Calcium dibenzoate

Endpoint summary

Description of key information

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422 (2016); GLP) with calcium dibenzoate was conducted in rats. Based on substance-related effects on body weight/body weight gain, food consumption, liver weight, kidney weight, T4 level, bile acid and histopathological findings (liver and kidney) in males as well as on organ weight (kidney) and histopathological findings (liver) in females at the 1000 mg/kg bw/day dose level, the no observed adverse effect level (NOAEL) is 300 mg/kg bw/day for males and females.

 

Furthermore, an extended one-generation reproductive toxicity study according to OECD 443 (2018; GLP) with benzoic acid was conducted in rats. In this higher tier study, no substance-related effects were observed for clinical signs, mortality, body weight, food consumption, haematology, clinical chemistry, T4 and TSH levels, urinalysis, organ weights, macroscopical examination and microscopically examinations and in the F1 generation immunotoxicity, neurotoxicity and neurobehavioural parameters up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL for male and female rats is considered to be greater than 1000 mg/kg bw/day.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-30 to 2021-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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_Rigo 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

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_Rigo 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

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.

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 (-5.9 %; p < 0.05).

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 +23.7 %; 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 +17.1 %; 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 +18.9 %; 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 (-5.9%; p<0.05 and -6.6%; p<0.01, respectively) compared to the control.

Heart:
- 100 and 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 +19.2%; p<0.01 and +11.3%; p<0.01, respectively) compared to the control. In addition, an increased body weight-normalized heart weight was noted in the low dose-treated males (+6.8 5; p < 0.05). 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 (+7.2%; 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 +8.0%; 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 +4.1 %; 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 +12.0%; p<0.05, respectively) and in the absolute and brain-weight normalized values of the low dose-treated females (+12.4%; p<0.05 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"

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

Critical 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).

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

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The data on this study comes from a publication and not from a study report. According to the publication, the study was conducted in accordance with the OECD guideline 443 in its most recent version (2018-06-25) and GLP guidelines by a well-known and experienced laboratory to fulfil a data commitment to the Joint FAO/WHO Expert Committee on Food Additives (JECFA), one of the most renowned expert committees for the evaluation of the safety of chemicals worldwide. It can therefore be assumed that the deficiencies pointed on in the following are not due to the way the study was conducted, but to the nature of the available source, as a publication cannot reflect the wealth of information contained in a full study report. Even if the publication does not address all requirements of an EOGRT study, it can be preconditioned that all required sampling and examinations were performed according to the guideline: Information on the detailed clinical examination conducted in the parental generation not given. The reference does also not included information on the number of parental animals and the kind of parameters investigated for haematology, clinical chemistry and urinalysis. There is no information on timing of measurement of body weight and food consumption for the parental generation. The authors did not list the organs that were investigated for weight and histopathology. Information on investigation of parental females for implantation sites, corpora lutea and growing follicles and examination of the presence of gross anomalies in pups is lacking. A description stating what happened to the surplus offspring after standardisation of the litters on postnatal days 4 and 22 is missing. Lastly, individual data is missing in the reference.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018-06-25

Deviations:

yes

Remarks:

Please refer to the field "Rationale for reliability incl. deficiencies" .

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: STBG4672V

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks
- Weight at study initiation (estimated): males: ca. 275 g; females: ca. 225 g
- Housing: housed 2 - 3 animals/cage in solid-bottom cages containing heat-treated aspen bedding material
- Diet (ad libitum): certified rodent feed (PMI Nutrition International Rodent LabDiet® 5002)
- Water (ad libitum): reverse osmosis-purified municipal water
- Acclimation period: acclimation was conducted, but length of acclimation was not stated.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 26 °C
- Humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test item was incorporated in the dry ground (meal) diet.

Dietary benzoic acid concentrations used in the current study were adjusted weekly for females during lactation (and also during gestation), based on body weight and food consumption data from age-matched historical controls from previous multigenerational studies conducted at the testing facility, to adhere to target dose level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical testing confirmed stability of benzoic acid and homogeneity in feed.

