body weight gains that were ascribed to the administration of the test item.

- 1000 mg/kg bw/day: in comparison with the control, statistically significantly mean higher body weight gain were noted from Day 0 to 7 (p<0.05) and from Day 0 to 13 (p<0.01) in the female high dose group. The statistical significances in the body weight gain data were considered incidental, not related to the test item.

Control group (Day 0 to 7): 8.4 ± 4.83 g
1000 mg/kg bw/day (Day 0 to 7): 17.6 ± 3.91 g
Control group (Day 0 to 13): 20.2 ± 6.42 g
1000 mg/kg bw/day (Day 0 to 13): 34.8 ± 7.26 g

Please also refer to the section "Overall remarks, attachments" below.

FOOD CONSUMPTION
- 100, 300 and 1000 mg/kg bw/day: there were no test item-related differences during the study in the mean daily food consumption in any test item treated groups compared to the control.

HAEMATOLOGY
- 100, 300 and 1000 mg/kg bw/day: there were no differences that were considered toxicologically significant between the control and test item-treated groups on haematology parameters.

Minor variations were noted in a few parameters, attaining occasionally statistical significance, as follows:

1) Males:
- 100 mg/kg bw/day: statistically significant higher eosinophil percentage in low dose males (p<0.05).
Control group: 0.6 ± 0.15 %
Treatment group: 0.8 ± 0.15 %

- 300 mg/kg bw/day: statistically significant higher basophil percentage in mid dose males (p<0.05).
Control group: 0.0 ± 0.09 %
Treatment group: 0.3 ± 0.17 %

- 1000 mg/kg bw/day: statistically significant higher neutrophil percentage in high dose males (p<0.01) and lower lympocyte percentage in high dose males (p<0.05)
Neutrophil percentage:
Control group: 14.9 ± 3.02 %
Treatment group: 21.1 ± 2.28 %
Lymphocyte percentage:
Control group: 79.0 ± 3.40 %
Treatment group: 73.4 ± 2.86 %

2) Females:
- 300 mg/kg bw/day: statistically significant higher basophil count and basophil percentage in mid dose females (p<0.01).
Basophil count
Control group: 0.00 ± 0.00 x10E9/L
Treatment group: 0.01 ± 0.01 x10E9/L
Basophil percentage
Control group: 0.0 ± 0.00 %
Treatment group: 0.2 ± 0.17 %

Evaluation of the results compared to the control did not reveal any test-item related cause of the changes and all parameters were within the historical control range, therefore these differences were considered to be incidental and not related to the test item.

Please also refer to the section "Overall remarks, attachments" below.

CLINICAL BIOCHEMISTRY FINDINGS
1) Females
- 300 and 1000 mg/kg bw/day: when compared to the controls, statistically significant differences were observed in the phosphorus (p<0.05), total protein (p<0.01), albumin (p<0.05) concentration in high dose females, or potassium concentration (p<0.05) in mid and high dose females, and in the sodium/potassium rate (p<0.05) in mid dose females. These differences did not show clear dose response or consistent patterns between genders. In addition, all parameters were within the historical control range; thus they are considered not toxicologically significant under the conditions of this study.

Phosphorus
Control group: 2.04 ± 0.30 mmol/L
1000 mg/kg bw/day: 2.49 ± 0.29 mmol/L

Potassium
Control group: 3.94 ± 0.28 mmol/L
300 mg/kg bw/day: 4.52 ± 0.38 mmol/L
1000 mg/kg bw/day: 4.40 ± 0.34 mmol/L

Sodium/potassium rate
Control group: 38.0 ± 3.08
300 mg/kg bw/day (mid dose): 33.4 ± 3.05

Total protein
Control group: 64.4 ± 0.55 g/L
1000 mg/kg bw/day: 61.8 ± 1.30 g/L

Albumin
Control group: 34.0 ± 1.41 g/L
1000 mg/kg bw/day: 31.6 ± 1.14 g/L

Please also refer to the section "Overall remarks, attachments" below.

GROSS PATHOLOGICAL FINDINGS
1) Males
- 300 and 1000 mg/kg bw/day: red multifocal discoloration of the glandular mucosa of the stomach was observed in 1/5 mid dose and in 1/5 high dose male animals. As the observation was restricted only to one animal in both dose, this finding was considered as incidental, not related to the test item.

2) Females
- 0, 100 and 1000 mg/kg bw/day: bilateral dilation of the uterine horns and body observed in three control, one low dose and one high dose female animals. As this is known as a common sign of oestrus, it was not considered as test item-related effect.

ORGAN WEIGHT FINDINGS
Males and females:
- 100 and 1000 mg/kg bw/day: other organs showed occasionally variations that attained statistical significance in the males and females as well, such as lower absolute (15.2 %; p<0.05) and relative prostate weight (15.3 %; p<0.05 adjusted to body weight or 17.8 %, p<0.01 adjusted to brain weight) in high dose males, lower relative thymus weight when adjusted to the body and brain weight (p<0.05; 19.1 % and 20.7 %, respectively) in high dose males, lower adrenal glands weight relative to the brain weight (14.8 %, p<0.05) in low dose males, lower absolute heart weight (15.5 %, p<0.01) in high dose males, lower relative heart weight adjusted to the body and brain weight in low dose (p<0.05, 12.3 % and 13.9 %, respectively) and in high dose males (p<0.01, 15.5 % and 17.9 %, respectively), higher absolute (17.8 %, p<0.01) and relative thyroid weight (p<0.01, 17.5 % adjusted to body weight or 14.4 % adjusted to brain weight) in high dose males, higher absolute and body weight related spleen weight (p<0.05, 12.1 % and 11.9 %, respectively) in high dose males; higher absolute kidney weight (p<0.05) in low and high dose females (5.2 % and 9.5 %, respectively), higher relative kidney weight adjusted to the brain weight (8.5 %, p<0.05) in low dose females, higher absolute and body weight adjusted relative heart weight (p<0.01, 26.3 % and 21.7 %, respectively) in high dose females and higher heart weight adjusted to the brain weight in low and high dose females (15.9 %, p<0.05 and 26.4 %, p<0.01, respectively). As no necropsy findings were observed and no dose response was noted and/or all data were within the historical control range, these differences were regarded unrelated to the treatment or incidental.