Duration of treatment / exposure:

FO (parental) generation:
- males: 2 weeks prior to mating and continuing until euthanasia (10 - 11 weeks)
- females: 2 weeks prior to mating and continuing throughout mating (2 weeks), gestation (3 weeks) and lactation (3 weeks) until euthanasia (10 - 11 weeks)
F1 genreration:
- up to post natal day 91

Frequency of treatment:

continuously

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

equivalent to 7500 ppm (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

equivalent to 11500 pmm (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 15000 ppm (nominal)

No. of animals per sex per dose:

30 males / 30 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: benzoic acid concentrations were selected based on the results of a dose range-finding (OECD 422-type; OECD 2016) study, supplemented by a 14-day palatability study in adult rats. In the palatability study, benzoic acid was generally well tolerated by both males and non-pregnant females at 7500, 11,500, 15,000 and 19,000 ppm in the diet. Based on these results, constant dietary benzoic acid concentrations of 11,500, 15,000, and 19,000 ppm were selected for the dose range-finding study. Male rats were administered benzoic acid continuously in the diet for 2 weeks prior to mating and continuing until euthanasia (4 weeks). Female rats were administered benzoic acid continuously in the diet for 2 weeks prior to mating and continuing throughout mating (2 weeks), gestation (3 weeks), and mid-lactation (2 weeks), until euthanasia (50–58 days). Dietary concentrations were selected to achieve target benzoic acid intake levels of 0, 750, 1000, and 1250 mg/kg bw/day. While these intake levels were generally achieved in males throughout the study, and in females prior to mating and during gestation, the increased food consumption associated with the increased metabolic demands of lactation resulted in elevated intake of benzoic acid (1539 mg/kg bw/day (Lactation Days (LD) 1–13); 1884 mg/kg bw/day (LD 1–13); and 1732 mg/kg bw/day (LD 1–7), in the 11,500, 15,000, and 19, 000 ppm groups, respectively). The maximum tolerated dose (MTD) for lactating females was exceeded for the 15,000 ppm and 19,000 ppm groups, with associated adverse effects observed including moribundity, mortality and adverse neuropathology (neuronal degeneration/necrosis in the hippocampus and amygdala) in the majority of females (10 of 12) in the 19,000 ppm groups. The neuropathological findings were considered the cause of death/moribundity.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Clinical signs: once daily
Mortality/morbundity: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study, and twice weekly during gestation and lactation for mated females

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: weekly throughout th study, and twice weekly during gestation and lactation for mated females
- Compound intake was calculated based on food consumption (g/kg bw/day) and the appropriate concentration of benzoic acid in the food (ppm or mg/kg)

FOOD EFFICIENCY:
- Body weight gained as a percentage of food consumed: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of termination
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of termination
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: not specified

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at the time of termination
- Animals fasted: not specified
- How many animals: 10 animals/sex/group
- Parameters checked: serum total thyroxine (T4) and thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of termination
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: not specified
- Parameters checked: not specified

Sacrifice and pathology:

GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGHTS
Following necropsy, tissues and organs were collected for organ weights and full histopathological evaluation.

Other examinations:

NEUROTOXICITY and NEUROBEHAVIOURAL EXAMINATION: assessed in F1 generation (for details see entry on same study in section 7.8)

IMMUNOLOGY: assessed in F1 generation (for details see entry on same study in section 7.8)

Statistics:

The animals were used as the experimental unit. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each benzoic acid group to the control group by sex.

Body weights, body weight changes, food consumption, food efficiency, absolute and relative organ weights, clinical pathology and thyroid hormone data were subjected to a parametric one-way ANOVA to determine intergroup differences; if the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett’s test (Dunnett, 1964)* was applied to compare the benzoic acid groups to the control group.

*Reference:

- Dunnett, C.W., 1964. New tables for multiple comparisons with a control. Biometrics 20 (3), 482–491.

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 specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS
- 500, 750 and 1000 mg/kg bw/day: no benzoic acid-related clinical signs were observed in males and females.

MORTALITY
- 500, 750 and 1000 mg/kg bw/day: the test item had no effects on survival in the P0 generation.

BODY WEIGHT AND WEIGHT CHANGES
- 500, 750 and 1000 mg/kg bw/day: the test item had no significant effects on male or female body weights.

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

FOOD CONSUMPTION AND COMPOUND INTAKE
1) Food consumption
- 500, 750 and 1000 mg/kg bw/day: the test item had no significant effects on food consumption
.
2) Compound intake
- 500, 750 and 1000 mg/kg bw/day: for the duration of the study, mean benzoic acid intake was maintained at dose levels within 10% of target levels. Exception for males was noted during the post-mating period where mean food consumption, and consequently test substance intake was 19 – 22 % lower, than target. Reduced food consumption following separation from the females is not uncommon in male rodents. The adjustments made to female dietary concentrations during gestation and lactation successfully allowed for maintenance of target dose levels.

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

HAEMATOLOGICAL FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related haematological findings in males and females at necropsy.

CLINICAL BIOCHEMISTRY FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related serum chemistry findings in males and females at necropsy. No test item related effect was observed for bile acids.