Please also refer to the section "Overall remarks, attachments" below.
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: see "Remarks"
Remarks:
No test item-related effects were observed in animals for clinical signs, mortality, body weight, food consumption or haematology. However, clinical chemistry measurements revealed that alanine aminotransferse was increased in males and females of the 1000 mg/kg bw/day dose group attaining statistical significance in males only. Furthermore, at the 1000 mg/kg bw/day dose level a test item-related effect in form of an increase of absolute and relative liver weight in females and absolute liver weights in males cannot be excluded.
Critical effects observed:
no
Conclusions:
In the current dose range finding study conducted prior to a combined repeated dose toxicity study with the reproduction/develomental toxicity screening test, groups of five males and five females were administered daily calcium dibenzoate in 1 % methyl cellulose solution via gavage at dose levels of 100, 300 and 1000 mg/kg bw/day for duration of 14 days. A vehicle control group was run concurrently.

No test item-related effects were observed in animals for clinical signs, mortality, body weight, food consumption or haematology. However, clinical chemistry measurements revealed that alanine aminotransferse was increased in males and females of the 1000 mg/kg bw/day dose group attaining statistical significance in males only. Furthermore, at the 1000 mg/kg bw/day dose level a test item-related effect in form of an increase of absolute and relative liver weight in females and absolute liver weights in males cannot be excluded.

In conclusion, the dose levels considered to be suitable for a subsequent combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat according to OECD guideline 422 (2016) are 100, 300 and 1000 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

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)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed on 2020-04-22
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium dibenzoate
EC Number:
218-235-4
EC Name:
Calcium dibenzoate
Cas Number:
2090-05-3
Molecular formula:
C7H6O2.1/2Ca
IUPAC Name:
calcium dibenzoate
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25 °C), tight closed container

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity and reproductive studies. Wistar rat was selected due to experience with this strain in toxicity and reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
- Females were nulliparous and non-pregnant: yes
- Age (at start of dosing): approx. 12 weeks old
- Age (at mating): 14 weeks old
- Weight (at start of dosing): males: 359 – 435 g; females: 202 – 245 g
- Housing: group-housed, up to 2 animals/sex/cage, with the exception of the mating and gestation/delivery/lactation period, when they will be paired (mating period only) or individually housed (with pups), respectively; cage type: T3H polycarbonate; “SAFE 3/4-S-FASERN” certified wooden chips (J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Cages were arranged in such a way that possible effects due to cage placement were minimised.
- Diet (ad libitum): SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable (manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
- Water (ad libitum): tap water from the municipal supply, as for human consumption
- Acclimation period: 12 days

A pre-exposure period of 14 days was included in the current study. During this period no treatment with the test item occurred.

DETAILS OF FOOD AND WATER QUALITY:
The food is not considered to contain any contaminants that could affect the purpose or integrity of the study.

Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24 °C (target: 22 ± 3 °C)
- Humidity: 36 – 64 % (target: 30 - 70 %)
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of human exposure.
Vehicle:
other: 1 % methyl cellulose solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (1% methyl cellulose in distilled water) by mixing in a pestle and mortar, as a visibly stable homogenous suspension at the appropriate concentrations. Formulations were prepared up to 2 days before use (formulation were kept closed, at room temperature until use).

Administration volume: 5 mL/kg

The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

The test item was administered in the morning hours, approximately at similar times each day.

VEHICLE
- Justification for use and choice of vehicle: based on results of trial formulation and a dose range finding toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: CaBenz_s_Kiss_2021_DRF) performed with the test item, 1% methyl cellulose solution was selected as vehicle for this study.

Components of vehicle:
Component 1:
Name: methylcellulosum
Batch number: 2104-4173/8075543
Manufacturer: Parma Product Kft./Shin-Etsu
Expiry date: 2021-11-19/2022-07-10
Storage: room temperature

Component 2:
Name: aqua purificata (distilled water)
Batch number: 2106-5502
Manufacturer: Parma Product Kft.
Expiry date: 2021-12-02
Storage: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test item formulations for concentration and homogeneity was performed using an HPLC-DAD method. Representative top, middle and bottom duplicate samples were taken from the test item formulations three times during the study (during the first and last weeks and approximately midway during the dosing period), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

According to an analytical method validation, acceptance criteria of the concentration analysis were set to be at 100 ± 15 % of the nominal value. Acceptance criteria of the homogeneity were that the CV of replicates (top, middle and bottom of test item formulations) must be less than 10%.

Stability of the test item in the vehicle was assessed under the conditions employed in the study during the analytical method validation. In that study, the formulation samples in the 1-200 mg/mL concentration range (using 1% methyl cellulose in distilled water as vehicle) were proven to be being stable for 2 days when stored at room temperature.

All test item formulations were shown to be homogeneous and in the range of 96 to 107 % of nominal concentrations, as shown below. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

1. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 19.14 ± 0.63 mg/mL (96 % of nominal concentration)
60 mg/mL: 63.52 ± 3.65 mg/mL (106 % of nominal concentration)
200 mg/mL: 207.70 ± 10.17 mg/mL (104 % of nominal concentration)

2. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 21.39 ± 0.29 mg/mL (107 % of nominal concentration)
60 mg/mL: 62.54 ± 1.34 mg/mL (104 % of nominal concentration)
200 mg/mL: 212.84 ± 3.21 mg/mL (106 % of nominal concentration)

3. Analytical sampling (measured concentration ± 95 % confidence interval):
Control: not detectable
20 mg/mL: 20.40 ± 0.42 mg/mL (102 % of nominal concentration)
60 mg/mL: 61.12 ± 2.58 mg/mL (102 % of nominal concentration)
200 mg/mL: 204.60 ± 6.91 mg/mL (102 % of nominal concentration)
Duration of treatment / exposure:
- males: 28 days (14 days pre-mating and 14 days mating/post-mating period)
- females: circa 53 days (14 days pre-mating, for up to five days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing))
Frequency of treatment:
daily; 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a Dose Range Finding (DRF) study (please refer to Section 7.5.1 Repeated dose toxicity: oral: CaBenz_s_Kiss_2021_DRF). Based on the results from the preliminary study, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

- Randomization: before start of dosing, the animals were assigned to their respective dose groups by randomisation based on body weights. It was checked that all animals were within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent body weight. Males and females were randomised separately.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Clinical signs: once a day
Mortality/morbidity: twice daily (at the beginning and end of each working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure, prior to the first dosing (to allow for within-subject comparisons) and at least weekly thereafter, in the morning hours

BODY WEIGHT: Yes
Time schedule for examinations:
- all parental animals: weekly during the pre-exposure period, on the first day of dosing (Day 0, prior to start of dosing), then afterwards at least weekly and prior to the scheduled necropsy (fasted)
- parental females: gestation days 0, 7, 14, 20, and on lactation days 0 (within 24 hours after parturition), 4, 13, and at termination (lactation day 14, fasted)

FOOD CONSUMPTION: Yes
- Time schedule: at least weekly (on body weight measurement days) during pre-mating, mating, pregnancy and lactation.

The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated.

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: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last dose, prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes, pentobarbital anaesthesia
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: total number of erythrocytes, hematocrit value, haemoglobin concentration, mean erythrocyte volume in total sample (MCV), mean haemoglobin volume per red blood cell (RBC) count (MCH), mean hemoglobin concentration of erythrocytes (MCHC), degree of variation in size of the erythrocyte population, reticulocyte (count and percent), total number of leukocytes, neutrophil (count and percent), lymphocyte (count and percent), monocyte (count and percent), eosinophil (count and percent), basophil (count and percent), total number of platelets, mean platelet volume, platelet distribution width, plateletcrit value, activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last dose, prior to scheduled necropsy
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, urea concentration, calcium, cholesterol (total), creatinine, chloride, gamma-glutamyltransferase, glucose, potassium, sodium, inorganic phosphate, total bilirubin, total protein and total bile acids

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: one day after the last dose, prior to scheduled necropsy (samples were taken before 11:30 AM each day, to avoid the diurnal variation of the hormone concentration among animals)
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: all animals
- Parameters checked: thyroxine (T4), triiodothyronine (T3; females only) and thyroid-stimulating hormone (TSH; females only)

URINALYSIS: Yes
- Time schedule for collection of urine: one day after the last dose, prior to scheduled necropsy
- Metabolism cages used for collection of urine: Yes, approximately 16 hours
- Animals fasted: Yes, overnight period of food deprivation
- How many animals per dose group: 5 males / 5 females
- Parameters checked: clarity, colour, volume, leukocyte, nitrite, urobilinogen, protein, pH, blood (occult), specific gravity, ketones, bilirubin, glucose, sediment microscopic examination (white blood cell, red blood cell, epithelial cell, crystals, bacteria and amorphous globlets)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- How many animals: 5 males / 5 females
- Battery of functions tested: sensory activity / grip strength /locomotor activity

Animals were subjected to the functional observation battery, including quantitative assessment of fore/hind grip strength and measurement of landing foot splay (hind limbs only).
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed (Irwin, 1968)*.

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 reflex, 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.

IMMUNOLOGY: No

*Reference:
- Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257, 1968
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy and macroscopic examination were performed on all animals, at the end of the dosing period (after the sample collection for thyroid hormone analysis). The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium).

After sacrifice, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

The following organs were trimmed of fat and weighed in all adult animals: brain, epididymides, heart, kidneys, liver, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, uterus including cervix, adrenal glands, ovaries and thyroid with parathyroid glands.

Testes, epididymides and the other paired organs were weighed individually, but reported together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

HISTOPATHOLOGY: Yes
On completion of the macroscopic examination, the following tissues and organs were retained from all adult animals: gross findings, adrenals, aorta (thoracic and abdominal), brain (representative regions including cerebrum, cerebellum and medulla/pons), epididymis, eye with the optic nerve, oesophagus, femur with marrow, heart (including both ventricles and atria, septum with papillary muscle), kidney, large intestine (caecum, colon and rectum), extraorbital lachrymal gland, Harderian gland, liver (lobes, left lateral, right medial, caudate), lungs with bronchi (was infused with formalin followed by immersion in fixative; 3 lobes, left, right cranial), lymph node (mandibular and mesenteric), ovary, oviduct, pancreas, pituitary, prostate, salivary gland (including mandibular, sublingual and parotid glands), sciatic nerve, seminal vesicle with coagulating gland, skin (subcutis with mammary gland (inguinal)), skeletal muscle (quadriceps), small intestine (duodenum, ileum and jejunum with Peyer’s patches), spinal cord (cervical, thoracic and lumbar), spleen, sternum with marrow, stomach, testis, thymus, thyroid with parathyroid gland, tongue, trachea, urinary bladder, uterus (horns, body and cervix) and vagina.

The eyes with the optic nerve, testes and epidymides were preserved in modified Davidson’s fixative, all other organs in 10 % buffered formalin solution.

In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:
- on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group),
- all macroscopic findings (abnormalities),
- on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups,
- on the liver of all low and mid dose male and female animals,
- on the liver and kidney of the remaining animals (not processed as per guideline) of the control and high dose groups (males and females),
- on the kidney of low and mid dose male animals.


Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, and bone marrow).

Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Statistics:
Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel.

Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:

The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: test item related effects on body weight and body weight gain were observed in the males of the high dose group. Daily administration of the test item caused reduced mean body weight gain in the male high-dose group in all four observation periods: Days 0 - 7 (-53.4 %; p<0.05), Days 7 - 14 (-40.2 %; p < 0.05), Days 14 - 21 (-36.7 %) and Days 21 - 27 (-19.1 %), the first two attaining statistical significance. Consequently, the mean body weight gain of the male high-dose group was also reduced for the entire observation period (Days 0 - 27) by -42.0 % (p < 0.01). The reduced body weight gain was insufficient to result in a significant reduction of the mean body weight, however in the high dose group, as of the first week the mean body weight decreased in comparison to the control steadily throughout the entire treatment period (-1.8 %, -3.3 %, -3.6 % and -3.8 % for the treatment days 7, 14, 21 and 27) resulting in a significant difference in the terminal (fasted) body weight (-6.5 %; p < 0.01).