ENDOCRINE FINDINGS
- 500, 750 and 1000 mg/kg bw/day: at termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in males and females.The test item groups were not statistically significant different from the control group.

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

URINALYSIS FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related effects on urinalysis parameters in males and females at necropsy.

ORGAN WEIGHT FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related effects on organ weight in males and females at necropsy.

GROSS PATHOLOGICAL FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related gross macroscopic observations in males and females at necropsy.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related microscopic observations in males and females at necropsy.

IMMUNOTOXICOLOGICAL FINDINGS
-- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related findings (assessed in F1 generation)

NEUROTOXICOLOGICAL and NEUROBEHAVIORAL FINDINGS
- 500, 750 and 1000 mg/kg bw/day: there were no benzoic acid related findings (assessed in F1 generation)

Key result

Dose descriptor:

NOEL

Remarks:

highest dose tested

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

other: lack of any adverse effects on any of the parameters evaluated

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

In an extended one generation reproductive toxicity study (Turnbull et al., 2021) according to OECD 443 (2018), benzoic acid was administered via diet to groups of 30 male and 30 female Sprague-Dawley rats at dose levels of 500, 750 and 1000 mg/kg bw/day. A control group receiving plain diet was run concurrently.

No test item-related effects were observed for clinical signs, mortality, body weight, food consumption, haematology, clinical chemistry, T4 and TSH levels, urinalysis, organ weights, macroscopical examination and microscopically examinations. Assessments in the F1 generation regarding immunotoxicity, neurotoxicity and neurobehavioral parameters were also without any findings.

In conclusion, the NOAEL for general toxicity was determined to be greater than 1000 mg/kg bw/day for male and female rats based on the lack of adverse toxic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Calcium dibenzoate

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (Rigo, 2022), 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. A statistically significant higher mean absolute, body weight-, and brain weight-normalized kidney weights 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.

 

Histological examination of the liver revealed vacuolation in the hepatocytes in 4/12 male animals at the 300 mg/kg bw/day dose level. 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 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) at the 300 mg/kg bw/day dose level, there are no significant changes in hepatic-derived plasma enzymes alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase or gamma-glutamyltransferase at the 300 and also not at the 1000 mg/kg bw/day dose level. 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 histopathological findings in the kidneys of the male rats could indicate the initial phase of CPN. CPN is a rodent specific disease with no apparent similar human kidney disease condition, and thus without toxicological relevance (Hard and Khan 2004, Hard et al (2009), Seely and Hard 2008)*. 

 

*References:

- 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

- Hard GC and Khan KN (2004), A contemporary overview of chronic progressive nephropathy in the laboratory rat, and its significance for human risk assessment, Toxicol Pathol. Mar-Apr 2004;32(2):171-80

- Hard GC et al (2009), A comparison of rat chronic progressive nephropathy with human renal disease-implications for human risk assessment, Crit Rev Toxicol. 39(4):332-46 

- 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

- Seely and Hard (2008), Review Chronic Progressive Nephropathy (CPN) in the Rat: Review of Pathology and Relationship to Renal Tumorigenesis, Journal of Toxicologic Pathology, Vol. 21 Issue 4, 199-205

 

Benzoic acid

 

In an extended one generation reproductive toxicity study (Turnbull et al., 2021) according to OECD 443 (2018), benzoic acid was administered via diet to groups of 30 male and 30 female Sprague-Dawley rats at dose levels of 500, 750 and 1000 mg/kg bw/day. A control group receiving plain diet was run concurrently. No test item-related effects were observed for clinical signs, mortality, body weight, food consumption, haematology, clinical chemistry, T4 and TSH levels, urinalysis, organ weights, macroscopical examination and microscopically examinations and in the F1 generation immunotoxicity, neurotoxicity and neurobehavioural parameters. Therefore, the NOAEL for general toxicity was determined to be greater than 1000 mg/kg bw/day for male and female rats based on the lack of adverse toxic effects.

 

Conclusion

 

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (Rigo, 2022) revealed test item related effects in rats. Based on substance-related effects on body weight/body weight gain, food consumption, liver weight, kidney weight, T4 level, bile acid and histopathological findings (liver and kidney) in males as well as on organ weight (kidney) and histopathological findings (liver) in females at the 1000 mg/kg bw/day dose level, the no observed adverse effect level (NOAEL) is 300 mg/kg bw/day for males and females.