Please also refer to section "Overall remarks, attachments".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: a test item-related effect on food consumption of the males of the high dose group was observed. A statistical significantly increased mean food consumption was observed in the high dose male group between Days 7 - 14 (+8.6 %; p < 0.01), and Days 21 - 27 (+9.6 %; p < 0.05) compared to the control. Although in the other treatment weeks the differences did not attain statistical significance, the mean food consumption steadily increased by the duration of the treatment compared to the control (+3.7 %, +8.6 %, +9.4 % and +9.6 % for the weeks 1, 2, 3 and 4) resulting in a significant overall effect for the entire period of Days 0 - 27 (+7.8 % p < 0.01). For the periods with significant differences to the control group, all values of food consumption were within the range of the historical control.

Please also refer to section "Overall remarks, attachments".
Food efficiency:
not examined
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):
Males:
- 300 and 1000 mg/kg bw/day: statistically significant increase of bile-acid levels were noted in the mid-, and high dose treated males (+234.2%; p<0.05 and +357.6%; p<0.01, respectively). The differences to the control in the low dose-treated males and in any of the treatment groups of the females did not attain statistical significance. However, a dose-dependency could be observed in the males and the findings was considered to be test item-related in males.

- 1000 mg/kg bw/day: statistically significantly decrease of the globulin levels ( -13.6 %; p < 0.0.1) and consequently an increased albumin/globulin ratio was noted in the high dose-treated male group (+21.8 %; p < 0.05) compared to the control. Furthermore, statistically significant increase of blood urea nitrogen/creatinine ratio was noted in the high dose-treated male group (+31.3 %; p < 0.05). In addition, a statistically significant decrease of total cholesterol was noted in the high dose-treated male group (-24.3 %; p < 0.05) compared to the control. All values were within the historical control data.

Please also refer to section "Overall remarks, attachments".
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
- 1000 mg/kg bw/day: a statistically significant decrease of the T4 level was noted in the high dose-treated males (-45.2%; p<0.01) comapred to the control, with a mean level (29 nmol/L) below of the range of historical controls (31 to 106 nmol/L).

Please also refer to section "Overall remarks, attachments".
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 1000 mg/kg bw/day: there was a statistical significant decrease in the pH of the urine in the high dose male group compared to the control group (control group: pH= 7.0; treatment group: pH = 5.8; p < 0.01).

2) Females
- 100, 300 and 1000 mg/kg bw/day: there was a statistical significant decrease in the pH of the urine in the low dose-, (pH=5.8; p<0.01), in the mid dose-, (pH=5.7; p<0.05) and in the high dose-treated female groups (pH=5.7; p<0.01) compared to the control group (pH = 6.4).

Please also refer to section "Overall remarks, attachments".

Overall, a reduction of the pH is a well-known effect of the benzoic acid and benzoates and therefore this can be regarded as a test item-related effect, but is without toxicological relevance (Lenis et al.,1998b and EC SCAN, 2020)*.

*References:
- Lenis NP, van Diepen JTM, van der Pasch BLCP, Jongbloed AW and Kogut J, 1998b, Dose-response relationship of dietary benzoic acid and buffering capacity on urinary pH in growing pigs, unpublished Report ID-DLO No. 98-77, , Department of Nutrition of Pigs and Poultry, Institute for Animal Science and Health, Lelystad, The Netherlands
- EC SCAN, Opinion of the Scientific Committee on Animal Nutrition on the use of benzoic acid in feedingstuffs for pigs for fattening (2020) https://ec.europa.eu/food/system/files/2020-12/sci-com_scan-old_report_out100.pdf

Please also refer to section "Overall remarks, attachments".
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) Males
- 1000 mg/kg bw/day: a statistical significant higher mean absolute, body weight-, and brain weight normalized, liver weights were noted in high dose-treated males (+16.2 %; p < 0.01 and +24.4 %; p < 0.01 and +15.5 %; p < 0.01, respectively) compared to the control. The findings of the liver in the males of the high dose group were considered as test item-related. Furthermore, a statistical significant higher mean absolute, body weight-, and brain weight-normalized kidney weights were noted in high dose-treated males (+9.8 %; p < 0.01 and +17.8 %; p < 0.01 and +9.1 %; p < 0.01, respectively) compared to the control. The findings can also be considered to be test item-related.

2) Females:
- 100, 300 and 1000 mg/kg bw/day: a statistical significant higher mean absolute, body weight-, and brain weight-normalized kidney weights were noted in the high dose treated females (+19.2 %; p < 0.01 and +23.3 %; p < 0.01 and 19.5 %; p < 0.01, respectively) and in addition also in the low dose treated females (+6.2 %; p < 0.05 and 5.6 %; p < 0.05 and 5.6 %; p < 0.05, respectively) compared to the control. In the mid dose-treated females, the enlargement of the kidney attained statistical significance in the absolute and body weight-normalized values (+9.4 %; p < 0.05 and +6.7 %; p < 0.05, respectively).

Please also refer to section "Overall remarks, attachments".
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1) Males
Liver:
- 300 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 4/12 of mid dose-treated male animals. This phenomenon, observed on the histological picture of hematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

- 1000 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 10/12 male of high dose-treated. This phenomenon, observed on the histological picture of hematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

In the present study, this phenomenon in the liver – based on the incidence – seems to be a test item related lesion. This could be considered as a slight reversible lesion in the liver.

Kidney:
- 300 mg/kg bw/day: histological examination revealed slight focal lymphocytic and histiocytic infiltration in the kidney in 1/12 mid dose male.

- 1000 mg/kg bw/day: histological examination revealed slight focal lymphocytic and histiocytic infiltration in the kidney in 3/12 high dose male animals.

The kidney finding could indicate the initial phase of chronic progressive nephropathy (CPN) of laboratory rats.

2) Females
- 1000 mg/kg bw/day: histological examination revealed vacuolation in the hepatocytes in 9/12 female of high dose-treated. This phenomenon, observed on the histological picture of hematoxylin eosin stained slides is indicative on potential hepatic lipidosis.

In the present study, this phenomenon in the liver – based on the incidence – seems to be a test item related lesion. This could be considered as a slight reversible lesion in the liver.

Please also refer to section "Overall remarks, attachments".
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: during the general clinical and detailed clinical observations, no clinical signs were observed in the current study.

MORTALITY
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: no mortality was seen during the study.

BODY WEIGHT AND WEIGHT CHANGES
1) Males:
- 100 and 300 mg/kg bw/day: no test item related changes were observed in body weight and body weight gain of the low and mid dose group animals compared to control data.

2) Females:
- 100 and 300 mg/kg bw/day: no test item related changes were observed in body weight and body weight gain of the low and mid dose group animals compared to control data.
- 1000 mg/kg bw/day: the female animals of the high dose group showed no statistically significant change in the mean body weight, and body weight gain if compared to the control in any of the observation periods with the exception of the lactation period 0-13 where the mean body weight gain of 39.2g was 23.32% above of the control group (p <0.01). In view of the fact, that one animal of the control group was a clear outlier (body weight gain of -6 g/d, instead of the average body weight gain of +35.6 g of the rest of this dose group) and as the mean body weight gain of 39.2 g is within the range of historical control data for the same period (15 to 68 g), this change is regarded as toxicologically not relevant.

Please also refer to section "Overall remarks, attachments".

FOOD CONSUMPTION
1) Males
- 100 mg/kg bw/day: statistical significant differences to control were observed between Days 21 - 27 (-6.5 %; p < 0.05) in low dose males. As these differences were below 10% and were without dose dependenc and caused no statistical significant changes in the mean body weights or body weight gains, these food intake differences can be regarded as incidental and not related to the test item.

2) Females:
- 100, 300 and 1000 mg/kg bw/day: statistical significant differences to control were observed occasionally, such as between Days 0 - 7 (-4.1 %; p < 0.05) in low dose females and between Days 0 - 7 (+9.6 %; p < 0.01), between Days 7 - 14 (+6.2 %; p < 0.05) and consequently between Days 0-14 (+7.9%; p <0.01) as well as lactation days 7 - 13 (+13.0 %; p < 0.05) in mid dose females and between Days 0 - 7 (+4.7 %; p < 0.01) and Days 0 - 14 (+5.7 %; p < 0.05) in high dose females. As these differences were below 10% and were without dose dependency and caused no statistical significant changes in the mean body weights or body weight gains, these food intake differences can be regarded as incidental and not related to the test item.

- 1000 mg/k bw/day: statistically significant lower mean food consumption was observed in the high dose female group between lactation days 0 - 7 (-24.4 %; p <0.05) and between lactation days 7 - 13 (-20.2 %; p < 0.05) and consequently between lactation days 0 - 13 (-22.1 %; p < 0.05) compared to the control. The reduced food intake of these dams correlates with the smaller mean litter size and reduced mean body weight of the pups in this group.

Please also refer to section "Overall remarks, attachments".

HAEMATOLOGICAL FINDINGS
1) Males
- 100, 300 and 1000 mg/kg bw/day: no test item-related effects were observed for haemtaology at any dose level compared to the control group.

- 100 mg/kg bw/day: compared to the control, the haematocrit and haemoglobin were elevated by +4.9 % (p<0.05) and +4.9 % (p < 0.05), respectively. Because the statistical significance is observed only in the low dose group compared to the control group and due to of the lack of dose dependency as well as these values were with the range of the historical data, these changes can be regarded as incidental.

- 1000 mg/kg bw/day: compared to the control, a statistically significant increase of white blood cells was noted (+67 %; p < 0.05) in high dose males. Due to the broad distribution of the individual data of the control animals and because the mean values were within the range of historical control, these changes can be regarded as incidental and not test item related.

In the high dose-treated males, the relative (RETIC %) and absolute number of reticulocytes (RETIC) were increased by +53.6 % (p < 0.01) and +52.0 % (p < 0.01) compared to the control. An increased reticulocyte number (reticulocytosis) is an indicator of potential blood loss, but in lack of any significant change in the red blood cell parameters (erythrocytes, heamatocrit and heamoglobin) and the lack of similar effect in the female animals and since the mean RETIC values of 4.30 (relative value) and 354.16 (absolute value) are well within the range of the historical control data (2.6 to 4.5 (relative value) and 207.0 to 408.4 (absolute value)) the increased RETIC [%] and RETIC [K/µL] values can be regarded as of no toxicological relevance.

In the high dose-treated males, the platelet distribution width was statistical significantly elevated (+8.4 %, p < 0.05) compared to the control. In view of the lack of dose dependency and lack of changes in the other platelet-related parameters (total number of platelets, mean platelet volume and plateletcrit value) and since the platelet distribution width value of 8.74 is well within the range of the historical control (7.2 to 9.6), the increased platelet distribution width value can be regarded as incidental and not test item related.

2) Females:
- 100, 300 and 1000 mg/kg bw/day: no test item-related effects were observed for haemtaology at any dose level compared to the control group.

- 1000 mg/kg bw/day: compared to the control, a statistically significant decrease of white blood cells was noted (-52.3 %; p < 0.05) in high dose females. Due to the broad distribution of the individual data of the control animals and lack of dose dependency and because the mean values were within the range of historical control, these changes can be regarded as incidental and not test item related.

In the high dose-treated females, the absolute number of neutrophils was decreased by -55.5 % (p < 0.05) compared to the control. In the lack of dose dependency and since the decrease in the relative number of neutrophils (%) was statistically not significant and since the mean value was within the historical control range, this change is regarded as toxicologically not relevant.