 

Although there are histopathological findings in the liver (vacuolation of hepatocytes; 4/12 males) at the 300 mg/kg bw/day dose level, there are no significant changes in hepatic-derived plasma enzymes alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase or gamma-glutamyltransferase at the 300 and also not at the 1000 mg/kg bw/day dose level. 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 histopathological findings in the kidneys of the male rats could indicate the initial phase of CPN. CPN is a rodent specific disease with no apparent similar human kidney disease condition, and thus without toxicological relevance (Hard and Khan 2004, Hard et al (2009), Seely and Hard 2008).

 

Calcium dibenzoate will dissociate into the two moieties calcium and benzoic acid respectively benzoate in the gastrointestinal tract (EFSA, 2016). Calcium is not known to cause any adverse health effects. Thus the moiety of concern if at all is the benzoate anion.

 

An extended-one generation reproduction toxicity (EOGRT) study with benzoic acid (Turnbull et al., 2021) showed no substance related effects in parental animals for clinical signs, mortality, body weight, food consumption, haematology, clinical chemistry, T4 and TSH levels, urinalysis, organ weights, macroscopical examination and microscopically examinations and in the F1 generation immunotoxicity, neurotoxicity and neurobehavioural parameters up to a dose level of 1000 mg/kg bw/day. Therefore, a NOAEL greater than 1000 mg/kg bw/day was derived for benzoic acid.

 

Overall, the study with calcium dibenzoate is mere a screening test with only limited information for hazard and risk assessment purposes. The available higher-tier EOGRT study does not confirm the above described findings. Calcium dibenzoate will dissociate into the moiety benzoic acid that showed no adverse effects up to a dose level of 1000 mg/kg bw/day in the EOGRT study. In the study, benzoic acid was given at a higher dosage as in the study with calcium dibenzoate, which comprises of 85.8% benzoate, and the EOGRT study uses a longer exposure period (exposure duration: 10 – 11 weeks both sexes) than the screening test (males: 4 weeks; females about 7.5 weeks), uses more animals and thus has more statistical significance and has a much wider scope of investigations. Therefore, the EOGTR study on benzoic acid is more relevant for assessing the repeated dose toxicity of the calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is greater than 1000 mg/kg bw/day (recalculated to calcium dibenzoate of 1166 mg/kg bw/day) and therefore  above the cut-off level for classification STOT RE of 300 mg/kg bw. In addition, calcium is also not known to cause any adverse health effects.

 

In conclusion, calcium dibenzoate is not classified and labelled according to regulation (EC) 1272/2008 and subsequent adaptations. Further testing is not required. For further information on the toxicity of the individual moieties, please refer to the relevant sections in the IUCLID and CSR.

 

References:

• EFSA (2016): Scientific Opinion on the re-evaluation of benzoic acid (E 210), sodium benzoate (E 211), potassium benzoate (E 212) and calcium benzoate (E 213) as food additives. EFSA Journal 14 (3): 4433.

Justification for classification or non-classification

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (Rigo, 2022) revealed test item related effects on body weight/body weight gain, food consumption, liver weight, kidney weight, T4 level, bile acid and histopathological findings (liver and kidney) in males as well as on organ weight (kidney) and histopathological findings (liver) in females at the 1000 mg/kg bw/day dose level. Therefore, the no observed adverse effect level (NOAEL) is 300 mg/kg bw/day for both sexes. However, calcium dibenzoate dissociates into the moieties calcium and benzoic acid. A higher tier study, an extended-one generation reproduction toxicity (EOGRT) study, with benzoic acid showed no substance related effects in parental animals up to a dose level of 1000 mg/kg bw/day. In addition, calcium is also not known to cause adverse health effects.

 

 

 

Overall, the study with calcium dibenzoate is mere a screening test with only limited information for hazard and risk assessment purposes. The available higher-tier EOGRT study does not confirm the above-described findings of the OECD 422 study. In the EOGRT study, benzoic acid was given at a higher dosage as in the study with calcium dibenzoate, which comprises of 85.8% benzoate, and the EOGRT study uses a longer exposure period (exposure duration: 10 – 11 weeks both sexes) than the screening test (males: 4 weeks; females about 7.5 weeks), uses more animals and thus has more statistical significance and has a much wider scope of investigations. Therefore, the EOGRT study on benzoic acid is more relevant for assessing repeated dose toxicity of calcium benzoate than a screening test on this substance. The NOAEL for benzoic acid is greater than 1000 mg/kg bw/day (recalculated to calcium dibenzoate of 1166 mg/kg bw/day). In addition, calcium is also not known to cause any adverse health effects. Therefore, calcium dibenzoate will not be classified and labelled according to regulation (EC) 1272/2008 and subsequent adaptations.