In the high dose-treated females, the platelet count and the total platelet volume (paletecrit) were found to be decreased by -22.7 % (p < 0.05) and -19.4 % (p < 0.01), respectively, compared to the control. Furthermore the prothrombin time was increased by +8.3 % (p < 0.05). As the mean values of these parameters were well within historical controls, these changes are regarded as toxicologically not relevant.

Please also refer to section "Overall remarks, attachments".

CLINICAL BIOCHEMISTRY FINDINGS
Females
- 100 and 1000 mg/kg bw/day: at the 1000 mg/kg bw/day dose level, globulin level was decreased by -7.7 % (p < 0.05) without statistically significant change in the albumin/globulin ratio compared to the control. The finding was not considered to be test item-related. Furthermore, a statistically significant decrease of alkaline phosphatase levels were noted in the low and high dose-treated female groups (-29.5 %; p < 0.05 and -30.2 %; p < 0.01, respectively). Since there is a lack of dose-dependency, no statistical significance in the mid dose group and all values were within the historical control data, these changes can be regarded as incidental and not related to the test item. Overall, a decreased level of alkaline phosphatase is not considered to be of toxicological relevance. Lastly, statistically significant increase of the mean natrium level and a decrease of the mean potassium level was noted in the low dose-treated female (+2.4%; p<0.05 and -21.9%; p<0.01, respectively) compared to the control, leading to an increased Na+/K+ ratio in this group (+28.8%; p<0.01). In addition, a statistically significant increase of the mean Na+ level was noted in the high dose female group (+2.4%; p<0.05). As the differences attained statistical significance only in the low dose group and/or no clear dose dependency was observed, these changes can be regarded as incidental and not related to the test item.

Please also refer to section "Overall remarks, attachments".

ENDOCRINE FINDINGS
- 100, 300 and 1000 mg/kg bw/day: the thyroid hormone levels (T3, T4 and TSH ) of the female animals (dams) of any of the dose groups were statistically not different to the control group.

URINALYSIS FINDINGS
Males and females
- 100, 300 and 1000 mg/kg bw/day: no test item related changes were observed in the urinalysis parameters clarity, colour, volume, leukocyte, nitrite, urobilinogen, protein, blood (occult), specific gravity, ketones, bilirubin, glucose and sediment microscopic examination (white blood cell, red blood cell, epithelial cell, crystals, bacteria and amorphous globlets) of males and females of any dose groups compared to the control group.

BEHAVIOUR
Males and females:
- 100, 300 and 1000 mg/kg bw/day: the modified Irwin test did not reveal any test item-related effect for males and females at any dose level. Furthermore, there was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity. The only statistical significant findings was made during the measurement of forelimb grip strength of the low dose result measured during the first trial with the forelimb of the low dose males (first trail only; -14.56%, p<0.05), which can be regarded as incidental.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20 - 30 minutes. The some isolated, statistically significant changes (males of the low and high dose groups at the 40 to 45 minute measurement: -42.3 % (p < 0.05) and -32.2 % (p < 0.05) compared to the control, respectively; females of the mid dose group at the 25 to 30 minute measurement: -61.5 % (p < 0.05) compared to the control group; females of the high dose group at the 0 to 5 minute and 10 to 15 minutes measurements: +27.9 % (p < 0.05) and +35.2 % (p < 0.05) compared to the control, respectively) were without a clear trend or dose response, and the pattern of the movements’ intensity under the whole duration of the test was similar in all dose groups, these changes were considered toxicologically irrelevant, and not test item related. Furthermore, these values were all within the historical control data range.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS
1) Males
Fasted body weight:
- 1000 mg/kg bw/day: a statistical significant lower mean absolute and brain weight normalized, fasted body weight was noted in high dose-treated males (-6.5%; p<0.01 and -7.2%; p<0.01, respectively) compared to the control.

Heart:
- 1000 mg/kg bw/day: a statistical significant higher mean absolute, body weight-, and brain weight normalized, heart weights were noted in high dose-treated males (+12.0%; p<0.01 and +20.0%; p<0.01 and +11.3%; p<0.01, respectively) compared to the control. All values of males were within the range of the historical control data and no findings were made during histopathology of the heart. The findings of the heart were not considered to be of toxicological relevance.

Brain:
- 1000 mg/kg bw/day: higher mean body weight adjusted brain weights were found in high-dose treated males (+7.8%; p<0.01) compared to the control. As the absolute values of the brain weights were almost identical to the control (+0.8%), this body weight-normalized increase is attributable to the reduced body weight of these animals. No significant changes in brain weights of the females were noted.

Epididymis:
- 100 mg/kg bw/day: statistically significant changes were observed for the absolute and body weight-normalized epididymis weights of the males of the low dose group (+6.5%; p<0.05 and+8.7%; p<0.01, respectively) compared to the control. In lack of dose dependency and since these values were within the range of historical control data, these differences can be regarded as incidental.

Thymus:
- 1000 mg/kg bw/day: statistically significant changes were observed for the brain weight-normalized thymus weights of males of the high dose group (-14.1%; p<0.05) compared to the control. In lack of dose dependency and since this value was within the range of historical control data, the difference can be regarded as incidental.

2) Females
Liver:
- 1000 mg/kg bw/day: a statistical significant higher mean body weight-normalized, liver weight (+10.9 %; p < 0.05) were noted in high dose-treated females compared to the control. Since the body weight of the females was decreased in the high dose group compared to the control group (not statistically significant), the difference in liver weight normalized to the body weight between the high dose group and control group was observed and, therefore, this finding was not considered to be test item-related.

Heart:
- 1000 mg/kg bw/day: statistical significant higher mean body weight-, and brain weight normalized, heart weights (+7.9 %; p < 0.05 and +4.6 %; p < 0.01, respectively) were noted compared to the control. In addition, an increased body weight-normalized heart weight was noted in the low dose-treated females (+7.5 %; p < 0.05) compared to the control. All values of females were within the range of the historical control data and no findings were made during histopathology of the heart. The findings of the heart were not considered to be of toxicological relevance.

Uterus:
- 1000 mg/kg bw/day: statistically significant increased uterus weights were noted in the high dose-treated females in the absolute and body weight-normalized values (+16.1%; p<0.01 and +20.4%; p<0.01, respectively) compared to the control. This finding was not considered to be test item-related due to the lack of a dose-dependency.

Thyroid and parathyroid:
- 100 and 1000 mg/kg bw/day: statistically significant increased thyroid and parathyroid weights were noted in the high dose-treated females in the absolute and body weight-, and brain weight-normalized values (+11.8%; p<0.01 and +14.9%; p<0.01 and +12.7%; p<0.05, respectively) and in the absolute and brain-weight normalized values of the low dose-treated females (+12.4%; p<0.01 05 and +12.3%; p<0.05, respectively) compared to the control. No significant change in the thyroid and parathyroid weights of any of the male treatment groups were noted. The findings in the females can be considered to be not test item-related due to the lack of a dose-dependency. Furthermore, all values of the females were within the historical control data.

Please also refer to section "Overall remarks, attachments".

GROSS PATHOLOGICAL FINDINGS
Males and females:
- 0, 100, 300 and 1000 mg/kg bw/day: no external findings were made in males and females of any dose group as well as control group during the macroscopic examination.

Males:
Kidney:
- 1000 mg/kg bw/day: in 1/12 high dose-treated male animal, bilateral multifocal pale discoloration of the kidney was observed. Furthermore, unilateral/bilateral pelvic dilation of the kidney was observed in 2/12 high dose-treated males.

Stomach:
- 1000 mg/kg bw/day: red/dark red multifocal discoloration of the glandular mucosa of the stomach was observed in in 4/12 male high dose animals.

Seminal vesicle with coagulating gland
- 100 mg/kg bw/day: in 1/12 low dose male, bilateral, small seminal vesicle with coagulating gland was noted.

Females:
Stomach:
- 0, 100, and 300 mg/kg bw/day: red/dark red focal or multifocal discoloration of the glandular mucosa of the stomach was observed in 2/11 female control, 2/10 female low dose, 1/11 female mid dose and in 4/12 male high dose animals.

Kidney:
- 300 and 1000 mg/kg bw/day: unilateral/bilateral pelvic dilation of the kidney was observed in 1/11 mid dose-treated female and in 1/10 high dose-treated female.

Uterus:
- 100 and 300 mg/kg bw/day: bilateral dilatation with clear fluid of the body and horns of the uterus observed in 1/2 non pregnant low dose female and in 1/1 mid dose female animal.

Please also refer to section "Overall remarks, attachments"

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
1) Males
Stomach:
- 1000 mg/kg bw/day: focal/multifocal red discoloration in the mucous membrane of the stomach (glandular region) was observed in 4/12 high dose males. Histological examination revealed focal congestion (2/4) or focal erosion (2/4). The focal congestion or focal erosion in the stomach in treated rats is most likely in connection with the treatment procedure (gastric tube) without toxicological significance.

Reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands):
- 0 and 1000 mg/kg bw/day: in the male animals belonging to the high-dose and control groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in all cases.

Kidney:
- 0, 100, 300 and 1000 mg/kg bw/day: pyelectasia in the kidneys (one side) (Control: 1/12, 100 mg/kg bw/day: 2/12; 300 mg/kg bw/day: No. 2/12; 1000 mg/kg bw/day: 2/12) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.

Thyroid:
- 0 and 1000 mg/kg bw/day: histological examination did not reveal pathological lesion (atrophy degeneration, pigmentation, mineralization, amyloidosis, inflammation, proliferation, follicular hyperplasia, C-cell hyperplasia, adenoma, and /or carcinoma) in the investigated thyroid glands of experimental animals belonging to the control and 1000 mg/kg bw/day treated groups.
No morphological difference was detectable in the histological picture of thyroid glands between the different treated and control groups.

2) Females
Stomach:
- 0, 100 and 300 mg/kg bw/day: focal/multifocal red discoloration in the mucous membrane of the stomach (glandular region) was observed in 2/12 control, 2/12 low dose and 1/12 mid dose female animals. Histological examination revealed focal congestion (300 mg/kg bw/day: 1/1) or focal erosion (Control No.: 2/2; 100 mg/kg bw/day: No.: 2/2). The focal congestion or focal erosion in the stomach in treated rats is most likely in connection with the treatment procedure (gastric tube) without toxicological significance.

Reproductive system (ovaries, uterus, cervix, vagina ):
- 0 and 1000 mg/kg bw/day: in the female animals belonging to the high-dose and control groups the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well. The endometrium of nursing mothers was normal as well. No degenerative, inflammatory or other histopathological lesions were detectable, including the high dose treated animals.

- 100 and 300 mg/kg bw/day: in two cases (one animal in the low and mid dose group each) the dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.

Kidney:
The pyelectasia in the kidneys (one side) (Control: 2/12; 300 mg/kg bw/day: 1/1; 1000 mg/kg bw/day: 1/12) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.

Thyroid:
- 0 and 1000 mg/kg bw/day: histological examination did not reveal pathological lesion (atrophy degeneration, pigmentation, mineralization, amyloidosis, inflammation, proliferation, follicular hyperplasia, C-cell hyperplasia, adenoma, and /or carcinoma) in the investigated thyroid glands of experimental animals belonging to the control and 1000 mg/kg bw/day treated groups. In one case of one high dose female single cyst formation (one side) was observed, which can be regarded as incidental.
No morphological difference was detectable in the histological picture of thyroid glands between the different treated and control groups.

Please also refer to section "Overall remarks, attachments"

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
serum/plasma biochemistry
serum/plasma hormone analyses
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422, calcium dibenzoate in 1 % methyl cellulose solution was administered via gavage to groups of male and female Han:WIST rats (n = 12 animals/sex/group) at dose levels of 100, 300 and 1000 mg/kg bw/day. A vehicle control group was run concurrently. The administration occurred daily on 7 days/week. The substance was administered to males for 14 days pre-mating and 14 days mating/post-mating period (28 days in total) and to females for 14 days pre-mating, for up to five days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing) (up to 53 days in total).

No test item related effects were observed in male and female animals for clinical signs, mortality, detailed clinical examination, haematology, behavioural examination, and gross pathology. However, test item related effects on body weight and body weight gain were observed in the males of the 1000 mg/kg bw/day dose group. Daily administration of the test item caused statistically significant reduced mean body weight gain in the males in all four observation periods: Days 0 - 7, Days 7 - 14, Days 14 – 21 (not significant) and Days 21 - 27 (not significant). Consequently, the mean body weight gain of the males was also statistically significant reduced for the entire observation period (Days 0 - 27) by -42.0 %. The reduced body weight gain was insufficient to result in a significant reduction of the mean body weight, however in the 1000 mg/kg bw/day dose group, as of the first week the mean body weight difference to the control steadily decreased throughout the entire treatment period (-1.8 %, -3.3 %, -3.6 % and -3.8 % for the treatment days 7, 14, 21 and 27) resulting in a statistically significant difference in the terminal (fasted) body weight. In addition, a test item-related effect on food consumption of the males of the 1000 mg/kg bw/day dose group was observed. A statistical significantly increased mean food consumption was observed in the males between Days 7 - 14, and Days 21 - 27 compared to the control. Although in the other treatment weeks the differences did not attain statistical significance, the mean food consumption steadily increased by the duration of the treatment compared to the control (+3.7 %, +8.6 %, +9.4 % and +9.6 % for the weeks 1, 2, 3 and 4) resulting in a statistically significant overall effect for the entire period of Days 0 - 27.

A statistically significant increase of bile acid levels was noted in the males of the 300 and 1000 mg/kg bw/day dose groups. The differences to the control in the 100 mg/kg bw/day dose-treated males and in any of the treatment groups of the females did not attain statistical significance. However, a dose-dependency could be observed in the males and the finding was considered to be test item-related in males. Also, at the 1000 mg/kg bw/day dose level a statistically significantly decrease of the globulin levels and a statistically significant increased albumin/globulin ratio was noted in males compared to the control. Furthermore, statistically significant increase of blood urea nitrogen/creatinine ratio was noted in the high dose-treated male group. In addition, a statistically significant decrease of total cholesterol was noted in the high dose-treated male group compared to the control. All values were within the historical control data and are therefore of questionable biological relevance.

A statistically significant decrease of the T4 level was noted in the 1000 mg/kg bw/day dose-treated males compared to the control, with a mean level (29 nmol/L) below the range of historical controls (31 to 106 nmol/L). This finding was considered to be test item related. Also, a test item related effect was observed regarding the pH of urine. The pH of females of any dose level and males of the 1000 mg/kg bw/day dose level were statistically significantly reduced. Overall, a reduction of the pH is a well-known effect of the benzoic acid and benzoates and therefore this can be regarded as a test item-related effect, but is without toxicological relevance (Lenis et al.,1998b and EC SCAN, 2020)*.

In males, a statistically significant higher liver and kidney weights (absolute, relative body-weight-normalised, relative brain-weight-normalized) were noted in males of the 1000 mg/kg bw/day dose group compared to the control. The findings of the liver in the males of the 1000 mg/kg bw/day dose group were considered as test item-related. In addition, a statistically significant higher kidney weight (absolute, relative body-weight-normalised, relative brain-weight-normalized) were noted in the females of the 1000 mg/kg bw/day dose group and also in females of the 100 mg/kg bw/day dose group compared to the control. In the females of the 300 mg/kg bw/day dose group, the enlargement of the kidney attained statistical significance in the absolute and body weight-normalized values.

Histopathological examination showed test item related effects in male and female animals. At the 300 mg/kg bw/day dose level, histological examination of the liver revealed vacuolation in the hepatocytes in 4/12 male animals. The same effect was observed in 10/12 males and 9/12 female of the 1000 mg/kg bw/day dose group. Histological examination of the kidneys revealed slight focal lymphocytic and histiocytic infiltration in the kidneys of 1/12 and 3/12 males of the 300 and 1000 mg/kg bw/day dose levels, respectively, which was also considered to be test item related. The kidney finding could indicate the inital phase of chronic progressive nephropathy (CPN) of laboratory rats, a common disease in aging rats.

Based on the histopathological findings in the liver (vacuolation of hepatocytes) and increased kidney weight (relative and absolute) at 1000 mg/kg bw/day, the No observed adverse effect level (NOAEL) for systemic toxicity is 300 mg/kg bw/day for female rats. Furthermore, based on decreased body weight gain/fasted body weight, increased food consumption, increased bile acid, decreased T4 levels, increased liver and kidney weights as well as histopathological findings in the liver (vacuolation of hepatocytes) and kidney (slight focal lymphocytic and histiocytic infiltration) at 1000 mg/kg bw/day, the NOAEL for systemic toxicity is 300 mg/kg bw/day for male rats.

Although there are histopathological findings in the liver (vacuolation of hepatocytes; 4/12 males) and the kidney (slight focal lymphocytic and histiocytic infiltration; 1/12) at the 300 mg/kg bw/day dose level, the NOAEL of the male rats is derived at 1000 mg/kg bw/day. The finding in liver at the 300 mg/kg bw/ day dose level is considered to indicate an adaptive response of the liver due to the low frequency of occurrence.

*References:
- Lenis NP, van Diepen JTM, van der Pasch BLCP, Jongbloed AW and Kogut J, 1998b, Dose-response relationship of dietary benzoic acid and buffering capacity on urinary pH in growing pigs, unpublished Report ID-DLO No. 98-77, , Department of Nutrition of Pigs and Poultry, Institute for Animal Science and Health, Lelystad, The Netherlands
- EC SCAN, Opinion of the Scientific Committee on Animal Nutrition on the use of benzoic acid in feedingstuffs for pigs for fattening (2020) https://ec.europa.eu/food/system/files/2020-12/sci-com_scan-old_report_out100.pdf